**Stem Cell Aging**

**Stem Cell Aging**

Primož Rožman, Katerina Jazbec and Mojca Jež Additional information is available at the end of the chapter

Primož Rožman, Katerina Jazbec and Mojca Jež

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71764

#### **Abstract**

Stem cells persist throughout life, replacing cells lost to homeostatic turnover, injury, and disease. However, their functions decline with age, which contributes to degeneration and dysfunction. The molecular mechanisms involved in the aging of stem cells are the same as the ones involved in the aging of somatic cells, including telomere shortening, oxidative stress, epigenetic dysregulation, miRNAs changes, alterations of DNA, RNA, proteome, and various cellular organelles. Aging impacts various pathways, such as insulin/insulin-like growth factor 1 (IGF-1), mTOR, FoxO, AMP-activated protein kinase (AMPK), sirtuin, and many others, resulting in senescent stem cells that exhibit functional and numerical impairment. Stem cells have developed special mechanisms to prevent age related damage accumulation and to sustain their stemness properties, however, these mechanisms lose their effectiveness over time. The most fatal consequence of this is found in the immune system, where both innate and adaptive immunity are affected, exhibiting a plethora of defects, including increased autoimmune disease occurrence, elevated tolerance to cancer and chronic inflammatory status. Stem cell therapies call for the best quality of stem cells grafts. Stem cell products should be devoid of cells containing a senescent phenotype, thus a comprehensive knowledge of the biology behind the senescence of stem cells should be taken into account in every cell based therapy.

DOI: 10.5772/intechopen.71764

**Keywords:** molecular mechanisms of aging, senescence, stem cell niche, epigenetic changes, telomere attrition, stem cell pool, mitochondrial changes, proteostasis, immune deterioration, shortened life span

### **1. Introduction – Stem cells aid regeneration and longevity**

An 70 kg adult human body consists of approximately 3.72 × 1013 cells [1]. These trillions of cells are not permanent and a majority of them are constantly renewed throughout our lifetime, although some of them – such as cells in the lenses of our eyes and some of the neurons

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

of our central nervous system – are thought to be an exception. The frequency of renewal depends on the function of the cells and may vary from several hours to several years. A collection of the replacement rates of different cells in our body is given in **Table 1**.

tissues, replacing cells lost to homeostatic turnover, injury, and disease. Stem cells reside in specific anatomic reservoirs, such as bone marrow, and circulate in the organism when needed. SCs represent a very small proportion in adult tissues. It is estimated that the bone marrow of a 70 kg adult human contains around 1.5–1.7 × 1012 cells, among them only 45–120 × 106 are true hematopoietic stem cells (HSCs) that give rise to more frequent progenitors (Jazbec et al. 2017, submitted). The frequencies of stem cells in other tissues are even lower and still

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Adult SCs can typically self-renew and differentiate into multiple cell types within a developing and adult body. Due to their self-renewal capacity they were regarded as immortal reservoirs of youth, however, they are nonetheless susceptible to the age related damages. To prevent or reverse the accumulation of age related damage and epigenetic changes, SCs developed special mechanisms to maintain long telomeres, enhance proteostasis, avoid ROS production and defend against toxic substances. In spite of that, their functions decline with age in a number of tissues, including blood, forebrain, skeletal muscle, skin and all the other

Declines in stem cell functions not only contribute to degeneration and dysfunction of aging tissues, but also negatively affect the life span of the organism [3, 4]. Some strong evidence for SCs as regulators of longevity comes from animal studies. For instance, if in *C. elegans* germline stem cells (GSCs) are eliminated, this almost doubles its lifespan [5] and such a phenomenon is highly conserved [6]. Similarly, if the fruit flies are modified with overexpression of a PGC-1α homolog or a heat-shock response transcription factor and moderate repression of insulin/IGF or JNK signaling, this directly extends their life span [7], implying that improved stem cell function leads to better tissue function, and that stem cell aging underlies the aging

In humans, there is considerable evidence supporting the fact that young stem cells perform better than old ones. Proof of this concept is best documented is the recent multicenter study on the success of hematopoietic stem cell transplantation, which is currently the most popular and efficient cell therapy for malignant diseases. In more than 6000 cases of allogeneic bone marrow transplantation between 2007 and 2011, it was clearly shown that patient survival was significantly better after grafts from young donors (aged 18–32 years) were used. For every 10-year increment in donor age, there was a 5.5% increase in the hazard ratio for overall mortality [8]. This is probably one of the most important findings in this field, suggesting that for regenerative purposes, and other stem cell therapies, grafted stem cells should be young

Adult stem cells express several characteristic features that are specific to stem cells, as well as certain features that are found in any other somatic cell in the body. They express telomerase – an enzyme required for telomere extension that is essential for repeated selfrenewal, they cycle between phases of quiescence and activation needed for the production

a matter of debate.

of tissues and organs.

and devoid of senescent defects.

**2. The biology of stem cell aging**

tissues as reviewed by Schultz et al. [2].

The renewal of adult tissues is enabled by specialized cells that function over the lifetime of an organism, i.e., the stem cells (SCs). They persist throughout life in numerous mammalian


**Table 1.** Cell renewal rates in different tissues of the human body.

tissues, replacing cells lost to homeostatic turnover, injury, and disease. Stem cells reside in specific anatomic reservoirs, such as bone marrow, and circulate in the organism when needed. SCs represent a very small proportion in adult tissues. It is estimated that the bone marrow of a 70 kg adult human contains around 1.5–1.7 × 1012 cells, among them only 45–120 × 106 are true hematopoietic stem cells (HSCs) that give rise to more frequent progenitors (Jazbec et al. 2017, submitted). The frequencies of stem cells in other tissues are even lower and still a matter of debate.

Adult SCs can typically self-renew and differentiate into multiple cell types within a developing and adult body. Due to their self-renewal capacity they were regarded as immortal reservoirs of youth, however, they are nonetheless susceptible to the age related damages. To prevent or reverse the accumulation of age related damage and epigenetic changes, SCs developed special mechanisms to maintain long telomeres, enhance proteostasis, avoid ROS production and defend against toxic substances. In spite of that, their functions decline with age in a number of tissues, including blood, forebrain, skeletal muscle, skin and all the other tissues as reviewed by Schultz et al. [2].

Declines in stem cell functions not only contribute to degeneration and dysfunction of aging tissues, but also negatively affect the life span of the organism [3, 4]. Some strong evidence for SCs as regulators of longevity comes from animal studies. For instance, if in *C. elegans* germline stem cells (GSCs) are eliminated, this almost doubles its lifespan [5] and such a phenomenon is highly conserved [6]. Similarly, if the fruit flies are modified with overexpression of a PGC-1α homolog or a heat-shock response transcription factor and moderate repression of insulin/IGF or JNK signaling, this directly extends their life span [7], implying that improved stem cell function leads to better tissue function, and that stem cell aging underlies the aging of tissues and organs.

In humans, there is considerable evidence supporting the fact that young stem cells perform better than old ones. Proof of this concept is best documented is the recent multicenter study on the success of hematopoietic stem cell transplantation, which is currently the most popular and efficient cell therapy for malignant diseases. In more than 6000 cases of allogeneic bone marrow transplantation between 2007 and 2011, it was clearly shown that patient survival was significantly better after grafts from young donors (aged 18–32 years) were used. For every 10-year increment in donor age, there was a 5.5% increase in the hazard ratio for overall mortality [8]. This is probably one of the most important findings in this field, suggesting that for regenerative purposes, and other stem cell therapies, grafted stem cells should be young and devoid of senescent defects.

### **2. The biology of stem cell aging**

of our central nervous system – are thought to be an exception. The frequency of renewal depends on the function of the cells and may vary from several hours to several years. A col-

The renewal of adult tissues is enabled by specialized cells that function over the lifetime of an organism, i.e., the stem cells (SCs). They persist throughout life in numerous mammalian

lection of the replacement rates of different cells in our body is given in **Table 1**.

**Cell type Turnover time** Small intestine epithelium 2–4 days Stomach 2–9 days Blood neutrophils 1-5 days White blood cells eosinophils 2-5 days Gastrointestinal colon crypt cells 3–4 days Cervix 6 days Lungs alveoli 8 days Tongue taste buds (rat) 10 days Platelets 10 days Bone osteoclasts 2 weeks Intestine Paneth cells 20 days Skin epidermis cells 10–30 days Pancreas beta cells (rat) 20–50 days Blood B cells 4–7 weeks Trachea 1–2 months Hematopoietic stem cells 2 months Sperm (male gametes) 2 months Bone osteoblasts 3 months Red blood cells 4 months Liver hepatocyte cells 0.5–1 year Fat cells 8 years

32 Stem Cells in Clinical Practice and Tissue Engineering

Cardiomyocytes 0.5–10% per year

Central nervous system life time Skeleton 10% per year Lens cells life time Oocytes (female gametes) life time Adapted from: http://bionumbers.hms.harvard.edu/bionumber.aspx?&id=107875

**Table 1.** Cell renewal rates in different tissues of the human body.

Adult stem cells express several characteristic features that are specific to stem cells, as well as certain features that are found in any other somatic cell in the body. They express telomerase – an enzyme required for telomere extension that is essential for repeated selfrenewal, they cycle between phases of quiescence and activation needed for the production of progeny, their chromatin exists in a bivalent state primed for self-renewal or differentiation, they have unique metabolic requirements, they distribute their macromolecules asymmetrically during asymmetric cell divisions, and they reside in niches that regulate their behavior [9].

**2.1. Formation of damaging reactive oxygen species (ROS) and oxidative stress**

and lipid molecules [14].

reviewed by Haines, et al. [18].

erative and survival signals [27].

The free radical theory of aging has been long accepted as the most plausible explanation for the aging process. It was first formulated in the 1950s by Harman who hypothesized that an accumulation of endogenous oxygen radicals (reactive oxygen species, or ROS) occurs, which in turn causes further mitochondrial deterioration and the global cellular damage responsible for the aging and death of all living beings [10]. This theory was then revised in 1972 when mitochondria were identified as being responsible for the initiation of most of the free radical reactions [11]. It was also postulated that life span was determined by the rate of free radical damage to the mitochondria. Mitochondrial respiration, the basis of energy production in all eukaryotes, generates ROS by leaking intermediates from the electron transport chain [12]. In all aerobic organisms, age-related oxidative stress is generated either by exposure to endogenous metabolites or exogenous sources such as radiation (UV, X-ray), and ROS accumulation is the result of an imbalance between free radical production and antioxidant defenses, such as superoxide dismutase that is responsible for scavenging superoxide anions [12, 13]. In fact, oxidative modifications have been shown to occur in DNA, protein,

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35

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Whereas young stem cells contain a spectrum of antioxidant mechanisms, aged stem cells display an inadequate anti-oxidant defense that is associated with functional impairment, including decreased responsiveness to physical environmental cues and decreased resistance to oxidative stress [15]. In several studies, aging stem cells from bone marrow and adipose tissue showed a significantly reduced capacity for coping with oxidative stress with increasing donor age [16, 17]. Therefore, oxidative stress is still recognized as the fundamental underlying component of the aging process, leading to dysregulation of various cellular pathways and the subsequent accumulation of toxic aggregates and cellular debris, and ultimately to the activation of cell death/survival pathways leading to apoptosis, necrosis, or autophagy, as

However, recent developments have forced an intense re-evaluation of the mitochondrial free radical theory of aging after the unexpected observation that increased ROS may paradoxically prolong the lifespan of yeast and *Caenorhabditis elegans* [19–21]. In mice, genetic manipulations, which increased mitochondrial ROS and oxidative damage, did not accelerate aging as one would expect [22, 23]. Furthermore, manipulations that increased antioxidant defenses did not extend longevity [24], and lastly, genetic manipulations that impaired mitochondrial function but did not increase ROS, accelerated aging [25, 26]. There has also been other solid evidence that in response to physiological signals and stress conditions, ROS triggered prolif-

The mitochondrial theory of aging has also been challenged as it has become clear that there exists a rather complicated interplay between various other cellular compartments [28]. Dysfunctional mitochondria can contribute to aging independently of ROS, as demonstrated by studies with mice deficient in DNA polymerase γ [29, 30]. This could happen through a number of mechanisms, for example, mitochondrial deficiencies may affect apoptotic signaling by increasing the propensity of cell's death through mitochondrial membrane

The molecular mechanisms that are involved in the aging of adult stem cells are the same as the ones involved in the aging of the somatic cells. Traits and mechanisms that are affected by aging are present in various populations of stem cells. The age-related decline of stem cells is mainly functional, but in some cases, a decline in stem cell numbers can also be observed. Since many of these mechanisms appear simultaneously, it is practically impossible to trace or determine a single initial damaging agent that causes the cascade of other detrimental sequences. Therefore authors agree that aging is probably the result of multifactorial derangements caused by several causative factors that act in parallel, including the formation of damaging reactive oxygen species (ROS), telomere attrition, DNA damage and mutations, epigenetic changes (alterations of histones, DNA and the consequent dysregulation of gene expression), mitochondrial DNA mutations with mitochondrial decline, changes of microRNAs, ribosomal changes and defects of RNA splicing, changes of proteostasis, changes in cellular polarity, changes in nutrient sensing and metabolism, niche deterioration, improper accumulation of various circulating factors, stem cell pool exhaustion, cellular senescence with cell cycle arrest, and altered intercellular communication (**Table 2**).


**Table 2.** Multifactorial causes of stem cell aging.

<sup>1.</sup> Formation of damaging reactive oxygen species (ROS)

<sup>2.</sup> Mitochondrial DNA mutations, decline of mitochondrial integrity and biogenesis

#### **2.1. Formation of damaging reactive oxygen species (ROS) and oxidative stress**

of progeny, their chromatin exists in a bivalent state primed for self-renewal or differentiation, they have unique metabolic requirements, they distribute their macromolecules asymmetrically during asymmetric cell divisions, and they reside in niches that regulate their

The molecular mechanisms that are involved in the aging of adult stem cells are the same as the ones involved in the aging of the somatic cells. Traits and mechanisms that are affected by aging are present in various populations of stem cells. The age-related decline of stem cells is mainly functional, but in some cases, a decline in stem cell numbers can also be observed. Since many of these mechanisms appear simultaneously, it is practically impossible to trace or determine a single initial damaging agent that causes the cascade of other detrimental sequences. Therefore authors agree that aging is probably the result of multifactorial derangements caused by several causative factors that act in parallel, including the formation of damaging reactive oxygen species (ROS), telomere attrition, DNA damage and mutations, epigenetic changes (alterations of histones, DNA and the consequent dysregulation of gene expression), mitochondrial DNA mutations with mitochondrial decline, changes of microRNAs, ribosomal changes and defects of RNA splicing, changes of proteostasis, changes in cellular polarity, changes in nutrient sensing and metabolism, niche deterioration, improper accumulation of various circulating factors, stem cell pool exhaustion, cellular senescence with cell cycle arrest, and altered intercel-

behavior [9].

34 Stem Cells in Clinical Practice and Tissue Engineering

lular communication (**Table 2**).

1. Formation of damaging reactive oxygen species (ROS)

3. Nuclear damage and nuclear DNA mutations

7. Changes of RNA splicing and ribosomal machinery

4. Telomere shortening /attrition

6. Changes of microRNAs

8. Changes of proteostasis 9. Changes of cell polarity

11. Niche deterioration

13. Stem cell pool exhaustion

10. Metabolism and nutrient sensing

12. Accumulation of various circulating factors

14. Cellular senescence – arrest of the cell cycle 15. Altered intercellular communication

**Table 2.** Multifactorial causes of stem cell aging.

2. Mitochondrial DNA mutations, decline of mitochondrial integrity and biogenesis

5. Epigenetic changes/alterations of histones and DNA and consequent dysregulation of gene expression

The free radical theory of aging has been long accepted as the most plausible explanation for the aging process. It was first formulated in the 1950s by Harman who hypothesized that an accumulation of endogenous oxygen radicals (reactive oxygen species, or ROS) occurs, which in turn causes further mitochondrial deterioration and the global cellular damage responsible for the aging and death of all living beings [10]. This theory was then revised in 1972 when mitochondria were identified as being responsible for the initiation of most of the free radical reactions [11]. It was also postulated that life span was determined by the rate of free radical damage to the mitochondria. Mitochondrial respiration, the basis of energy production in all eukaryotes, generates ROS by leaking intermediates from the electron transport chain [12]. In all aerobic organisms, age-related oxidative stress is generated either by exposure to endogenous metabolites or exogenous sources such as radiation (UV, X-ray), and ROS accumulation is the result of an imbalance between free radical production and antioxidant defenses, such as superoxide dismutase that is responsible for scavenging superoxide anions [12, 13]. In fact, oxidative modifications have been shown to occur in DNA, protein, and lipid molecules [14].

Whereas young stem cells contain a spectrum of antioxidant mechanisms, aged stem cells display an inadequate anti-oxidant defense that is associated with functional impairment, including decreased responsiveness to physical environmental cues and decreased resistance to oxidative stress [15]. In several studies, aging stem cells from bone marrow and adipose tissue showed a significantly reduced capacity for coping with oxidative stress with increasing donor age [16, 17]. Therefore, oxidative stress is still recognized as the fundamental underlying component of the aging process, leading to dysregulation of various cellular pathways and the subsequent accumulation of toxic aggregates and cellular debris, and ultimately to the activation of cell death/survival pathways leading to apoptosis, necrosis, or autophagy, as reviewed by Haines, et al. [18].

However, recent developments have forced an intense re-evaluation of the mitochondrial free radical theory of aging after the unexpected observation that increased ROS may paradoxically prolong the lifespan of yeast and *Caenorhabditis elegans* [19–21]. In mice, genetic manipulations, which increased mitochondrial ROS and oxidative damage, did not accelerate aging as one would expect [22, 23]. Furthermore, manipulations that increased antioxidant defenses did not extend longevity [24], and lastly, genetic manipulations that impaired mitochondrial function but did not increase ROS, accelerated aging [25, 26]. There has also been other solid evidence that in response to physiological signals and stress conditions, ROS triggered proliferative and survival signals [27].

The mitochondrial theory of aging has also been challenged as it has become clear that there exists a rather complicated interplay between various other cellular compartments [28]. Dysfunctional mitochondria can contribute to aging independently of ROS, as demonstrated by studies with mice deficient in DNA polymerase γ [29, 30]. This could happen through a number of mechanisms, for example, mitochondrial deficiencies may affect apoptotic signaling by increasing the propensity of cell's death through mitochondrial membrane permeabilization in response to stress [31], and trigger inflammatory reactions by favoring ROS-mediated and/or permeabilization- facilitated activation of inflammasomes [32]. Also, mitochondrial dysfunction may directly impact cellular signaling and interorganellar crosstalk, by affecting mitochondrion-associated membranes that constitute an interface between the outer mitochondrial membrane and the endoplasmic reticulum [33].

including reduced biogenesis of mitochondria. For instance, in telomerase-deficient mice, it can be a consequence of telomere attrition with subsequent p53-mediated repression of PGC-1α and PGC-1β (peroxisome proliferator-activated receptor gamma coactivator 1 – alpha and –beta, which are the master regulators of mitochondrial biogenesis) [41]. This mitochondrial decline also occurs during physiological aging in wild-type mice and can be partially reversed by telomerase activation [42]. Sirtuin 1 (SIRT1) modulates mitochondrial biogenesis through a process involving the transcriptional co-activator PGC-1α [43] and the removal of damaged mitochondria by autophagy [44]. SIRT3, which is the main mitochondrial deacetylase [45], targets many enzymes involved in energy metabolism, including components of the respiratory chain, tricarboxylic acid cycle, ketogenesis and fatty acid β-oxidation pathways [46]. SIRT3 may also directly control the rate of ROS production by deacetylating manganese superoxide dismutase, a major mitochondrial antioxidant enzyme [47]. Collectively, these results support the idea that sirtuins may act as metabolic sensors to control mitochondrial

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37

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Interestingly, endurance training and alternate-day-fasting may improve healthspan through the capacity to avoid mitochondrial degeneration [50, 51]. It is tempting to speculate that these beneficial effects are mediated, at least in part, through the induction of autophagy, for which both endurance training and fasting constitute potent triggers [52]. However, autophagy induction is probably not the sole mechanism through which a healthy lifestyle can retard aging, since, depending on the precise diet reduction regime, additional longevity pathways

The combination of increased damage and reduced turnover in mitochondria, due to lower biogenesis and reduced clearance, may contribute to the aging process [48]. Some other mechanisms can also affect the mitochondrial bioenergetics and contribute to the aging mitochondrial phenotype, among them the mutations and deletions in mtDNA, oxidation of mitochondrial proteins, destabilization of the macromolecular organization of respiratory chain, defects of the lipid membranes, and defective autophagy that targets deficient

In conclusion we could say that the importance of mitochondria in the basic biology of aging and the pathogenesis of age-associated diseases is stronger than ever, although the emphasis has moved from ROS to other causative aspects. Obviously, besides the mitochondrial dysfunction due to ROS, there exists a complex interplay of several other factors of aging, such as mDNA mutations, changes of lysosome processing, endoplasmic reticulum stress, genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, altered intercellular communication, mitochondrial biogenesis and turnover, energy sensing, apoptosis, senescence, and calcium dynamics. Mitochondria do play one of the key roles in the pathophysiology of aging and events that lead to the aged phenotype, therefore they will increasingly be targeted to prevent and treat chronic diseases and to promote healthy aging [48, 55, 56]. We expect that future studies will determine whether genetic manipulations that decrease the load of mtDNA mutations and other damaging factors, are

function and play a protective role against age-associated diseases [48, 49].

can be activated [53].

mitochondria [54].

able to extend lifespan.

The mitochondrial ROS that were considered the main cause of age related defects actually contribute positively to various signaling pathways and normal cellular responses, such as adaptation to hypoxia, cellular differentiation, autophagy, inflammation, and immune responses, as reviewed recently [28, 34], meaning that ROS are also beneficial for cellular biology.

### **2.2. Mitochondrial DNA mutations, the decline of mitochondrial integrity and biogenesis**

Mitochondrial function has a profound impact on the aging process. Mitochondrial dysfunction can accelerate aging in mammals. It was generally believed that age-related pathology was caused by defects of mitochondria related to oxidative stress, leading to the accumulation of irreparable changes of nucleic acids, proteins, and lipid molecules [14, 35]. But there are also other defects of mitochondria that develop during normal aging. Similar to the nuclear DNA, mitochondrial DNA (mtDNA) is exposed to mutations and deletions in aged cells, which are not found in nuclear DNA, and which also contribute to aging [36]. This is aggravated by the oxidative microenvironment of the mitochondria and the limited efficiency of the mtDNA repair mechanisms [37].

The mutations that can lead to mitochondrial dysfunction and death are now detectable in generated induced pluripotent stem cell (iPSC) lines, i.e., expanded clones from each individual skin or blood cell. As a result, every cell in the iPSC line contains the same mitochondrial DNA (mtDNA) mutations as the original adult cell, and can for this reason be easily sequenced. We now know that to ensure healthy mitochondrial genes, we must screen stem cells for mutations or collect them at a younger age. This may help illuminate the role of mutated mitochondria in degenerative diseases and to assess the patient-derived regenerative products destined for clinical applications [38].

Interestingly, most mtDNA mutations in adult or aged cells appear to be caused by replication errors early in life, rather than by oxidative damage. These mutations may undergo polyclonal expansion and cause respiratory chain dysfunction in different tissues [39]. Studies of accelerated aging in HIV-infected patients treated with anti-retroviral drugs, which interfere with mtDNA replication, have supported the concept of clonal expansion of mtDNA mutations that originated early in life [40].

Aging also affects the biogenesis of mitochondria. Mitochondrial biogenesis is the process by which cells increase their individual mitochondrial mass and copy their number to increase the production of ATP, as a response to greater energy needs. With aging, the reduced efficiency of mitochondrial bioenergetics may be a result of multiple converging mechanisms, including reduced biogenesis of mitochondria. For instance, in telomerase-deficient mice, it can be a consequence of telomere attrition with subsequent p53-mediated repression of PGC-1α and PGC-1β (peroxisome proliferator-activated receptor gamma coactivator 1 – alpha and –beta, which are the master regulators of mitochondrial biogenesis) [41]. This mitochondrial decline also occurs during physiological aging in wild-type mice and can be partially reversed by telomerase activation [42]. Sirtuin 1 (SIRT1) modulates mitochondrial biogenesis through a process involving the transcriptional co-activator PGC-1α [43] and the removal of damaged mitochondria by autophagy [44]. SIRT3, which is the main mitochondrial deacetylase [45], targets many enzymes involved in energy metabolism, including components of the respiratory chain, tricarboxylic acid cycle, ketogenesis and fatty acid β-oxidation pathways [46]. SIRT3 may also directly control the rate of ROS production by deacetylating manganese superoxide dismutase, a major mitochondrial antioxidant enzyme [47]. Collectively, these results support the idea that sirtuins may act as metabolic sensors to control mitochondrial function and play a protective role against age-associated diseases [48, 49].

permeabilization in response to stress [31], and trigger inflammatory reactions by favoring ROS-mediated and/or permeabilization- facilitated activation of inflammasomes [32]. Also, mitochondrial dysfunction may directly impact cellular signaling and interorganellar crosstalk, by affecting mitochondrion-associated membranes that constitute an interface between

The mitochondrial ROS that were considered the main cause of age related defects actually contribute positively to various signaling pathways and normal cellular responses, such as adaptation to hypoxia, cellular differentiation, autophagy, inflammation, and immune responses, as reviewed recently [28, 34], meaning that ROS are also beneficial for cellular

Mitochondrial function has a profound impact on the aging process. Mitochondrial dysfunction can accelerate aging in mammals. It was generally believed that age-related pathology was caused by defects of mitochondria related to oxidative stress, leading to the accumulation of irreparable changes of nucleic acids, proteins, and lipid molecules [14, 35]. But there are also other defects of mitochondria that develop during normal aging. Similar to the nuclear DNA, mitochondrial DNA (mtDNA) is exposed to mutations and deletions in aged cells, which are not found in nuclear DNA, and which also contribute to aging [36]. This is aggravated by the oxidative microenvironment of the mitochondria and the limited efficiency of the

The mutations that can lead to mitochondrial dysfunction and death are now detectable in generated induced pluripotent stem cell (iPSC) lines, i.e., expanded clones from each individual skin or blood cell. As a result, every cell in the iPSC line contains the same mitochondrial DNA (mtDNA) mutations as the original adult cell, and can for this reason be easily sequenced. We now know that to ensure healthy mitochondrial genes, we must screen stem cells for mutations or collect them at a younger age. This may help illuminate the role of mutated mitochondria in degenerative diseases and to assess the patient-derived regenerative

Interestingly, most mtDNA mutations in adult or aged cells appear to be caused by replication errors early in life, rather than by oxidative damage. These mutations may undergo polyclonal expansion and cause respiratory chain dysfunction in different tissues [39]. Studies of accelerated aging in HIV-infected patients treated with anti-retroviral drugs, which interfere with mtDNA replication, have supported the concept of clonal expansion of mtDNA muta-

Aging also affects the biogenesis of mitochondria. Mitochondrial biogenesis is the process by which cells increase their individual mitochondrial mass and copy their number to increase the production of ATP, as a response to greater energy needs. With aging, the reduced efficiency of mitochondrial bioenergetics may be a result of multiple converging mechanisms,

the outer mitochondrial membrane and the endoplasmic reticulum [33].

**2.2. Mitochondrial DNA mutations, the decline of mitochondrial integrity** 

biology.

**and biogenesis**

mtDNA repair mechanisms [37].

36 Stem Cells in Clinical Practice and Tissue Engineering

products destined for clinical applications [38].

tions that originated early in life [40].

Interestingly, endurance training and alternate-day-fasting may improve healthspan through the capacity to avoid mitochondrial degeneration [50, 51]. It is tempting to speculate that these beneficial effects are mediated, at least in part, through the induction of autophagy, for which both endurance training and fasting constitute potent triggers [52]. However, autophagy induction is probably not the sole mechanism through which a healthy lifestyle can retard aging, since, depending on the precise diet reduction regime, additional longevity pathways can be activated [53].

The combination of increased damage and reduced turnover in mitochondria, due to lower biogenesis and reduced clearance, may contribute to the aging process [48]. Some other mechanisms can also affect the mitochondrial bioenergetics and contribute to the aging mitochondrial phenotype, among them the mutations and deletions in mtDNA, oxidation of mitochondrial proteins, destabilization of the macromolecular organization of respiratory chain, defects of the lipid membranes, and defective autophagy that targets deficient mitochondria [54].

In conclusion we could say that the importance of mitochondria in the basic biology of aging and the pathogenesis of age-associated diseases is stronger than ever, although the emphasis has moved from ROS to other causative aspects. Obviously, besides the mitochondrial dysfunction due to ROS, there exists a complex interplay of several other factors of aging, such as mDNA mutations, changes of lysosome processing, endoplasmic reticulum stress, genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, altered intercellular communication, mitochondrial biogenesis and turnover, energy sensing, apoptosis, senescence, and calcium dynamics. Mitochondria do play one of the key roles in the pathophysiology of aging and events that lead to the aged phenotype, therefore they will increasingly be targeted to prevent and treat chronic diseases and to promote healthy aging [48, 55, 56]. We expect that future studies will determine whether genetic manipulations that decrease the load of mtDNA mutations and other damaging factors, are able to extend lifespan.

#### **2.3. Nuclear damage and nuclear DNA mutations**

It is clear that in aged humans and model organisms, somatic mutations accumulate over time within all cells [57]. Other forms of DNA damage, such as chromosomal aneuploidies, copy-number variations and increased clonal mosaicism for large chromosomal anomalies have also been found to be associated with aging [58, 59]. Each time a stem cell replicates its DNA and divides, the likelihood of DNA defects and oncogenic transformations increases. Therefore the lifetime risk of cancer development in a tissue correlates with the number of divisions the stem cells of this particular tissue have undergone [60]. A variety of these DNA alterations can finally affect the essential genes that control the key transcriptional pathways. Such defect cells should be normally eliminated by apoptosis or senescence, however, if this does not happen it may jeopardize tissue and organismal homeostasis. This is especially important in stem cells because the DNA damage has a detrimental impact on their functional competence, i.e., on their role in tissue renewal [61, 62].

to undergo apoptosis, senescence or differentiation, although it is not yet confirmed whether

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It is also known that deficiencies in DNA repair mechanisms cause accelerated aging in mice and underlie several human progeroid syndromes such as Werner syndrome, Bloom syndrome, xeroderma pigmentosum, trichothiodystrophy, Cockayne syndrome, or Seckel syndrome [74–76]. Moreover, transgenic mice overexpressing multidomain protein kinase BUBR1 (budding uninhibited by benzimidazole-related 1), a mitotic checkpoint component that ensures the accurate segregation of chromosomes, exhibit an increased protection against aneuploidy and cancer, and display an extended healthy lifespan [77]. These experimental data prove that

In addition to genomic damage affecting nuclear or mitochondrial DNA, there is evidence that certain defects in the nuclear lamina can also change nuclear architecture and thereby cause genomic instability [78]. Nuclear lamins participate in genome maintenance by providing a scaffold for tethering chromatin and protein complexes that regulate genomic stability [79, 80]. Mutations in genes encoding protein components of this structure, or factors affecting their maturation and dynamics, cause accelerated aging syndromes such as the Hutchinson-Gilford and the Néstor-Guillermo progeria syndromes [81–83]. Alterations of the nuclear lamina and production of an aberrant prelamin A isoform called progerin have also been detected during normal human aging [84]. Since telomere dysfunction also promotes progerin production in normal human fibroblasts upon prolonged *in vitro* culture, this suggests that there exist intimate links between telomere maintenance and progerin expression during normal aging [85]. In addition to these age-associated changes in A-type lamins, lamin B1 levels decline

artificial reinforcement of nuclear DNA repair mechanisms could delay aging [48].

during cell senescence, pointing to its utility as a biomarker of this process [86].

division, and are thus markers for cellular aging and replicative capacity [90].

telomeres and mortality risk, particularly at younger ages [95].

Although accumulation of DNA damage affects the genome near-to-randomly, there are some chromosomal regions that are particularly susceptible to age-related deterioration [87]. Telomeres are repetitive TTAGGG sequences and associated nucleoproteins at the ends of a chromosome that play a critical role in protecting chromosomes from degradation, undesirable recombination, and chromosome fusion [88, 89]. With each somatic cell division, telomeres shorten and this exposes cells to the aging phenotype. Due to inability of the normal DNA replication machinery to completely replicate the telomeric sequences, telomeres in somatic cells shorten with each cell

Mice with shortened or lengthened telomeres exhibit decreased or increased lifespans, respectively [91–93]. Telomere shortening is observed during normal aging both in humans and mice [94]. In humans, recent meta-analyses have indicated a strong relation between short

In contrast to somatic cells, embryonic and adult stem cells express telomerase, a reverse transcriptase enzyme, which catalyzes the extension of telomeric sequences, thereby avoiding telomere attrition and prolonging cellular proliferative life span. While the telomerases are normally absent from most somatic cells, they are active in the stem cells and most cancer

**2.4. Telomere shortening**

these effects are due to an increase in stem cell longevity [2].

An accumulation in DNA damage and mutations leading to stem cell aging has been one of the earliest theories of aging [63]. DNA damage can be caused by external factors (ionizing radiation, ultraviolet radiation or environmental toxins), or by internal factors (ROS and errors in DNA replication). These factors can lead to various DNA lesions such as modifications of bases or sugar residues, the formation of DNA adducts, cross-linking of the DNA strands or the appearance of single and double-strand breaks. Among these lesions, DNA double-strand breaks (DSBs) are particularly lethal because they result in physical cleavage of the DNA backbone. DSBs can occur through replication fork collapse, during the processing of interstrand crosslinks, or following exposure to ionizing radiation [64, 65]. In spite of the fact that cells have evolved at least six different DNA repair pathways to deal with these distinct types of DNA damage [66], there is convincing evidence that with aging, stem cell DNA is also subject to damage. In HSCs, histone H2AX phosphorylation and comet tails, both of which are measures of DNA damage, increase with age [67, 68]. Phosphorylation of *H2AX*, one of several genes coding for histone H2A (one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells), accumulates with age in satellite cells, i.e., stem cells of the muscles [69]. Moreover, aged HSCs display a history of replication stress and decreased expression of DNA helicases, further sensitizing them to future replication challenges [70].

Since mutations are a common daily occurrence, our cells could not survive without DNA repair mechanisms. There are two groups of repairing mechanisms, the first acting to repair DNA single-strand breaks (mismatch repair mechanism, base excision repair mechanism, nucleotide excision mechanism), and the second acting to synchronously repair DNA doublestrand breaks, i.e. homologous recombination and non-homologous end joining (NHEJ). In spite of the repair mechanisms our DNA accumulates mutations, since the genes of repair mechanisms are themselves subject to mutations [71].

Evidence that DNA damage plays a causal role in the aging process includes the observation that mice with defects in DNA damage repair display some aspects of premature aging [72], whereas enhancing DNA repair through increased expression of *SIRT6* increases lifespan [73]. In some situations, DNA damage may also reduce stem cell numbers by causing them to undergo apoptosis, senescence or differentiation, although it is not yet confirmed whether these effects are due to an increase in stem cell longevity [2].

It is also known that deficiencies in DNA repair mechanisms cause accelerated aging in mice and underlie several human progeroid syndromes such as Werner syndrome, Bloom syndrome, xeroderma pigmentosum, trichothiodystrophy, Cockayne syndrome, or Seckel syndrome [74–76]. Moreover, transgenic mice overexpressing multidomain protein kinase BUBR1 (budding uninhibited by benzimidazole-related 1), a mitotic checkpoint component that ensures the accurate segregation of chromosomes, exhibit an increased protection against aneuploidy and cancer, and display an extended healthy lifespan [77]. These experimental data prove that artificial reinforcement of nuclear DNA repair mechanisms could delay aging [48].

In addition to genomic damage affecting nuclear or mitochondrial DNA, there is evidence that certain defects in the nuclear lamina can also change nuclear architecture and thereby cause genomic instability [78]. Nuclear lamins participate in genome maintenance by providing a scaffold for tethering chromatin and protein complexes that regulate genomic stability [79, 80]. Mutations in genes encoding protein components of this structure, or factors affecting their maturation and dynamics, cause accelerated aging syndromes such as the Hutchinson-Gilford and the Néstor-Guillermo progeria syndromes [81–83]. Alterations of the nuclear lamina and production of an aberrant prelamin A isoform called progerin have also been detected during normal human aging [84]. Since telomere dysfunction also promotes progerin production in normal human fibroblasts upon prolonged *in vitro* culture, this suggests that there exist intimate links between telomere maintenance and progerin expression during normal aging [85]. In addition to these age-associated changes in A-type lamins, lamin B1 levels decline during cell senescence, pointing to its utility as a biomarker of this process [86].

#### **2.4. Telomere shortening**

**2.3. Nuclear damage and nuclear DNA mutations**

38 Stem Cells in Clinical Practice and Tissue Engineering

competence, i.e., on their role in tissue renewal [61, 62].

mechanisms are themselves subject to mutations [71].

replication challenges [70].

It is clear that in aged humans and model organisms, somatic mutations accumulate over time within all cells [57]. Other forms of DNA damage, such as chromosomal aneuploidies, copy-number variations and increased clonal mosaicism for large chromosomal anomalies have also been found to be associated with aging [58, 59]. Each time a stem cell replicates its DNA and divides, the likelihood of DNA defects and oncogenic transformations increases. Therefore the lifetime risk of cancer development in a tissue correlates with the number of divisions the stem cells of this particular tissue have undergone [60]. A variety of these DNA alterations can finally affect the essential genes that control the key transcriptional pathways. Such defect cells should be normally eliminated by apoptosis or senescence, however, if this does not happen it may jeopardize tissue and organismal homeostasis. This is especially important in stem cells because the DNA damage has a detrimental impact on their functional

An accumulation in DNA damage and mutations leading to stem cell aging has been one of the earliest theories of aging [63]. DNA damage can be caused by external factors (ionizing radiation, ultraviolet radiation or environmental toxins), or by internal factors (ROS and errors in DNA replication). These factors can lead to various DNA lesions such as modifications of bases or sugar residues, the formation of DNA adducts, cross-linking of the DNA strands or the appearance of single and double-strand breaks. Among these lesions, DNA double-strand breaks (DSBs) are particularly lethal because they result in physical cleavage of the DNA backbone. DSBs can occur through replication fork collapse, during the processing of interstrand crosslinks, or following exposure to ionizing radiation [64, 65]. In spite of the fact that cells have evolved at least six different DNA repair pathways to deal with these distinct types of DNA damage [66], there is convincing evidence that with aging, stem cell DNA is also subject to damage. In HSCs, histone H2AX phosphorylation and comet tails, both of which are measures of DNA damage, increase with age [67, 68]. Phosphorylation of *H2AX*, one of several genes coding for histone H2A (one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells), accumulates with age in satellite cells, i.e., stem cells of the muscles [69]. Moreover, aged HSCs display a history of replication stress and decreased expression of DNA helicases, further sensitizing them to future

Since mutations are a common daily occurrence, our cells could not survive without DNA repair mechanisms. There are two groups of repairing mechanisms, the first acting to repair DNA single-strand breaks (mismatch repair mechanism, base excision repair mechanism, nucleotide excision mechanism), and the second acting to synchronously repair DNA doublestrand breaks, i.e. homologous recombination and non-homologous end joining (NHEJ). In spite of the repair mechanisms our DNA accumulates mutations, since the genes of repair

Evidence that DNA damage plays a causal role in the aging process includes the observation that mice with defects in DNA damage repair display some aspects of premature aging [72], whereas enhancing DNA repair through increased expression of *SIRT6* increases lifespan [73]. In some situations, DNA damage may also reduce stem cell numbers by causing them Although accumulation of DNA damage affects the genome near-to-randomly, there are some chromosomal regions that are particularly susceptible to age-related deterioration [87]. Telomeres are repetitive TTAGGG sequences and associated nucleoproteins at the ends of a chromosome that play a critical role in protecting chromosomes from degradation, undesirable recombination, and chromosome fusion [88, 89]. With each somatic cell division, telomeres shorten and this exposes cells to the aging phenotype. Due to inability of the normal DNA replication machinery to completely replicate the telomeric sequences, telomeres in somatic cells shorten with each cell division, and are thus markers for cellular aging and replicative capacity [90].

Mice with shortened or lengthened telomeres exhibit decreased or increased lifespans, respectively [91–93]. Telomere shortening is observed during normal aging both in humans and mice [94]. In humans, recent meta-analyses have indicated a strong relation between short telomeres and mortality risk, particularly at younger ages [95].

In contrast to somatic cells, embryonic and adult stem cells express telomerase, a reverse transcriptase enzyme, which catalyzes the extension of telomeric sequences, thereby avoiding telomere attrition and prolonging cellular proliferative life span. While the telomerases are normally absent from most somatic cells, they are active in the stem cells and most cancer cells [88, 96]. Mammalian telomerase consists of a telomerase RNA component (TERC) and a telomerase reverse transcriptase (TERT) component. The latter catalyzes the synthesis of new telomeric repeats. Nevertheless, consistent decline in telomere length with age does occur in adult stem cells, suggesting that telomerase activity is insufficient to maintain the replication of these cells indefinitely [90]. So the telomeres of various stem cells, i.e., hematopoietic, neural, germinal and other, do shorten with age [97, 98].

As already mentioned, of all other different types of DNA damage, the one that has the greatest lasting effect on chromatin is the double-strand breaks, which cause a dramatic redistribution of chromatin factors. This is a part of the response to damage that is not fully restored after the repair [110]. Thus, changes in chromatin caused by DNA damage might underlie the

Stem Cell Aging

41

http://dx.doi.org/10.5772/intechopen.71764

The epigenetic changes have now been cataloged. In mice, it has been observed that the level of histone deacetylase SIRT1 decreases with age and that decrease of SIRT1 expression cor-

The expression levels of chromatin modifiers, including components of the SWI-SNF (switch/ sucrose non-fermentable) and PRC (polycomb repressive complex) complexes, histone deacetylases (HDACs) including sirtuins, and DNA methyltransferases, also change with age in stem cells [113, 114]. These changes may underpin declining stem cell function. Indeed, the overexpression of enhancer of zeste homolog 2 (EZH2), a component of PRC2, improves long-term repopulating potential in HSCs [115]. Additionally, in aged HSCs, clusters of genes increase in expression levels based on chromosomal location, suggesting that epigenetic dysregulation engenders regional loss of transcriptional silencing [113]. Taken together, these findings suggest that changes in epigenetic

It is interesting that with aging appear changes that reinforce self-renewal. Sun et al. conducted a comprehensive integrated genomic analysis of young (4 mo) and old (24 mo) murine HSCs by profiling the transcriptome, DNA methylome, and histone modifications. Transcriptome analysis indicated reduced transforming growth factor beta (TGF-β) signaling and perturbation of genes involved in HSC proliferation and differentiation. Aged HSCs showed increased DNA methylation at transcription factor binding sites associated with differentiation-promoting genes, combined with a reduction at genes associated with HSC maintenance. When they profiled the principal regulatory chromatin marks with the use of chromatin immunoprecipitation sequencing (ChIP-seq) they found that the H3K4me3 mark, an activating histone modification, increases with age at loci that regulate HSC self-renewal, potentially underlying

In satellite cells of muscles, H3K4me3 levels modestly decrease with age, whereas levels of the repressive modification H3K27me3 significantly increase with age. It has also been shown that the expression levels of histones themselves decrease with age [117]. The levels of H4K16Ac, another activating modification, decrease with age in HSCs; inhibition of cell division control protein 42 homolog (CDC42) restores H4K16Ac levels to that of young HSCs and reverses

It is not known whether the epigenetic changes in stem cell products affect their clinical efficiency. In our recent study we intended to gain insight into the methylation status of CD34+ enriched cell products intended for autologous CD34+ cell transplantation in patients with cardiomyopathy. We found that the global DNA methylation and hydroxymethylation status as well as the target methylation profile of 94 stem cell transcription factor genes in CD34+ enriched cell products did not differ significantly as compared to initial leukapheresis products. The epigenetic landscape of different cell products can tell us little about the

functional capacity and regenerative properties of CD34+ cells (Rozman et al. [108]).

skewed lineage phenotypes exhibited by aged stem cells [111].

relates with premature aging in mice with increased p53 activity [112].

modifications are a general trait of stem cell aging, which impacts their function.

the increase in HSC number observed with aging [116].

phenotypes of HSC aging in transplantation assays [118].

Telomere exhaustion also explains the limited proliferative capacity of some types of *in vitro* cultured cells, the so-called replicative senescence or Hayflick limit [99, 100]. Indeed, as shown already in the 1990s, ectopic expression of telomerase confers immortality to otherwise mortal cells, without causing oncogenic transformation [101]. Similarly, telomerase deficiency in humans is associated with the premature development of diseases, such as pulmonary fibrosis, dyskeratosis congenita and aplastic anemia, which involve the loss of the regenerative capacity of different tissues [102].

Recent evidence also indicates that aging can be reverted by telomerase activation. In particular, the premature aging of telomerase-deficient mice can be reverted when telomerase is genetically reactivated in these aged mice [103]. Moreover, normal physiological aging can be delayed without increasing the incidence of cancer in adult wild-type mice by pharmacological activation or systemic viral transduction of telomerase [104].

This correlation between telomere length, telomerase activity and age is not completely clear. For example, while telomere length is negatively correlated with age in humans up to 75 years, it is positively correlated with age in the elderly, suggesting that long telomeres contribute to survival in old age [105]. Furthermore, telomere length predicted survival in elderly twins, suggesting that telomeres contribute to longevity in humans even when controlling for the influence of genetic background [106].

There is a good correlation between the expression of human TERT mRNA and the presence of telomerase activity in extracts from tissue culture cells, and normal and cancer tissues, suggesting that human TERT expression is the primary and rate-limiting determinant of telomerase activity [107]. This is important in stem cell therapies, so we have already investigated the importance of longer telomere length of the CD34+ cell grafts used for cell therapy and found that longer telomere length and higher telomerase expression agree with CD34+ cell's increased functional capacity, however the patients with longer CD34+ telomere length did not favorably respond to autologous CD34+ cell transplantation therapy [108].

#### **2.5. Epigenetic changes and consequent dysregulation of gene expression**

The regulation of the chromatin state is important for stem cell function. In Waddington's epigenetic landscape theory, stem cells stand at an undifferentiated epigenetic summit above multiple cell fates [109]. During the differentiation and aging of cells, numerous chromatin and gene expression changes appear progressively in response to cell stress, most notably in response to DNA damage signals. The changes in epigenetic modification of chromatin and histones lead to dysregulation of gene expression. The epigenetic modifications that are observed to change during aging are histone acetylation, histone methylation, and DNA methylation.

As already mentioned, of all other different types of DNA damage, the one that has the greatest lasting effect on chromatin is the double-strand breaks, which cause a dramatic redistribution of chromatin factors. This is a part of the response to damage that is not fully restored after the repair [110]. Thus, changes in chromatin caused by DNA damage might underlie the skewed lineage phenotypes exhibited by aged stem cells [111].

cells [88, 96]. Mammalian telomerase consists of a telomerase RNA component (TERC) and a telomerase reverse transcriptase (TERT) component. The latter catalyzes the synthesis of new telomeric repeats. Nevertheless, consistent decline in telomere length with age does occur in adult stem cells, suggesting that telomerase activity is insufficient to maintain the replication of these cells indefinitely [90]. So the telomeres of various stem cells, i.e., hematopoietic, neu-

Telomere exhaustion also explains the limited proliferative capacity of some types of *in vitro* cultured cells, the so-called replicative senescence or Hayflick limit [99, 100]. Indeed, as shown already in the 1990s, ectopic expression of telomerase confers immortality to otherwise mortal cells, without causing oncogenic transformation [101]. Similarly, telomerase deficiency in humans is associated with the premature development of diseases, such as pulmonary fibrosis, dyskeratosis congenita and aplastic anemia, which involve the loss of the regenerative

Recent evidence also indicates that aging can be reverted by telomerase activation. In particular, the premature aging of telomerase-deficient mice can be reverted when telomerase is genetically reactivated in these aged mice [103]. Moreover, normal physiological aging can be delayed without increasing the incidence of cancer in adult wild-type mice by pharmacologi-

This correlation between telomere length, telomerase activity and age is not completely clear. For example, while telomere length is negatively correlated with age in humans up to 75 years, it is positively correlated with age in the elderly, suggesting that long telomeres contribute to survival in old age [105]. Furthermore, telomere length predicted survival in elderly twins, suggesting that telomeres contribute to longevity in humans even when controlling for

There is a good correlation between the expression of human TERT mRNA and the presence of telomerase activity in extracts from tissue culture cells, and normal and cancer tissues, suggesting that human TERT expression is the primary and rate-limiting determinant of telomerase activity [107]. This is important in stem cell therapies, so we have already investigated the importance of longer telomere length of the CD34+ cell grafts used for cell therapy and found that longer telomere length and higher telomerase expression agree with CD34+ cell's increased functional capacity, however the patients with longer CD34+ telomere length did not favorably respond to autologous CD34+ cell transplantation

The regulation of the chromatin state is important for stem cell function. In Waddington's epigenetic landscape theory, stem cells stand at an undifferentiated epigenetic summit above multiple cell fates [109]. During the differentiation and aging of cells, numerous chromatin and gene expression changes appear progressively in response to cell stress, most notably in response to DNA damage signals. The changes in epigenetic modification of chromatin and histones lead to dysregulation of gene expression. The epigenetic modifications that are observed to change

**2.5. Epigenetic changes and consequent dysregulation of gene expression**

during aging are histone acetylation, histone methylation, and DNA methylation.

ral, germinal and other, do shorten with age [97, 98].

cal activation or systemic viral transduction of telomerase [104].

capacity of different tissues [102].

40 Stem Cells in Clinical Practice and Tissue Engineering

the influence of genetic background [106].

therapy [108].

The epigenetic changes have now been cataloged. In mice, it has been observed that the level of histone deacetylase SIRT1 decreases with age and that decrease of SIRT1 expression correlates with premature aging in mice with increased p53 activity [112].

The expression levels of chromatin modifiers, including components of the SWI-SNF (switch/ sucrose non-fermentable) and PRC (polycomb repressive complex) complexes, histone deacetylases (HDACs) including sirtuins, and DNA methyltransferases, also change with age in stem cells [113, 114]. These changes may underpin declining stem cell function. Indeed, the overexpression of enhancer of zeste homolog 2 (EZH2), a component of PRC2, improves long-term repopulating potential in HSCs [115]. Additionally, in aged HSCs, clusters of genes increase in expression levels based on chromosomal location, suggesting that epigenetic dysregulation engenders regional loss of transcriptional silencing [113]. Taken together, these findings suggest that changes in epigenetic modifications are a general trait of stem cell aging, which impacts their function.

It is interesting that with aging appear changes that reinforce self-renewal. Sun et al. conducted a comprehensive integrated genomic analysis of young (4 mo) and old (24 mo) murine HSCs by profiling the transcriptome, DNA methylome, and histone modifications. Transcriptome analysis indicated reduced transforming growth factor beta (TGF-β) signaling and perturbation of genes involved in HSC proliferation and differentiation. Aged HSCs showed increased DNA methylation at transcription factor binding sites associated with differentiation-promoting genes, combined with a reduction at genes associated with HSC maintenance. When they profiled the principal regulatory chromatin marks with the use of chromatin immunoprecipitation sequencing (ChIP-seq) they found that the H3K4me3 mark, an activating histone modification, increases with age at loci that regulate HSC self-renewal, potentially underlying the increase in HSC number observed with aging [116].

In satellite cells of muscles, H3K4me3 levels modestly decrease with age, whereas levels of the repressive modification H3K27me3 significantly increase with age. It has also been shown that the expression levels of histones themselves decrease with age [117]. The levels of H4K16Ac, another activating modification, decrease with age in HSCs; inhibition of cell division control protein 42 homolog (CDC42) restores H4K16Ac levels to that of young HSCs and reverses phenotypes of HSC aging in transplantation assays [118].

It is not known whether the epigenetic changes in stem cell products affect their clinical efficiency. In our recent study we intended to gain insight into the methylation status of CD34+ enriched cell products intended for autologous CD34+ cell transplantation in patients with cardiomyopathy. We found that the global DNA methylation and hydroxymethylation status as well as the target methylation profile of 94 stem cell transcription factor genes in CD34+ enriched cell products did not differ significantly as compared to initial leukapheresis products. The epigenetic landscape of different cell products can tell us little about the functional capacity and regenerative properties of CD34+ cells (Rozman et al. [108]).

#### **2.6. Changes of microRNA**

Impairments in stem cell function that occur during aging are globally mirrored in the epigenome and transcriptome of HSCs, including the microRNAs. MicroRNAs (miRNAs) are small noncoding evolutionarily conserved RNAs that regulate gene expression primarily at the posttranscriptional level. They act by binding to specific sequences in the 3′ untranslated region of their target genes and causing the transcripts to be degraded by the RNA-induced silencing complex (RISC). The human genome encodes over 1000 miRNAs that appear to target about 60% of other genes. MiRNAs are important posttranscriptional regulators of gene expression and play important and diverse roles in almost all biological and metabolic processes, including early development, cell proliferation, cell cycle regulation, apoptosis, fat metabolism, signal transduction, aging and diseases, as reviewed recently [119].

On the other hand, miRNAs also modulate development of other tissues, such as cardiovascular differentiation of cardiomyocyte progenitor cells and stem cells, including the differentiation of cardiomyocytes, vascular smooth muscle cells, and endothelial cells. They are involved in the regulation of cardiovascular differentiation of human-derived cardiomyocyte progenitor cells, the cardiovascular differentiation of ESCs and iPSCs, in cardiac differentiation of ESCs after myocardial infarction, vascular endothelial growth factor (VEGF) signaling and angiogenesis, which has great therapeutic value for the future regenerative medicine, as reviewed recently by Li et al. [119]. Some other observations comment on the important role miRNAs play in brain development, as well as in later stages of mammalian neuronal maturation and synapse development. Conversely, dysregulation of miRNAs expression has been implicated in developmental defects, cancers and nervous system diseases, as recently

Stem Cell Aging

43

http://dx.doi.org/10.5772/intechopen.71764

Lee et al. have measured the expression levels of 521 small regulatory miRNAs in young and old animals of six mouse strains and found that expression levels of three miRNAs (miR-203-3p, miR-664-3p, and miR-708-5p) were associated with lifespan. Pathway analysis of binding sites for these three miRNAs revealed enrichment of key target genes involved in aging and longevity pathways including mechanistic target of rapamycin (mTOR), forkhead box protein O (FOXO) and mitogen-activated protein kinase (MAPK), most of which also

In conclusion, one could infer that miRNAs have critical roles in stem cell reprogramming, pluripotency maintenance and differentiation, as well as some other important cellular functions. In the future, miRNAs may greatly contribute to stem cell clinical therapy and have

RNA splicing is the editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA). After splicing, introns are removed and exones are joined together. Splicing usually takes place immediately after transcription, and is carried out in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). This results in an mRNA molecule, which can be translated into protein. Splicing enables one gene to generate multiple proteins allowing organisms to generate complexity from

In healthy aging, splicing homeostasis takes place, while deregulation of the splicing machinery is linked to several age-related chronic illnesses. Certain studies point out that defective splicing machinery and de-regulation of RNA splicing acts as a driver of the aging process itself. Studies on the roundworm *C. elegans* show that with age they lose muscle mass, their cutickle thickens, they wrinkle, and they experience declines in fertility and immune functions. The pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in this worm. Recently, Heintz and her colleagues found that splicing could also play a major role in the aging process of humans. Using transcriptomics and in-depth splicing analysis in young and old animals they found defects in global pre-mRNA splicing

reviewed by Murashov [121].

demonstrated associations with longevity [136].

potential applications in regenerative medicine.

a relatively limited number of genes.

**2.7. Changes in RNA splicing and ribosomal machinery**

In stem cells, miRNAs influence properties such as potency, differentiation, self-renewal, and senescence. Different kinds of stem cells possess distinct miRNA expression profiles. Among other things, miRNAs regulate a number of cell functions such as defense mechanisms against ROS, DNA repair, and apoptosis. These properties, and the assumption that miRNAs act as some kind of general switch, make them highly relevant in research on aging [120], especially since specific miRNA expression profiles could be used to terminally differentiate cells from stem cells in order to treat various diseases, including myocardial infarction, neurodegenerative diseases, blood diseases, and muscle diseases [121].

miRNAs regulate the state of stem cells by directly targeting three prime untranslated region (3′-UTR) of pluripotency factors in the section of messenger RNA. For instance, miR-145 miRNA represses the pluripotency of human embryonic stem cells (ESCs) through targeting octamer-binding transcription factor 4 (*Oct4*; also known as *Pou5f1*), sex determining region Y-box 2 (*Sox2*), and kruppel-like factor 4 (*Klf4*) [122]. In addition, miRNAs target the coding regions of transcription factors to modulate stem cell differentiation. miR-296, miR-470, and miR-134 regulate mouse ESC differentiation by targeting the coding regions of *Nanog*, *Oct4*, and *Sox2* [123]. Other classified miRNAs also regulate pluripotency, self-renewal, reprogramming, and differentiation of stem cells [124–128].

miRNAs act as key regulators of hematopoiesis during the proliferation and differentiation of HSCs in mammals. Ectopic expression of AAAGUGC seed-containing miRNAs enhance the primary hematopoietic progenitors [129]. miR-181, miR-223, and miR-142 are preferentially expressed in hematopoietic tissues, with miR-181 significantly promoting B-lymphocyte differentiation [130]. miR-125a is conservatively expressed in long-term HSCs and can increase the number of HSCs by targeting the apoptosis factor Bax1 [131]. Furthermore, overexpression of miR- 125b leads to lethal myeloid leukemia in mice [132]. See the recent review of Li et al. [119].

Besides regulating the ESCs, miRNAs exert several other actions that indirectly impact stem cells and regeneration. For instance, let-7 family and miR-15a/16-1 cluster function as regulators of the cell cycle and tumor suppressors. While miR-29a and miR-29b regulate progression through the cell cycle [133], miR-9 and miR-124a play a critical role in specification of the neural progenitors from ESCs [134, 135].

On the other hand, miRNAs also modulate development of other tissues, such as cardiovascular differentiation of cardiomyocyte progenitor cells and stem cells, including the differentiation of cardiomyocytes, vascular smooth muscle cells, and endothelial cells. They are involved in the regulation of cardiovascular differentiation of human-derived cardiomyocyte progenitor cells, the cardiovascular differentiation of ESCs and iPSCs, in cardiac differentiation of ESCs after myocardial infarction, vascular endothelial growth factor (VEGF) signaling and angiogenesis, which has great therapeutic value for the future regenerative medicine, as reviewed recently by Li et al. [119]. Some other observations comment on the important role miRNAs play in brain development, as well as in later stages of mammalian neuronal maturation and synapse development. Conversely, dysregulation of miRNAs expression has been implicated in developmental defects, cancers and nervous system diseases, as recently reviewed by Murashov [121].

Lee et al. have measured the expression levels of 521 small regulatory miRNAs in young and old animals of six mouse strains and found that expression levels of three miRNAs (miR-203-3p, miR-664-3p, and miR-708-5p) were associated with lifespan. Pathway analysis of binding sites for these three miRNAs revealed enrichment of key target genes involved in aging and longevity pathways including mechanistic target of rapamycin (mTOR), forkhead box protein O (FOXO) and mitogen-activated protein kinase (MAPK), most of which also demonstrated associations with longevity [136].

In conclusion, one could infer that miRNAs have critical roles in stem cell reprogramming, pluripotency maintenance and differentiation, as well as some other important cellular functions. In the future, miRNAs may greatly contribute to stem cell clinical therapy and have potential applications in regenerative medicine.

#### **2.7. Changes in RNA splicing and ribosomal machinery**

**2.6. Changes of microRNA**

42 Stem Cells in Clinical Practice and Tissue Engineering

Impairments in stem cell function that occur during aging are globally mirrored in the epigenome and transcriptome of HSCs, including the microRNAs. MicroRNAs (miRNAs) are small noncoding evolutionarily conserved RNAs that regulate gene expression primarily at the posttranscriptional level. They act by binding to specific sequences in the 3′ untranslated region of their target genes and causing the transcripts to be degraded by the RNA-induced silencing complex (RISC). The human genome encodes over 1000 miRNAs that appear to target about 60% of other genes. MiRNAs are important posttranscriptional regulators of gene expression and play important and diverse roles in almost all biological and metabolic processes, including early development, cell proliferation, cell cycle regulation, apoptosis,

fat metabolism, signal transduction, aging and diseases, as reviewed recently [119].

tive diseases, blood diseases, and muscle diseases [121].

ming, and differentiation of stem cells [124–128].

See the recent review of Li et al. [119].

neural progenitors from ESCs [134, 135].

In stem cells, miRNAs influence properties such as potency, differentiation, self-renewal, and senescence. Different kinds of stem cells possess distinct miRNA expression profiles. Among other things, miRNAs regulate a number of cell functions such as defense mechanisms against ROS, DNA repair, and apoptosis. These properties, and the assumption that miRNAs act as some kind of general switch, make them highly relevant in research on aging [120], especially since specific miRNA expression profiles could be used to terminally differentiate cells from stem cells in order to treat various diseases, including myocardial infarction, neurodegenera-

miRNAs regulate the state of stem cells by directly targeting three prime untranslated region (3′-UTR) of pluripotency factors in the section of messenger RNA. For instance, miR-145 miRNA represses the pluripotency of human embryonic stem cells (ESCs) through targeting octamer-binding transcription factor 4 (*Oct4*; also known as *Pou5f1*), sex determining region Y-box 2 (*Sox2*), and kruppel-like factor 4 (*Klf4*) [122]. In addition, miRNAs target the coding regions of transcription factors to modulate stem cell differentiation. miR-296, miR-470, and miR-134 regulate mouse ESC differentiation by targeting the coding regions of *Nanog*, *Oct4*, and *Sox2* [123]. Other classified miRNAs also regulate pluripotency, self-renewal, reprogram-

miRNAs act as key regulators of hematopoiesis during the proliferation and differentiation of HSCs in mammals. Ectopic expression of AAAGUGC seed-containing miRNAs enhance the primary hematopoietic progenitors [129]. miR-181, miR-223, and miR-142 are preferentially expressed in hematopoietic tissues, with miR-181 significantly promoting B-lymphocyte differentiation [130]. miR-125a is conservatively expressed in long-term HSCs and can increase the number of HSCs by targeting the apoptosis factor Bax1 [131]. Furthermore, overexpression of miR- 125b leads to lethal myeloid leukemia in mice [132].

Besides regulating the ESCs, miRNAs exert several other actions that indirectly impact stem cells and regeneration. For instance, let-7 family and miR-15a/16-1 cluster function as regulators of the cell cycle and tumor suppressors. While miR-29a and miR-29b regulate progression through the cell cycle [133], miR-9 and miR-124a play a critical role in specification of the RNA splicing is the editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA). After splicing, introns are removed and exones are joined together. Splicing usually takes place immediately after transcription, and is carried out in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). This results in an mRNA molecule, which can be translated into protein. Splicing enables one gene to generate multiple proteins allowing organisms to generate complexity from a relatively limited number of genes.

In healthy aging, splicing homeostasis takes place, while deregulation of the splicing machinery is linked to several age-related chronic illnesses. Certain studies point out that defective splicing machinery and de-regulation of RNA splicing acts as a driver of the aging process itself. Studies on the roundworm *C. elegans* show that with age they lose muscle mass, their cutickle thickens, they wrinkle, and they experience declines in fertility and immune functions. The pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in this worm. Recently, Heintz and her colleagues found that splicing could also play a major role in the aging process of humans. Using transcriptomics and in-depth splicing analysis in young and old animals they found defects in global pre-mRNA splicing with age that are reduced by caloric restriction via one particular component of the splicing apparatus, called splicing factor 1 (SFA-1)—a factor also present in humans. They also showed that SFA-1 is specifically required for lifespan extension by caloric restriction and by modulation of the target of rapamycin complex 1 (TORC1) pathway components 5′ AMPactivated protein kinase (AMPK), RAGA-1, and ribosomal protein S6 kinase (RSKS-1/S6 kinase), and demonstrated that overexpression of splicing factor 1 (SFA-1) extends lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in caloric restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy aging [137].

During the evolution the cells developed a variety of mechanisms that maintain and promote proteostasis and slow down the aging. This is performed by an array of quality control mechanisms that preserve the stability and functionality of the proteome. Various mechanisms for the correction of folded proteins have developed, such as the heat-shock family of proteins, as well as the corrective mechanisms for the degradation of misfolded proteins in proteasome or the lysosome [140, 143]. Moreover, there are regulators of age-related proteotoxicity, such as modifier of protein aggregation (MOAG-4), that act through an alternative pathway distinct from molecular chaperones and proteases [144]. The stress-induced synthesis of cytosolic and organelle-specific chaperones is significantly impaired in aging [145]. All these systems function in a coordinated fashion to restore the structure of misfolded polypeptides or to remove and degrade them completely, thus preventing the accumulation of damaged components

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http://dx.doi.org/10.5772/intechopen.71764

As previously mentioned, there are several approaches for maintaining or enhancing proteostasis aimed at activating protein folding and stability mediated by chaperones. A number of animal models support a causative impact of chaperone decline on longevity. In particular, transgenic worms and flies overexpressing chaperones are long-lived [146, 147]. Also, mutant mice deficient in a co-chaperone of the heat-shock family exhibit accelerated aging phenotypes, whereas long-

Moreover, activation of the master regulator of the heat-shock response, the transcription factor heat shock factor 1 (HSF-1), increases longevity and thermotolerance in nematodes [149], while amyloid-binding components can maintain proteostasis during aging and extend lifespan [150]. Pharmacological induction of the heat-shock protein Hsp72 preserves muscle function and delays progression of dystrophic pathology in mouse models of muscular dys-

Small molecules may be also employed as pharmacological chaperones to assure the refolding of damaged proteins and to improve age-related phenotypes in model organisms [152]. For the degradation of unneeded and misfolded proteins there are special protein complexes, named proteasomes, which degrade them with proteolysis, a chemical reaction that breaks peptide bonds. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in syn-

Stem cells can also maintain high levels of autophagy and proteasome activity to clear damaged proteins. For example, autophagy is greater in HSCs and skin stem cells than in surrounding differentiated cells [153]. Although proteasome activity has yet to be characterized in adult stem cells, it has been shown that human ESCs exhibit high proteasome activity [142]. Fly oocytes, which require similar long-term proteome-protection mechanisms as stem cells, maintain high activity of large multi-protein complex 26S proteasome with age, despite the

The activities of the two principal proteolytic systems implicated in protein quality control, namely, the autophagy-lysosomal system and the ubiquitin-proteasome system, decline with

lived mouse strains show a marked up-regulation of some heat-shock proteins [148].

and assuring the continuous renewal of intracellular proteins.

trophy [151].

thesis of new proteins.

decline of its activity in the somatic cells [154].

The ribosomal machinery that is responsible for protein synthesis (translation), i.e., linking amino acids in the order specified by mRNA molecules, consists of two major components: the small ribosomal subunit, which reads the RNA, and the large subunit, which joins amino acids to form a polypeptide chain. Ribosomes contain ribosomal RNA (rRNA) molecules and a variety of highly conserved ribosomal proteins, and similar to other cellular compartments, these are particular targets of aging. After a comprehensive integrated genomic analysis of young and aged cells, consisting of the profiling of transcriptome, DNA methylome, and histone modifications of young and old murine HSCs, Sun et al. found an increased transcription of ribosomal protein and RNA genes, and hypomethylation of rRNA genes [116]. Indeed, inhibition of ribosomal proteins or their regulators has been shown to extend life span in yeast and worms [138, 139]. Although the research has not been focused on the plicing in stem cells we can expect that the splicing homeostasis in stem cells is similarly affected by aging.

#### **2.8. Proteostasis**

The proteostasis or homeostasis of the proteome is a complex system that takes care of the proper folding, functioning, and degradation of cellular proteins. Mechanisms, by which proteostasis is ensured, include regulated protein translation, chaperone assisted protein folding, and protein degradation pathways. Adjusting each of these mechanisms to the requirements of proteins, which need to be correctly folded, is essential for maintaining all cellular functions.

In previous paragraphs it has been already explained that in aged subjects, stem cells display a thoroughly altered proteome. Many studies have demonstrated that proteostasis is altered with aging and that accumulation of misfolded or damaged proteins is an important determinant of the aging process [140]. Indeed, many different proteins involved in cytoskeletal organization, anti-oxidant defense, and other functions are age-dependent and associated with functional impairment of the cell functions, including decreased responsiveness to physical environmental cues and decreased resistance to oxidative stress [15]. Chronic expression of unfolded, misfolded or aggregated proteins contributes to the development of some age-related pathologies, such as Alzheimer's disease, Parkinson's disease and cataracts [141]. Since the passage of altered proteins to progenitor cells during asymmetric division could compromise development and cause aging, proteostasis maintenance in stem cells has an important role in organismal aging [142].

During the evolution the cells developed a variety of mechanisms that maintain and promote proteostasis and slow down the aging. This is performed by an array of quality control mechanisms that preserve the stability and functionality of the proteome. Various mechanisms for the correction of folded proteins have developed, such as the heat-shock family of proteins, as well as the corrective mechanisms for the degradation of misfolded proteins in proteasome or the lysosome [140, 143]. Moreover, there are regulators of age-related proteotoxicity, such as modifier of protein aggregation (MOAG-4), that act through an alternative pathway distinct from molecular chaperones and proteases [144]. The stress-induced synthesis of cytosolic and organelle-specific chaperones is significantly impaired in aging [145]. All these systems function in a coordinated fashion to restore the structure of misfolded polypeptides or to remove and degrade them completely, thus preventing the accumulation of damaged components and assuring the continuous renewal of intracellular proteins.

with age that are reduced by caloric restriction via one particular component of the splicing apparatus, called splicing factor 1 (SFA-1)—a factor also present in humans. They also showed that SFA-1 is specifically required for lifespan extension by caloric restriction and by modulation of the target of rapamycin complex 1 (TORC1) pathway components 5′ AMPactivated protein kinase (AMPK), RAGA-1, and ribosomal protein S6 kinase (RSKS-1/S6 kinase), and demonstrated that overexpression of splicing factor 1 (SFA-1) extends lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in caloric restriction longevity and suggest that modulation of specific spliceosome components may prolong

The ribosomal machinery that is responsible for protein synthesis (translation), i.e., linking amino acids in the order specified by mRNA molecules, consists of two major components: the small ribosomal subunit, which reads the RNA, and the large subunit, which joins amino acids to form a polypeptide chain. Ribosomes contain ribosomal RNA (rRNA) molecules and a variety of highly conserved ribosomal proteins, and similar to other cellular compartments, these are particular targets of aging. After a comprehensive integrated genomic analysis of young and aged cells, consisting of the profiling of transcriptome, DNA methylome, and histone modifications of young and old murine HSCs, Sun et al. found an increased transcription of ribosomal protein and RNA genes, and hypomethylation of rRNA genes [116]. Indeed, inhibition of ribosomal proteins or their regulators has been shown to extend life span in yeast and worms [138, 139]. Although the research has not been focused on the plicing in stem cells we can expect that the splicing homeostasis in stem cells is similarly affected by aging.

The proteostasis or homeostasis of the proteome is a complex system that takes care of the proper folding, functioning, and degradation of cellular proteins. Mechanisms, by which proteostasis is ensured, include regulated protein translation, chaperone assisted protein folding, and protein degradation pathways. Adjusting each of these mechanisms to the requirements of proteins, which need to be correctly folded, is essential for maintaining all

In previous paragraphs it has been already explained that in aged subjects, stem cells display a thoroughly altered proteome. Many studies have demonstrated that proteostasis is altered with aging and that accumulation of misfolded or damaged proteins is an important determinant of the aging process [140]. Indeed, many different proteins involved in cytoskeletal organization, anti-oxidant defense, and other functions are age-dependent and associated with functional impairment of the cell functions, including decreased responsiveness to physical environmental cues and decreased resistance to oxidative stress [15]. Chronic expression of unfolded, misfolded or aggregated proteins contributes to the development of some age-related pathologies, such as Alzheimer's disease, Parkinson's disease and cataracts [141]. Since the passage of altered proteins to progenitor cells during asymmetric division could compromise development and cause aging, proteostasis maintenance in stem cells has an

healthy aging [137].

44 Stem Cells in Clinical Practice and Tissue Engineering

**2.8. Proteostasis**

cellular functions.

important role in organismal aging [142].

As previously mentioned, there are several approaches for maintaining or enhancing proteostasis aimed at activating protein folding and stability mediated by chaperones. A number of animal models support a causative impact of chaperone decline on longevity. In particular, transgenic worms and flies overexpressing chaperones are long-lived [146, 147]. Also, mutant mice deficient in a co-chaperone of the heat-shock family exhibit accelerated aging phenotypes, whereas longlived mouse strains show a marked up-regulation of some heat-shock proteins [148].

Moreover, activation of the master regulator of the heat-shock response, the transcription factor heat shock factor 1 (HSF-1), increases longevity and thermotolerance in nematodes [149], while amyloid-binding components can maintain proteostasis during aging and extend lifespan [150]. Pharmacological induction of the heat-shock protein Hsp72 preserves muscle function and delays progression of dystrophic pathology in mouse models of muscular dystrophy [151].

Small molecules may be also employed as pharmacological chaperones to assure the refolding of damaged proteins and to improve age-related phenotypes in model organisms [152].

For the degradation of unneeded and misfolded proteins there are special protein complexes, named proteasomes, which degrade them with proteolysis, a chemical reaction that breaks peptide bonds. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesis of new proteins.

Stem cells can also maintain high levels of autophagy and proteasome activity to clear damaged proteins. For example, autophagy is greater in HSCs and skin stem cells than in surrounding differentiated cells [153]. Although proteasome activity has yet to be characterized in adult stem cells, it has been shown that human ESCs exhibit high proteasome activity [142]. Fly oocytes, which require similar long-term proteome-protection mechanisms as stem cells, maintain high activity of large multi-protein complex 26S proteasome with age, despite the decline of its activity in the somatic cells [154].

The activities of the two principal proteolytic systems implicated in protein quality control, namely, the autophagy-lysosomal system and the ubiquitin-proteasome system, decline with aging [155, 156], supporting the idea that collapsing proteostasis constitutes a common feature of old age. In relation to the proteasome, activation of epidermal growth factor (EGF) signaling extends longevity in nematodes by increasing the expression of various components of the ubiquitin-proteasome system activators accelerates the clearance of toxic proteins in human cultured cells [157]. Moreover, increased expression of the 26S proteasome subunit RPN-6 by the FOXO transcription factor DAF-16 confers proteotoxic stress resistance and extends lifespan in *C. elegans* [158].

The asymmetric division of stem cells first requires that a cell be polarized and several studies demonstrate that aged germinal stem cells (GSCs) and HSCs are less able to perform such polarized divisions, suggesting that loss of polarity contributes to stem cell aging [169]. Other data on HSCs suggest that changes in age-related Wnt signaling are a cause of this loss of polarity [170]. This process also appears to occur in satellite cells [171]. There is certain disagreement as to whether polarized division occurs in other stem cell populations, such as intestinal, hair follicle, neural or germline stem cells, as reviewed by Yennek and Tajbakhsh in 2013 [172].

Stem Cell Aging

47

http://dx.doi.org/10.5772/intechopen.71764

Metabolic status plays an important role in stem cell aging [2]. Similar to other cells, stem cells generate energy via glycolysis or oxidative phosphorylation. Quiescent stem cells generally rely upon glycolysis, perhaps because this reduces the abundance of ROS [142]. Many adult stem cells also reside in hypoxic niches, perhaps as a part of a mechanism to limit ROS production [173].

For the provision of necessary energy, proliferating stem cells rely on the oxidative phosphorylation, which predisposes them to oxidative damage and cellular dysfunction. Therefore the molecules that scavenge ROS or enable the overexpression of the transcription factor NRF2, which regulates the response to oxidative stress, reduce the aged phenotype of old cell.

The most robust longevity-extending intervention across species is caloric restriction (CR). For example, CR increases the abundance of satellite cells in muscles [174] and improves the function of many stem cell populations, including HSCs in mice [175] and GSCs in flies [176]. CR also promotes ISC self-renewal in mice by induction of the enzyme BST1 in Paneth cells, which form the niche. BST1 then converts NAD+ to the paracrine signal cyclic ADP ribose (cADPR), which is sensed by the ISCs [177]. Pathways and factors implicated in mediating the response of stem cells to CR that extend lifespan, include insulin and IGF-1 signaling (IIS) pathway, target of rapamycin (TOR) signaling, AMPK, sirtuins and FOXO transcription factors [178].

Recent studies also show that HSCs and satellite cells increase glucose and glutamine metabolism during activation [179] — an alteration that mimics the Warburg effect in cancer cells. Similarly, in skeletal muscle, aging is associated with pseudohypoxia and Warburg-like metabolism, which compromise cellular function [180] and promote oncogenic transformation [181]. Glucose is the main nutrient in the cell, whereas insulin informs cells about the presence of glucose. The intracellular signaling pathway that governs insulin is the same as that elicited by IGF-1, which is, together with the growth hormone (GH), produced by the anterior pituitary, and is the secondary mediator of the somatotrophic axis in mammals. For this reason, IGF-1 and insulin signaling are known as the "insulin and IGF-1 signaling" (IIS) pathway. GH and IGF-1 levels decline during normal aging, as well as in mouse models of premature aging [182]. Remarkably, the IIS pathway is the most conserved aging-controlling pathway

**2.10. Changes in metabolism and nutrient sensing**

*2.10.1. Caloric restriction*

*2.10.2. Glucose metabolism*

Regarding autophagy, transgenic mice with an extra copy of the chaperone-mediated autophagy receptor lysosome-associated membrane protein 2a (LAMP2a) do not experience agingassociated decline in autophagic activity and preserve improved hepatic function with aging [159]. This is a promising example of genetic manipulations that improve proteostasis and delay aging in mammals [159]. Functional decline in the cellular proteolytic machinery leads to the formation of an autofluorescent protein called lipofuscin, which can be used as a biomarker of aging [160]. Based on the given data it is obvious that SCs are a subject of age related changes of proteostasis and further studies will probably focus on proteostasis maintanance in SCs.

#### **2.9. Changes of cell polarity**

In order to prevent the accumulation of damaged components, stem cells developed diverse mechanisms such as the asymmetric segregation of damaged proteins and enhanced proteostasis. After a symmetric division, stem cells produce two daughter cells with the same fate, whereas after asymmetric division they produce one daughter stem cell and one differentiating daughter cell. During the asymmetric division, damaged components such as damaged DNA, replicating circular DNA, carbonylated proteins and damaged organelles are distributed into the differentiating cell, whereas the daughter stem cell remains youthful [161, 162]. In a similar way, stem cells have been shown to asymmetrically segregate damaged proteins and mitochondria into the progeny, which retains the stemness of the mother cell [163, 164]. A similar evolutionary principle enables that the parental strand of DNA is always sequestered in the daughter stem cell, whereas the strand synthesized during S phase, which might contain errors from replication, is directed to the differentiating daughter cell [165]. In this way the non-random strand segregation serves to avoid mutations and to control the inheritance of epigenetic state [166]. It was shown that the distribution of epigenetic modifications on mitotic chromosomes differs, which means that the bias is generated non-randomly during chromatid segregation. In *Drosophila* male GSCs, the histone modifications present in the stem cells are distinct from those in the differentiating daughter cells, which helps to retain pre-existing histones in the mother stem cell while imparting newly synthesized histones to the daughter cell. This retention of pre-existing histones in the stem cells is a prerequisite for maintaining their ability to self-renew. Different epigenetic modifications potentially lead to variations in the otherwise equivalent chromatids that segregate during asymmetric cell divisions [167].

There is accumulating evidence that other organelles are also non-randomly distributed between daughter cells. Numerous organelles have been widely studied for their asymmetric segregation in non-mammals and mammals, such as mitochondria, centrioles of the centrosome, and midbody, as well as different protein complexes [168].

The asymmetric division of stem cells first requires that a cell be polarized and several studies demonstrate that aged germinal stem cells (GSCs) and HSCs are less able to perform such polarized divisions, suggesting that loss of polarity contributes to stem cell aging [169]. Other data on HSCs suggest that changes in age-related Wnt signaling are a cause of this loss of polarity [170]. This process also appears to occur in satellite cells [171]. There is certain disagreement as to whether polarized division occurs in other stem cell populations, such as intestinal, hair follicle, neural or germline stem cells, as reviewed by Yennek and Tajbakhsh in 2013 [172].

#### **2.10. Changes in metabolism and nutrient sensing**

Metabolic status plays an important role in stem cell aging [2]. Similar to other cells, stem cells generate energy via glycolysis or oxidative phosphorylation. Quiescent stem cells generally rely upon glycolysis, perhaps because this reduces the abundance of ROS [142]. Many adult stem cells also reside in hypoxic niches, perhaps as a part of a mechanism to limit ROS production [173].

For the provision of necessary energy, proliferating stem cells rely on the oxidative phosphorylation, which predisposes them to oxidative damage and cellular dysfunction. Therefore the molecules that scavenge ROS or enable the overexpression of the transcription factor NRF2, which regulates the response to oxidative stress, reduce the aged phenotype of old cell.

#### *2.10.1. Caloric restriction*

aging [155, 156], supporting the idea that collapsing proteostasis constitutes a common feature of old age. In relation to the proteasome, activation of epidermal growth factor (EGF) signaling extends longevity in nematodes by increasing the expression of various components of the ubiquitin-proteasome system activators accelerates the clearance of toxic proteins in human cultured cells [157]. Moreover, increased expression of the 26S proteasome subunit RPN-6 by the FOXO transcription factor DAF-16 confers proteotoxic stress resistance and

Regarding autophagy, transgenic mice with an extra copy of the chaperone-mediated autophagy receptor lysosome-associated membrane protein 2a (LAMP2a) do not experience agingassociated decline in autophagic activity and preserve improved hepatic function with aging [159]. This is a promising example of genetic manipulations that improve proteostasis and delay aging in mammals [159]. Functional decline in the cellular proteolytic machinery leads to the formation of an autofluorescent protein called lipofuscin, which can be used as a biomarker of aging [160]. Based on the given data it is obvious that SCs are a subject of age related changes of proteostasis and further studies will probably focus on proteostasis maintanance in SCs.

In order to prevent the accumulation of damaged components, stem cells developed diverse mechanisms such as the asymmetric segregation of damaged proteins and enhanced proteostasis. After a symmetric division, stem cells produce two daughter cells with the same fate, whereas after asymmetric division they produce one daughter stem cell and one differentiating daughter cell. During the asymmetric division, damaged components such as damaged DNA, replicating circular DNA, carbonylated proteins and damaged organelles are distributed into the differentiating cell, whereas the daughter stem cell remains youthful [161, 162]. In a similar way, stem cells have been shown to asymmetrically segregate damaged proteins and mitochondria into the progeny, which retains the stemness of the mother cell [163, 164]. A similar evolutionary principle enables that the parental strand of DNA is always sequestered in the daughter stem cell, whereas the strand synthesized during S phase, which might contain errors from replication, is directed to the differentiating daughter cell [165]. In this way the non-random strand segregation serves to avoid mutations and to control the inheritance of epigenetic state [166]. It was shown that the distribution of epigenetic modifications on mitotic chromosomes differs, which means that the bias is generated non-randomly during chromatid segregation. In *Drosophila* male GSCs, the histone modifications present in the stem cells are distinct from those in the differentiating daughter cells, which helps to retain pre-existing histones in the mother stem cell while imparting newly synthesized histones to the daughter cell. This retention of pre-existing histones in the stem cells is a prerequisite for maintaining their ability to self-renew. Different epigenetic modifications potentially lead to variations in the otherwise equivalent chromatids that

There is accumulating evidence that other organelles are also non-randomly distributed between daughter cells. Numerous organelles have been widely studied for their asymmetric segregation in non-mammals and mammals, such as mitochondria, centrioles of the centro-

extends lifespan in *C. elegans* [158].

46 Stem Cells in Clinical Practice and Tissue Engineering

**2.9. Changes of cell polarity**

segregate during asymmetric cell divisions [167].

some, and midbody, as well as different protein complexes [168].

The most robust longevity-extending intervention across species is caloric restriction (CR). For example, CR increases the abundance of satellite cells in muscles [174] and improves the function of many stem cell populations, including HSCs in mice [175] and GSCs in flies [176].

CR also promotes ISC self-renewal in mice by induction of the enzyme BST1 in Paneth cells, which form the niche. BST1 then converts NAD+ to the paracrine signal cyclic ADP ribose (cADPR), which is sensed by the ISCs [177]. Pathways and factors implicated in mediating the response of stem cells to CR that extend lifespan, include insulin and IGF-1 signaling (IIS) pathway, target of rapamycin (TOR) signaling, AMPK, sirtuins and FOXO transcription factors [178].

#### *2.10.2. Glucose metabolism*

Recent studies also show that HSCs and satellite cells increase glucose and glutamine metabolism during activation [179] — an alteration that mimics the Warburg effect in cancer cells. Similarly, in skeletal muscle, aging is associated with pseudohypoxia and Warburg-like metabolism, which compromise cellular function [180] and promote oncogenic transformation [181].

Glucose is the main nutrient in the cell, whereas insulin informs cells about the presence of glucose. The intracellular signaling pathway that governs insulin is the same as that elicited by IGF-1, which is, together with the growth hormone (GH), produced by the anterior pituitary, and is the secondary mediator of the somatotrophic axis in mammals. For this reason, IGF-1 and insulin signaling are known as the "insulin and IGF-1 signaling" (IIS) pathway. GH and IGF-1 levels decline during normal aging, as well as in mouse models of premature aging [182]. Remarkably, the IIS pathway is the most conserved aging-controlling pathway in evolution and among its multiple targets are the FOXO family of transcription factors and the mTOR complexes, which are also involved in aging and conserved through evolution. Similarly, genetic polymorphisms or mutations that reduce the functions of GH, IGF-1 receptor, insulin receptor or downstream intracellular effectors such as protein kinase B (PKB), also known as AKT, mTOR and FOXO, influence longevity both in humans and in model organisms, further illustrating the major impact these pathways have on longevity [53].

to determine the extent to which beneficial and damaging effects of TOR inhibition can be sepa-

Stem Cell Aging

49

http://dx.doi.org/10.5772/intechopen.71764

There are two another nutrient sensors, AMPK and sirtuins, which act in the completely opposite direction of the IIS and mTOR. Instead of signaling nutrient abundance and anabolism, they signal nutrient scarcity and catabolism. Accordingly, their up-regulation promotes a healthy aging. AMPK activation has multiple effects on metabolism and, remarkably, shuts off mTORC1 [193]. There is evidence indicating that AMPK activation may mediate lifespan-

The role of sirtuins in lifespan regulation has been discussed above (see section 2.2 on DNA mutations). In addition, SIRT1 can deacetylate and activate the PPARγ co-activator 1α (PGC-1α) [43]. PGC-1α orchestrates a complex metabolic response that includes mitochondriogenesis, enhanced anti-oxidant defenses, and improved fatty acid oxidation [196]. Moreover, SIRT1 and AMPK can engage in a positive feedback loop, thus connecting both

Collectively, currently available evidence strongly supports the idea that anabolic signaling accelerates aging, and decreased nutrient signaling extends longevity [183]. Consistent with the relevance of deregulated nutrient-sensing as a hallmark of aging, CR increases lifespan or healthspan in all investigated eukaryote species, which are unicellular and multicellular organisms of several distinct phyla, including non-human primates [198]. What is more, a pharmacological manipulation that mimics a state of limited nutrient availability, such as rapamycin, can extend longevity in mice (Harrison et al. [189]). All of these reflects in stem cells, however,

In the context of a tissue, adult stem cells reside in a special microenvironment referred to as the "niche". The niche allows interaction between the stem cells and different extrinsic signals. In some instances, these signals are mediated via direct cell to cell communication or cell to matrix interaction. Another category of signals comprises of diffusible signaling ligands which regulate various transcription programs in the stem cells. These interactions are crucial, as they are able to regulate whether stem cells are quiescent, self-renew, or commit

Similarly to the stem cells themselves, the BM niche changes substantially with age. The niche consists of mesenchymal stem cells (MSCs), stromal cells, osteoblasts, adipocytes, and other cells, as well as extracellular matrix. The proliferative capacity of human MSCs has been shown to decline with age [200]. Certain other authors noticed a prominent increase in adipo-

Mechanisms of niche aging are probably the same as in other cells. Khatri et al. recently showed that accumulation of excessive ROS in BM stromal cells suppress BM cellularity by affecting microenvironment in aged mice. Treatment of these mice with a polyphenolic antioxidant curcumin has quenched ROS, rescued stromal cells from oxidative stress-dependent cellular injury, and improved hematopoietic reconstitution in old (18 months) mice. This

cytes in the aged BM, which is associated with lower HSC potential [201].

the exact mechanisms in the metabolism of stem cells awaits further clarification.

rated from each other, it will be crucial to understand the mechanisms involved.

extension following metformin administration to worms and mice [194, 195].

sensors of low-energy states into a unified response [197].

**2.11. Niche deterioration**

to differentiation [199].

Multiple genetic manipulations of the IIS pathway, which attenuate signaling intensity at different levels, consistently extend the lifespan of worms, flies and mice. Genetic analyses indicate that this pathway mediates part of the beneficial effects of CR on longevity [183].

Mice with an increased dosage of the tumor suppressor protein phosphatase and tensin homolog (PTEN) exhibit a general down-modulation of the IIS pathway and an increased energy expenditure that is associated with improved mitochondrial oxidative metabolism, as well as with an enhanced activity of the brown adipose tissue [184]. In line with other mouse models with decreased IIS activity, PTEN-overexpressing mice, as well as hypomorphic phosphatidylinositol-3-kinase (PI3K) mice show an increased longevity [185].

Organisms with a constitutively decreased IIS pathway can live longer because they have lower rates of cell growth and metabolism, and a lower rates of cellular damage. Similarly, the aged organisms decrease their IIS pathway in an attempt to extend their lifespan. However, defensive responses against aging eventually exhaust and later on they even aggravate aging [186].

#### *2.10.3. Other nutrient-sensing systems: mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK) and sirtuins*

Besides the IIS pathway, three additional related and interconnected nutrient-sensing systems that participate in glucose –sensing: mammalian TOR (mTOR), for the sensing of high amino acid concentrations; AMPK that senses low energy states by detecting high AMP levels; and sirtuins, which sense the low energy states by detecting high NAD+ levels [187].

The mTOR kinase is part of two multiprotein complexes, mTORC1 and mTORC2, that regulate essentially all aspects of anabolic metabolism. Genetic down-regulation of mTORC1 activity in yeast, worms and flies extends longevity and attenuates further longevity benefits from CR, suggesting that mTOR inhibition phenocopies CR [188]. In mice, treatment with rapamycin also extends longevity in what is considered the most robust chemical intervention to increase lifespan in mammals [189].

Genetically-modified mice with low levels of mTORC1 activity, but normal levels of mTORC2 activity, have an increased lifespan [190], and mice deficient in ribosomal protein S6 kinase beta-1 (S6 K1), which is a main mTORC1 substrate, are also long-lived [191]. This means that the downregulation of mTORC1/S6 K1 acts as the critical mediator of longevity in relation to mTOR.

It seems that the intense trophic and anabolic activity, signaled through the IIS or the mTORC1 pathways, is a major accelerator of aging. Although inhibition of TOR activity clearly has beneficial effects during aging, it also has some undesirable side-effects, such as impaired wound healing, insulin resistance, cataract formation and testicular degeneration in mice [192]. In order to determine the extent to which beneficial and damaging effects of TOR inhibition can be separated from each other, it will be crucial to understand the mechanisms involved.

There are two another nutrient sensors, AMPK and sirtuins, which act in the completely opposite direction of the IIS and mTOR. Instead of signaling nutrient abundance and anabolism, they signal nutrient scarcity and catabolism. Accordingly, their up-regulation promotes a healthy aging. AMPK activation has multiple effects on metabolism and, remarkably, shuts off mTORC1 [193]. There is evidence indicating that AMPK activation may mediate lifespanextension following metformin administration to worms and mice [194, 195].

The role of sirtuins in lifespan regulation has been discussed above (see section 2.2 on DNA mutations). In addition, SIRT1 can deacetylate and activate the PPARγ co-activator 1α (PGC-1α) [43]. PGC-1α orchestrates a complex metabolic response that includes mitochondriogenesis, enhanced anti-oxidant defenses, and improved fatty acid oxidation [196]. Moreover, SIRT1 and AMPK can engage in a positive feedback loop, thus connecting both sensors of low-energy states into a unified response [197].

Collectively, currently available evidence strongly supports the idea that anabolic signaling accelerates aging, and decreased nutrient signaling extends longevity [183]. Consistent with the relevance of deregulated nutrient-sensing as a hallmark of aging, CR increases lifespan or healthspan in all investigated eukaryote species, which are unicellular and multicellular organisms of several distinct phyla, including non-human primates [198]. What is more, a pharmacological manipulation that mimics a state of limited nutrient availability, such as rapamycin, can extend longevity in mice (Harrison et al. [189]). All of these reflects in stem cells, however, the exact mechanisms in the metabolism of stem cells awaits further clarification.

#### **2.11. Niche deterioration**

in evolution and among its multiple targets are the FOXO family of transcription factors and the mTOR complexes, which are also involved in aging and conserved through evolution. Similarly, genetic polymorphisms or mutations that reduce the functions of GH, IGF-1 receptor, insulin receptor or downstream intracellular effectors such as protein kinase B (PKB), also known as AKT, mTOR and FOXO, influence longevity both in humans and in model organisms, further illustrating the major impact these pathways have on longevity [53].

Multiple genetic manipulations of the IIS pathway, which attenuate signaling intensity at different levels, consistently extend the lifespan of worms, flies and mice. Genetic analyses indicate that this pathway mediates part of the beneficial effects of CR on longevity [183].

Mice with an increased dosage of the tumor suppressor protein phosphatase and tensin homolog (PTEN) exhibit a general down-modulation of the IIS pathway and an increased energy expenditure that is associated with improved mitochondrial oxidative metabolism, as well as with an enhanced activity of the brown adipose tissue [184]. In line with other mouse models with decreased IIS activity, PTEN-overexpressing mice, as well as hypomorphic phos-

Organisms with a constitutively decreased IIS pathway can live longer because they have lower rates of cell growth and metabolism, and a lower rates of cellular damage. Similarly, the aged organisms decrease their IIS pathway in an attempt to extend their lifespan. However, defensive responses against aging eventually exhaust and later on they even aggravate aging [186].

*2.10.3. Other nutrient-sensing systems: mammalian target of rapamycin (mTOR), AMP-activated* 

Besides the IIS pathway, three additional related and interconnected nutrient-sensing systems that participate in glucose –sensing: mammalian TOR (mTOR), for the sensing of high amino acid concentrations; AMPK that senses low energy states by detecting high AMP levels; and

The mTOR kinase is part of two multiprotein complexes, mTORC1 and mTORC2, that regulate essentially all aspects of anabolic metabolism. Genetic down-regulation of mTORC1 activity in yeast, worms and flies extends longevity and attenuates further longevity benefits from CR, suggesting that mTOR inhibition phenocopies CR [188]. In mice, treatment with rapamycin also extends longevity in what is considered the most robust chemical interven-

Genetically-modified mice with low levels of mTORC1 activity, but normal levels of mTORC2 activity, have an increased lifespan [190], and mice deficient in ribosomal protein S6 kinase beta-1 (S6 K1), which is a main mTORC1 substrate, are also long-lived [191]. This means that the downregulation of mTORC1/S6 K1 acts as the critical mediator of longevity in relation to mTOR.

It seems that the intense trophic and anabolic activity, signaled through the IIS or the mTORC1 pathways, is a major accelerator of aging. Although inhibition of TOR activity clearly has beneficial effects during aging, it also has some undesirable side-effects, such as impaired wound healing, insulin resistance, cataract formation and testicular degeneration in mice [192]. In order

sirtuins, which sense the low energy states by detecting high NAD+ levels [187].

phatidylinositol-3-kinase (PI3K) mice show an increased longevity [185].

*protein kinase (AMPK) and sirtuins*

48 Stem Cells in Clinical Practice and Tissue Engineering

tion to increase lifespan in mammals [189].

In the context of a tissue, adult stem cells reside in a special microenvironment referred to as the "niche". The niche allows interaction between the stem cells and different extrinsic signals. In some instances, these signals are mediated via direct cell to cell communication or cell to matrix interaction. Another category of signals comprises of diffusible signaling ligands which regulate various transcription programs in the stem cells. These interactions are crucial, as they are able to regulate whether stem cells are quiescent, self-renew, or commit to differentiation [199].

Similarly to the stem cells themselves, the BM niche changes substantially with age. The niche consists of mesenchymal stem cells (MSCs), stromal cells, osteoblasts, adipocytes, and other cells, as well as extracellular matrix. The proliferative capacity of human MSCs has been shown to decline with age [200]. Certain other authors noticed a prominent increase in adipocytes in the aged BM, which is associated with lower HSC potential [201].

Mechanisms of niche aging are probably the same as in other cells. Khatri et al. recently showed that accumulation of excessive ROS in BM stromal cells suppress BM cellularity by affecting microenvironment in aged mice. Treatment of these mice with a polyphenolic antioxidant curcumin has quenched ROS, rescued stromal cells from oxidative stress-dependent cellular injury, and improved hematopoietic reconstitution in old (18 months) mice. This implicates the role of ROS in perturbation of stromal cells function upon aging, which in turn affects BM's reconstitution ability in aged mice. Rejuvenation therapy using curcumin, prior to transplantation of HSCs and progenitor cells could be an efficient strategy for successful marrow reconstitution in older mice [202].

the niche in which the HSCs reside and that the clonality of HSCs may largely be influenced

Stem Cell Aging

51

http://dx.doi.org/10.5772/intechopen.71764

Various studies utilizing heterochronic transplantation and parabiosis experiments showed that aging can be also be caused by extrinsic mechanisms, i.e., it is caused by factors external to the cell itself. This was shown in satellite cells [217], NSCs [218, 219], and GSCs [220]. In flies, the cells that form the niche of the GSCs themselves decline in abundance with age, pos-

Aged niche cells can also fail to send proper signals to stem cells, namely through morphogen and growth factor signaling, thereby affecting cell fate decisions. For example, increased fibroblast growth factor 2 (FGF2) in the aged satellite cell niche of mouse muscle impairs selfrenewal [223]. Markers of inflammation also increase in the aging niche, for example in hair

Taken together, stem cells require support cells that constitute the niche to maintain proper function. Thus, aging of the stem cell niche can also critically modulate stem cell function.

The concentrations of various circulating factors exerts important influences on stem cell aging. Many of these factors have been identified by rejuvenating effects of blood or plasma derived from either young or calorically restricted animals. Among such factors are insulin and IGF-1, which have been already discussed (see paragraph 2.10.). Reduced signaling from these molecules is believed to mediate much of the longevity-extending effects of CR in mice. An opposite example is the TGF- β molecule, the levels of which increase during aging in mouse and human sera, which impairs the function of satellite muscle cells and NSCs [225]. By contrast, growth differentiation factor 11 (GDF11) has been suggested to improve the function of satellite cells and NSCs, and its levels appear to decrease during aging [218]. The validity of the effects of GDF11 on satellite cells, however, has been questioned by other studies, although it is worth noting that the dose of GDF11 and the skeletal muscle injury models used in the various studies differed [226]. Whether GDF11 actually declines with age has also been questioned, based in part on the argument that GDF11 detection methods cross-react with myostatin (ibid.), although a recent study using additional methods and controls also reports that GDF11 declines with age in mice [227]. Finally, the latest reports infer that high levels of GDF11 cause reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect with the conclusion that elevating blood levels of GDF11 in the aged might

An important debate regarding the decline in stem-cell function is the relative role of cell-intrinsic pathways compared to cell-extrinsic ones [229]. Recent work has provided strong support for the latter. In particular, CR increases intestinal and muscle stem functions through cell-extrinsic mechanisms [174]. Similarly, when muscle-derived stem cells from young mice are transplanted to progeroid mice, this extends their lifespan and improves degenerative changes even in tissues where donor cells are not detected, suggesting that their therapeutic benefit may derive from systemic effects caused by secreted

by specific niche cells at different anatomical sites [216].

sibly because of decreased self-renewal [221, 222].

follicle stem cells, and impair stem cell function [224].

**2.12. Influence of various circulating factors**

cause more harm than good [228].

The question remains as to whether aged BM niche cells induce age-related changes in HSCs. Evidence suggests that aging in the microenvironment influences HSC engraftment, as aged HSCs demonstrate a lower engraftment after transplantation [203]. Hematopoietic cells engrafted in subcutaneous implantation of BM stroma from both aged and young mice exhibit lower spleen colony-forming units (CFU-S) capacity [204]. Furthermore, young HSCs transplanted to aged niches exhibit impairment in homing and decreased potential for differentiation, failing to efficiently repopulate an old niche [205].

Another characteristic of aged HSCs is an altered differentiation potential tending toward higher myeloid/platelet output and lower lymphoid output. Skewing toward myeloid differentiation is attributed to the niche microenvironment, since the transplantation of young HSCs to aged recipients resulted in a tendency toward higher myeloid output [206]. Transplantation experiments on old recipients show that granulocyte-macrophage progenitor (GMP) expansion is comparable regardless of donor age. Also, the differentiation of B-cells depends on the BM microenvironment [207] and it was shown that aged HSCs occupy different niches to young HSCs [208].

One of the mechanisms of aging in the hematopoietic system are the changes in adhesion between HSCs and niche cells. Expression of various adhesion molecules in HSCs alters with age so the aged HSCs express low levels of integrin α4, integrin α5 and VCAM-1, and high levels of P-selectin and integrin α6 compared to young HSCs [209]. In *Drosophila*, the age-dependent E-cadherin decline in the stem cell-niche junction that regulates the adhesion of GSCs to the niche was shown to contribute to the aging of stem cells [210]. Another authors similarly showed that the aged HSCs exhibit less adhesion to the stromal cells compared to the young ones [211]. Another group has shown that an overexpression of CDC42, a small Rho GTPase that is involved in adhesion signaling, causes premature aging phenotypes in these cells [212].

Age-related changes in niche cells may also be attributed to changes in their metabolic state. MSCs obtained from old human BM have an elevated level of ROS along with p21 and p53 expression, indicating cellular senescence [17]. As already mentioned, high oxygen tension causes senescence in cultured human BM MSCs, whereas the continuous hypoxia make the human MSCs to exhibit higher self-renewal divisions without cellular senescence [213]. Compared to MSCs cultured in low oxygen, MSCs cultured in higher oxygen levels utilize oxidative phosphorylation, suggesting that the generation of ROS might influence MSC senescence.

Age-related changes in the stem cell niches can influence HSC mobilization from the BM, which is extremely important in the clinical settings. Several authors, including ourselves, have noted that the collection of stem cells from aged patients results in low yields of mobilized HSCs intended for therapy [214, 215]. It is interesting that in various animal models an opposite effect was demonstrated since the granulocyte colony-stimulating factor (G-CSF) induced mobilization resulted in increased numbers of HSCs in aged mice [211]. The authors deduce that differences in mobilization potential according to age are influenced mainly by the niche in which the HSCs reside and that the clonality of HSCs may largely be influenced by specific niche cells at different anatomical sites [216].

Various studies utilizing heterochronic transplantation and parabiosis experiments showed that aging can be also be caused by extrinsic mechanisms, i.e., it is caused by factors external to the cell itself. This was shown in satellite cells [217], NSCs [218, 219], and GSCs [220]. In flies, the cells that form the niche of the GSCs themselves decline in abundance with age, possibly because of decreased self-renewal [221, 222].

Aged niche cells can also fail to send proper signals to stem cells, namely through morphogen and growth factor signaling, thereby affecting cell fate decisions. For example, increased fibroblast growth factor 2 (FGF2) in the aged satellite cell niche of mouse muscle impairs selfrenewal [223]. Markers of inflammation also increase in the aging niche, for example in hair follicle stem cells, and impair stem cell function [224].

Taken together, stem cells require support cells that constitute the niche to maintain proper function. Thus, aging of the stem cell niche can also critically modulate stem cell function.

#### **2.12. Influence of various circulating factors**

implicates the role of ROS in perturbation of stromal cells function upon aging, which in turn affects BM's reconstitution ability in aged mice. Rejuvenation therapy using curcumin, prior to transplantation of HSCs and progenitor cells could be an efficient strategy for successful

The question remains as to whether aged BM niche cells induce age-related changes in HSCs. Evidence suggests that aging in the microenvironment influences HSC engraftment, as aged HSCs demonstrate a lower engraftment after transplantation [203]. Hematopoietic cells engrafted in subcutaneous implantation of BM stroma from both aged and young mice exhibit lower spleen colony-forming units (CFU-S) capacity [204]. Furthermore, young HSCs transplanted to aged niches exhibit impairment in homing and decreased potential for dif-

Another characteristic of aged HSCs is an altered differentiation potential tending toward higher myeloid/platelet output and lower lymphoid output. Skewing toward myeloid differentiation is attributed to the niche microenvironment, since the transplantation of young HSCs to aged recipients resulted in a tendency toward higher myeloid output [206]. Transplantation experiments on old recipients show that granulocyte-macrophage progenitor (GMP) expansion is comparable regardless of donor age. Also, the differentiation of B-cells depends on the BM microenvironment [207] and it was shown that aged HSCs occupy different niches to young HSCs [208].

One of the mechanisms of aging in the hematopoietic system are the changes in adhesion between HSCs and niche cells. Expression of various adhesion molecules in HSCs alters with age so the aged HSCs express low levels of integrin α4, integrin α5 and VCAM-1, and high levels of P-selectin and integrin α6 compared to young HSCs [209]. In *Drosophila*, the age-dependent E-cadherin decline in the stem cell-niche junction that regulates the adhesion of GSCs to the niche was shown to contribute to the aging of stem cells [210]. Another authors similarly showed that the aged HSCs exhibit less adhesion to the stromal cells compared to the young ones [211]. Another group has shown that an overexpression of CDC42, a small Rho GTPase that is involved in adhesion signaling, causes premature aging phenotypes in these cells [212].

Age-related changes in niche cells may also be attributed to changes in their metabolic state. MSCs obtained from old human BM have an elevated level of ROS along with p21 and p53 expression, indicating cellular senescence [17]. As already mentioned, high oxygen tension causes senescence in cultured human BM MSCs, whereas the continuous hypoxia make the human MSCs to exhibit higher self-renewal divisions without cellular senescence [213]. Compared to MSCs cultured in low oxygen, MSCs cultured in higher oxygen levels utilize oxidative phosphoryla-

Age-related changes in the stem cell niches can influence HSC mobilization from the BM, which is extremely important in the clinical settings. Several authors, including ourselves, have noted that the collection of stem cells from aged patients results in low yields of mobilized HSCs intended for therapy [214, 215]. It is interesting that in various animal models an opposite effect was demonstrated since the granulocyte colony-stimulating factor (G-CSF) induced mobilization resulted in increased numbers of HSCs in aged mice [211]. The authors deduce that differences in mobilization potential according to age are influenced mainly by

tion, suggesting that the generation of ROS might influence MSC senescence.

marrow reconstitution in older mice [202].

50 Stem Cells in Clinical Practice and Tissue Engineering

ferentiation, failing to efficiently repopulate an old niche [205].

The concentrations of various circulating factors exerts important influences on stem cell aging. Many of these factors have been identified by rejuvenating effects of blood or plasma derived from either young or calorically restricted animals. Among such factors are insulin and IGF-1, which have been already discussed (see paragraph 2.10.). Reduced signaling from these molecules is believed to mediate much of the longevity-extending effects of CR in mice. An opposite example is the TGF- β molecule, the levels of which increase during aging in mouse and human sera, which impairs the function of satellite muscle cells and NSCs [225]. By contrast, growth differentiation factor 11 (GDF11) has been suggested to improve the function of satellite cells and NSCs, and its levels appear to decrease during aging [218]. The validity of the effects of GDF11 on satellite cells, however, has been questioned by other studies, although it is worth noting that the dose of GDF11 and the skeletal muscle injury models used in the various studies differed [226]. Whether GDF11 actually declines with age has also been questioned, based in part on the argument that GDF11 detection methods cross-react with myostatin (ibid.), although a recent study using additional methods and controls also reports that GDF11 declines with age in mice [227]. Finally, the latest reports infer that high levels of GDF11 cause reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect with the conclusion that elevating blood levels of GDF11 in the aged might cause more harm than good [228].

An important debate regarding the decline in stem-cell function is the relative role of cell-intrinsic pathways compared to cell-extrinsic ones [229]. Recent work has provided strong support for the latter. In particular, CR increases intestinal and muscle stem functions through cell-extrinsic mechanisms [174]. Similarly, when muscle-derived stem cells from young mice are transplanted to progeroid mice, this extends their lifespan and improves degenerative changes even in tissues where donor cells are not detected, suggesting that their therapeutic benefit may derive from systemic effects caused by secreted factors [230]. Furthermore, parabiosis experiments have demonstrated that the decline in neural and muscle stem cell function in old mice can be reversed by systemic factors from young mice [231, 232].

were offspring of only two HSC clones that were still active in her old age. The telomeres of her white blood cells were significantly shorter than the telomeres from other tissues, suggesting that the HSCs have a finite lifespan, which is the cause of hematopoietic clonal evolution

Stem Cell Aging

53

http://dx.doi.org/10.5772/intechopen.71764

Several recent studies have confirmed that clonal hematopoiesis is almost a "normal" part of aging, with recent reports showing 0.8%, 11% and 19.5% of normal individuals aged <60, >80 and >90 years, respectively, having demonstrable clonal hematopoiesis – so called age-related clonal hematopoiesis [240, 241]. Clonal hematopoiesis (CH) arises when a substantial proportion of mature blood cells is derived from a single dominant hematopoietic stem cell lineage. It was recently shown, in the study on 11,262 elderly Icelanders which used whole-genome sequencing, that somatic mutations in candidate driver genes are thought to be responsible

At the same time there is ample evidence that there exist many dormant HSCs, and even some other and more "primitive" types of stem cells, such as for instance the VSEL stem cells with "primitive" embryonic characteristics, which co-inhabit the BM [243]. These VSEL cells exhibit some characteristics of long-term repopulating HSCs (LT-HSCs), they may differentiate into organ-specific cells (e.g., cardiomyocytes), and probably have a role in aging since the number of these cells positively correlates with longevity in several murine models [244]. Along with others, we have found similar cells in the reproductive

It is now becoming obvious that maintaining robust stem cell pools seems to extend not only

Cellular senescence can be defined as a stable arrest of the cell cycle coupled to typical phenotypic changes [246]. This phenomenon was originally described by Hayflick in human fibroblasts serially passaged in culture [99]. The senescence that was observed by Hayflick was caused by telomere shortening [101] and some other aging-associated stimuli that trigger senescence independently of the telomeric process. It is for instance well known that the non-telomeric DNA damage and de-repression of the *INK4/ARF* locus, both of which progres-

The accumulation of senescent cells with age is a simple mathematical result of the increase in the rate of generation of senescent cells and/or a decrease in their rate of clearance. In normal physiology this has detrimental consequences, but in some circumstances it also has useful effects. For instance, there is good evidence that the senescent tumor cells are subjected to

Among other functions, the senescent cells manifest dramatic alterations in their secretome, which is particularly enriched in pro-inflammatory cytokines and matrix metalloproteinases, which is referred to as the "senescence-associated secretory phenotype" [249, 250]. This pro-inflammatory secretome may contribute to aging (see paragraph 2.15. Intercellular

sively occur with chronological aging, are also capable of inducing senescence [247].

strict immune surveillance and are efficiently removed by phagocytosis [248].

at extreme ages [239].

organs [245].

Communication).

for at least some cases of CH [242].

lifespan but also healthspan [49].

**2.14. Cellular senescence – A stable arrest of the cell cycle**

There is also an ancient system in each cell that relates to the homeostasis of intracellular calcium (Ca2+), which in normal cell sustains a 20,000 fold concentration gradient to the exterior of the cell, resulting in the extracellular Ca2+ acting as cellular regulator when it enters the cell via the Ca2+ channels. This gradient is sustained by specific pumping and transporting mechanisms consisting of protein molecules [233]. Anomalies of these proteins results in an increase of intracellular calcium which can cause various diseases. With age, the hampered calcium homeostasis can lead to different muscle, immune and neural related defects [234].

#### **2.13. Stem cell exhaustion**

Although stem cells are regarded as immortal, as they are not subject to replicative senescence, they are susceptible to damage accumulation over time. Besides many other changes, a decline in their relative numbers and changes in subpopulations were observed. The group of dormant and active stem cells, existing in the niches of an organism that can be considered a pool of regenerative reserve, plays an important role in prevention of disease, in regeneration and aging. For instance, a decline in CD34+ circulating progenitor cells was reported with advancing age. When 100 octogenarians were observed for 7 and 10 years it was demonstrated that the number of their circulating CD34+ cells better predicted their lifespan and cardiovascular (CV) issues related mortality then the classic cardiovascular risk factors (hypertension, smoking, hypercholesterolemia), levels of inflammatory markers, or levels of cholesterol, or some other traditional cardiovascular indexes such as FRS and CVFRs The chances of reaching an older age depended on higher numbers of CD34+ cells at baseline, thus the number of CD34+ cells could be considered as a biomarker of longevity in the elderly over 80 years [235].

On the other hand, there are reports that in certain tissues the numbers of adult stem cells even increase with age, however the number of their parent clones decreases, meaning that fewer pluripotent stem cells give rise to more frequent progeny, in order to compensate for the decrease of numbers [236]. Ruzankina and Brown suggest that mammals in fact do have a finite number of stem cell replications per life and that aging of the hematopoietic system, which is due to a finite doubling capacity of stem cells, degrades its regenerative potential as well as the potential for preventing cancer [237].

Verovskaya used cellular barcoding combined with multiplex high-throughput sequencing to demonstrate clonal behavior of young HSCs transplanted to older organisms. In their study, the majority of transplanted clones steadily contributed to hematopoiesis in the long-term, although the clonal output in granulocytes, T cells, and B cells was substantially different. The final pool of old HSCs was composed of multiple small clones, whereas the young HSC pool was dominated by fewer, but larger, clones [238].

Holstege et al. have showed that the contents of a stem cell compartment actually deplete with old age. In the nonrepetitive genome of a 115-year-old centenarian woman they found approximately 450 somatic mutations that accumulated in the last years of her life, and the distribution of these mutations suggested that the majority of her peripheral white blood cells were offspring of only two HSC clones that were still active in her old age. The telomeres of her white blood cells were significantly shorter than the telomeres from other tissues, suggesting that the HSCs have a finite lifespan, which is the cause of hematopoietic clonal evolution at extreme ages [239].

Several recent studies have confirmed that clonal hematopoiesis is almost a "normal" part of aging, with recent reports showing 0.8%, 11% and 19.5% of normal individuals aged <60, >80 and >90 years, respectively, having demonstrable clonal hematopoiesis – so called age-related clonal hematopoiesis [240, 241]. Clonal hematopoiesis (CH) arises when a substantial proportion of mature blood cells is derived from a single dominant hematopoietic stem cell lineage. It was recently shown, in the study on 11,262 elderly Icelanders which used whole-genome sequencing, that somatic mutations in candidate driver genes are thought to be responsible for at least some cases of CH [242].

At the same time there is ample evidence that there exist many dormant HSCs, and even some other and more "primitive" types of stem cells, such as for instance the VSEL stem cells with "primitive" embryonic characteristics, which co-inhabit the BM [243]. These VSEL cells exhibit some characteristics of long-term repopulating HSCs (LT-HSCs), they may differentiate into organ-specific cells (e.g., cardiomyocytes), and probably have a role in aging since the number of these cells positively correlates with longevity in several murine models [244]. Along with others, we have found similar cells in the reproductive organs [245].

It is now becoming obvious that maintaining robust stem cell pools seems to extend not only lifespan but also healthspan [49].

#### **2.14. Cellular senescence – A stable arrest of the cell cycle**

factors [230]. Furthermore, parabiosis experiments have demonstrated that the decline in neural and muscle stem cell function in old mice can be reversed by systemic factors from

There is also an ancient system in each cell that relates to the homeostasis of intracellular calcium (Ca2+), which in normal cell sustains a 20,000 fold concentration gradient to the exterior of the cell, resulting in the extracellular Ca2+ acting as cellular regulator when it enters the cell via the Ca2+ channels. This gradient is sustained by specific pumping and transporting mechanisms consisting of protein molecules [233]. Anomalies of these proteins results in an increase of intracellular calcium which can cause various diseases. With age, the hampered calcium homeostasis can lead to different muscle, immune and neural related defects [234].

Although stem cells are regarded as immortal, as they are not subject to replicative senescence, they are susceptible to damage accumulation over time. Besides many other changes, a decline in their relative numbers and changes in subpopulations were observed. The group of dormant and active stem cells, existing in the niches of an organism that can be considered a pool of regenerative reserve, plays an important role in prevention of disease, in regeneration and aging. For instance, a decline in CD34+ circulating progenitor cells was reported with advancing age. When 100 octogenarians were observed for 7 and 10 years it was demonstrated that the number of their circulating CD34+ cells better predicted their lifespan and cardiovascular (CV) issues related mortality then the classic cardiovascular risk factors (hypertension, smoking, hypercholesterolemia), levels of inflammatory markers, or levels of cholesterol, or some other traditional cardiovascular indexes such as FRS and CVFRs The chances of reaching an older age depended on higher numbers of CD34+ cells at baseline, thus the number of CD34+

cells could be considered as a biomarker of longevity in the elderly over 80 years [235].

well as the potential for preventing cancer [237].

was dominated by fewer, but larger, clones [238].

On the other hand, there are reports that in certain tissues the numbers of adult stem cells even increase with age, however the number of their parent clones decreases, meaning that fewer pluripotent stem cells give rise to more frequent progeny, in order to compensate for the decrease of numbers [236]. Ruzankina and Brown suggest that mammals in fact do have a finite number of stem cell replications per life and that aging of the hematopoietic system, which is due to a finite doubling capacity of stem cells, degrades its regenerative potential as

Verovskaya used cellular barcoding combined with multiplex high-throughput sequencing to demonstrate clonal behavior of young HSCs transplanted to older organisms. In their study, the majority of transplanted clones steadily contributed to hematopoiesis in the long-term, although the clonal output in granulocytes, T cells, and B cells was substantially different. The final pool of old HSCs was composed of multiple small clones, whereas the young HSC pool

Holstege et al. have showed that the contents of a stem cell compartment actually deplete with old age. In the nonrepetitive genome of a 115-year-old centenarian woman they found approximately 450 somatic mutations that accumulated in the last years of her life, and the distribution of these mutations suggested that the majority of her peripheral white blood cells

young mice [231, 232].

52 Stem Cells in Clinical Practice and Tissue Engineering

**2.13. Stem cell exhaustion**

Cellular senescence can be defined as a stable arrest of the cell cycle coupled to typical phenotypic changes [246]. This phenomenon was originally described by Hayflick in human fibroblasts serially passaged in culture [99]. The senescence that was observed by Hayflick was caused by telomere shortening [101] and some other aging-associated stimuli that trigger senescence independently of the telomeric process. It is for instance well known that the non-telomeric DNA damage and de-repression of the *INK4/ARF* locus, both of which progressively occur with chronological aging, are also capable of inducing senescence [247].

The accumulation of senescent cells with age is a simple mathematical result of the increase in the rate of generation of senescent cells and/or a decrease in their rate of clearance. In normal physiology this has detrimental consequences, but in some circumstances it also has useful effects. For instance, there is good evidence that the senescent tumor cells are subjected to strict immune surveillance and are efficiently removed by phagocytosis [248].

Among other functions, the senescent cells manifest dramatic alterations in their secretome, which is particularly enriched in pro-inflammatory cytokines and matrix metalloproteinases, which is referred to as the "senescence-associated secretory phenotype" [249, 250]. This pro-inflammatory secretome may contribute to aging (see paragraph 2.15. Intercellular Communication).

Studies on aged mice have revealed an overall decrease in HSC cell cycle activity, with old HSCs undergoing fewer cell divisions than young HSCs [251]. This correlates with the accumulation of DNA damage and with the overexpression of cell cycle-inhibitory proteins such as p16INK4a [252]. In fact, old p16INK4a−/− HSCs exhibit better engraftment capacity and increased cell cycle activity compared with old wild-type HSCs (ibid.). Telomere shortening is also an important cause of stem cell decline with aging in multiple tissues [253].

that are able to induce senescence [260]. The number of mechanisms that implement senescence in response to this variety of oncogenic insults has also grown, but still, the originally reported p16INK4a/Rb and p19ARF/p53 pathways remain, in general, the most important ones [261]. The relevance of these pathways for aging becomes even more striking when considering that the levels of p16INK4a (and to a lesser extent also p19ARFARF) correlate with the chronological age of essentially all tissues analyzed, both in mice and humans [262, 263]. *INK4a/ARF* locus was actually determined as being genetically linked to the highest number of age-associated pathologies, including several types of cardiovascular diseases, diabetes, glaucoma, and Alzheimer's disease [264]. Although the activation of p53 and *INK4a/ARF* is a beneficial compensatory response that prevents the propagation of damaged cells, under the stress conditions the p53

Stem Cell Aging

55

http://dx.doi.org/10.5772/intechopen.71764

Taken together, cellular senescence is a beneficial compensatory response to damage, but it becomes deleterious and accelerates aging when tissues exhaust their regenerative capacity. A moderate enhancement of the senescence-inducing tumor suppressor pathways may extend longevity [265], whereas at the same time, elimination of senescent cells in an experimental progeria model delays age-related pathologies [266]. Therefore, two interventions that are

Beyond intrinsic cellular alterations, aging also involves changes at the level of intercellular endocrine, neuroendocrine or neuronal communication [267, 268]. As during the aging inflammatory reactions increase, immunosurveillance against pathogens and premalignant cells declines, and the composition of the peri- and extracellular environment changes, neurohormonal signaling (i.e., renin-angiotensin, adrenergic, insulin/IGF-1 signaling) is consequently deregulated, which affects various mechanical and functional properties of all

An important age-associated pathological finding in the intercellular communication in mammals is so called "inflammaging," i.e., an appearance of pro-inflammatory phenotype that accompanies aging. Several authors proposed that aging is accompanied by a chronic up-regulation of several pro-inflammatory responses. [35, 269, 270]. Inflammaging may result from multiple causes such as the accumulation of pro-inflammatory substances, tissue damage, the failure of the aged immune system to effectively clear pathogens and remove dysfunctional host cells, the secretion of pro-inflammatory cytokines by aged immune cells, the enhanced activation of the NF-κB transcription factor, or from a defective autophagy response. These defects and alterations result in an enhanced activation of the NLRP3 inflammasome and other pro-inflammatory pathways, finally leading to increased production of interleukin 1ß (IL-1ß), tumor necrosis factor and interferons [271]. Inflammation is also involved in the pathogenesis of obesity and type 2 diabetes, two conditions that contribute to, and correlate with aging in the human population [272]. Likewise, defective inflammatory responses play

Another link between inflammation and aging derives from the finding that inflammatory and stress responses activate NF-κB in the hypothalamus and induce a signaling pathway

and *INK4a/ARF* responses can become deleterious and even accelerate aging [2].

conceptually opposite are able to extend healthspan.

**2.15. Altered intercellular communication**

a critical role in atherosclerosis [273].

tissues [48].

The accumulation of senescent cells in aged tissues has been often inferred using surrogate markers such as DNA damage. Some studies have directly used senescence-associated β-galactosidase (SABG) to identify senescence in tissues [254]. Of note, a detailed and parallel quantification of SABG and DNA damage in liver produced comparable quantitative data, yielding a total of ~8% senescent cells in young mice and ~17% in very old mice [255]. Similar results were obtained in the skin, lung and spleen, but no changes were observed in heart, skeletal muscle and kidney. Based on these data, it is clear that cellular senescence is not a generalized property of all tissues in aged organisms.

Some authors think that the amount of senescent cells increases with age and that senescence contributes to aging, but this probably undervalues the primary purpose of senescence, which is to prevent the propagation of damaged cells and to trigger their removal by the immune system. They explain that senescence is a beneficial compensatory response that contributes to clearing tissues of damaged and potentially oncogenic cells. This however requires an efficient cell replacement system that involves clearance of senescent cells and mobilization of stem cells and their progenitors to re-establish cell numbers. In aged organisms, this turnover system may become exhausted, resulting in the accumulation of senescent cells that aggravate the damage and contribute to aging [48].

Deficient proliferation of stem and progenitor cells is obviously detrimental for the long-term maintenance of the organism, but excessive proliferation of stem and progenitor cells can also be deleterious by accelerating the exhaustion of stem cell niches, which can be compensated by stem cell quiescence over the long-term. This has been demonstrated in *Drosophila* ISCs, where excessive proliferation leads to exhaustion and premature aging [256] and in p21-null mice, which present premature exhaustion of HSCs and NSCs [257].

Recent studies have shown that an increase in FGF2 signaling in the aged muscle stem cell niche results in the loss of quiescence, stem cell depletion and diminished regenerative capacity, whereas the suppression of this signaling pathway reverses these defects [223]. This opens up the possibility of designing strategies aimed at inhibiting FGF2 signaling to reduce stem cell exhaustion during aging.

As a mechanism to protect themselves from acquiring damage, many stem cells are resting for a long time in a quiescent state. During this time they are protected from replicative damage, but they are more susceptible to mutations [258]. However, although proliferating stem cells are more likely to encounter DNA damages [259], they repair that damages more accurately than do quiescent stem cells.

In addition to DNA damage, excessive mitogenic signaling is the other stress most robustly associated with senescence. A recent account listed more than 50 oncogenic or mitogenic alterations that are able to induce senescence [260]. The number of mechanisms that implement senescence in response to this variety of oncogenic insults has also grown, but still, the originally reported p16INK4a/Rb and p19ARF/p53 pathways remain, in general, the most important ones [261]. The relevance of these pathways for aging becomes even more striking when considering that the levels of p16INK4a (and to a lesser extent also p19ARFARF) correlate with the chronological age of essentially all tissues analyzed, both in mice and humans [262, 263]. *INK4a/ARF* locus was actually determined as being genetically linked to the highest number of age-associated pathologies, including several types of cardiovascular diseases, diabetes, glaucoma, and Alzheimer's disease [264]. Although the activation of p53 and *INK4a/ARF* is a beneficial compensatory response that prevents the propagation of damaged cells, under the stress conditions the p53 and *INK4a/ARF* responses can become deleterious and even accelerate aging [2].

Taken together, cellular senescence is a beneficial compensatory response to damage, but it becomes deleterious and accelerates aging when tissues exhaust their regenerative capacity. A moderate enhancement of the senescence-inducing tumor suppressor pathways may extend longevity [265], whereas at the same time, elimination of senescent cells in an experimental progeria model delays age-related pathologies [266]. Therefore, two interventions that are conceptually opposite are able to extend healthspan.

#### **2.15. Altered intercellular communication**

Studies on aged mice have revealed an overall decrease in HSC cell cycle activity, with old HSCs undergoing fewer cell divisions than young HSCs [251]. This correlates with the accumulation of DNA damage and with the overexpression of cell cycle-inhibitory proteins such as p16INK4a [252]. In fact, old p16INK4a−/− HSCs exhibit better engraftment capacity and increased cell cycle activity compared with old wild-type HSCs (ibid.). Telomere shortening is also an

The accumulation of senescent cells in aged tissues has been often inferred using surrogate markers such as DNA damage. Some studies have directly used senescence-associated β-galactosidase (SABG) to identify senescence in tissues [254]. Of note, a detailed and parallel quantification of SABG and DNA damage in liver produced comparable quantitative data, yielding a total of ~8% senescent cells in young mice and ~17% in very old mice [255]. Similar results were obtained in the skin, lung and spleen, but no changes were observed in heart, skeletal muscle and kidney. Based on these data, it is clear that cellular senescence is not a

Some authors think that the amount of senescent cells increases with age and that senescence contributes to aging, but this probably undervalues the primary purpose of senescence, which is to prevent the propagation of damaged cells and to trigger their removal by the immune system. They explain that senescence is a beneficial compensatory response that contributes to clearing tissues of damaged and potentially oncogenic cells. This however requires an efficient cell replacement system that involves clearance of senescent cells and mobilization of stem cells and their progenitors to re-establish cell numbers. In aged organisms, this turnover system may become exhausted, resulting in the accumulation of senescent cells that aggravate

Deficient proliferation of stem and progenitor cells is obviously detrimental for the long-term maintenance of the organism, but excessive proliferation of stem and progenitor cells can also be deleterious by accelerating the exhaustion of stem cell niches, which can be compensated by stem cell quiescence over the long-term. This has been demonstrated in *Drosophila* ISCs, where excessive proliferation leads to exhaustion and premature aging [256] and in p21-null

Recent studies have shown that an increase in FGF2 signaling in the aged muscle stem cell niche results in the loss of quiescence, stem cell depletion and diminished regenerative capacity, whereas the suppression of this signaling pathway reverses these defects [223]. This opens up the possibility of designing strategies aimed at inhibiting FGF2 signaling to reduce stem

As a mechanism to protect themselves from acquiring damage, many stem cells are resting for a long time in a quiescent state. During this time they are protected from replicative damage, but they are more susceptible to mutations [258]. However, although proliferating stem cells are more likely to encounter DNA damages [259], they repair that damages more accurately

In addition to DNA damage, excessive mitogenic signaling is the other stress most robustly associated with senescence. A recent account listed more than 50 oncogenic or mitogenic alterations

mice, which present premature exhaustion of HSCs and NSCs [257].

important cause of stem cell decline with aging in multiple tissues [253].

generalized property of all tissues in aged organisms.

the damage and contribute to aging [48].

54 Stem Cells in Clinical Practice and Tissue Engineering

cell exhaustion during aging.

than do quiescent stem cells.

Beyond intrinsic cellular alterations, aging also involves changes at the level of intercellular endocrine, neuroendocrine or neuronal communication [267, 268]. As during the aging inflammatory reactions increase, immunosurveillance against pathogens and premalignant cells declines, and the composition of the peri- and extracellular environment changes, neurohormonal signaling (i.e., renin-angiotensin, adrenergic, insulin/IGF-1 signaling) is consequently deregulated, which affects various mechanical and functional properties of all tissues [48].

An important age-associated pathological finding in the intercellular communication in mammals is so called "inflammaging," i.e., an appearance of pro-inflammatory phenotype that accompanies aging. Several authors proposed that aging is accompanied by a chronic up-regulation of several pro-inflammatory responses. [35, 269, 270]. Inflammaging may result from multiple causes such as the accumulation of pro-inflammatory substances, tissue damage, the failure of the aged immune system to effectively clear pathogens and remove dysfunctional host cells, the secretion of pro-inflammatory cytokines by aged immune cells, the enhanced activation of the NF-κB transcription factor, or from a defective autophagy response. These defects and alterations result in an enhanced activation of the NLRP3 inflammasome and other pro-inflammatory pathways, finally leading to increased production of interleukin 1ß (IL-1ß), tumor necrosis factor and interferons [271]. Inflammation is also involved in the pathogenesis of obesity and type 2 diabetes, two conditions that contribute to, and correlate with aging in the human population [272]. Likewise, defective inflammatory responses play a critical role in atherosclerosis [273].

Another link between inflammation and aging derives from the finding that inflammatory and stress responses activate NF-κB in the hypothalamus and induce a signaling pathway that results in reduced production of gonadotropin-releasing hormone (GnRH) by neurons [274]. This GnRH decline can contribute to numerous aging-related changes such as bone fragility, muscle weakness, skin atrophy, and reduced neurogenesis. These findings suggest that the hypothalamus may modulate systemic aging by integrating NF-kB-driven inflammatory responses with GnRH-mediated neuroendocrine effects.

plethora of mechanisms that prevent or delay aging and age-related pathology. Over recent decades we have witnessed an immense increase in advanced therapies. The cells used in therapeutic products must meet stringent standards of quality. The huge increase in stem cell based therapies especially demands that we use the most advanced analysis of stem cell grafts

Stem Cell Aging

57

http://dx.doi.org/10.5772/intechopen.71764

The aging of stem cells is an important biological factor that contributes to the general aging of an organism. Therefore, senescence and the age related status of grafted stem cells have to be taken into account in every stem cell based therapy, as well as in tissue engineering procedure. Further research on the cellular mechanisms leading to the aging of stem cells will not only answer various burning questions related to current cell based therapies, but also pave

The work was supported by the Slovenian Research Agency (Grant No. P3-0371).

[1] Bianconi E, et al. An estimation of the number of cells in the human body. Annals of

[2] Schultz MB, Sinclair DA. When stem cells grow old: Phenotypes and mechanisms of

[3] Sharpless NE, Depinho RA. How stem cells age and why this makes us grow old. Nature

[4] Sharpless NE, Schatten G. Stem cell aging. The Journals of Gerontology. Series A,

[5] Wang MC, O'Rourke EJ, Ruvkun G. Fat metabolism links germline stem cells and lon-

[6] Kenyon C. A pathway that links reproductive status to lifespan in *Caenorhabditis elegans*.

to ensure optimal performance.

**Acknowledgements**

**Author details**

**References**

the way to designing future counter-aging procedures.

Primož Rožman\*, Katerina Jazbec and Mojca Jež

Human Biology. 2013;**40**(6):463-471

\*Address all correspondence to: primoz.rozman@ztm.si Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia

stem cell aging. Development. 2016;**143**(1):3-14

Reviews. Molecular Cell Biology. 2007;**8**:703-713

gevity in *C. elegans*. Science. 2008;**322**(5903):957-960

Biological Sciences and Medical Sciences. 2009;**64**(2):202-204

Annals of the New York Academy of Sciences. 2010;**1204**:156-162

Besides chronic inflammation, aged immune cells are prone to a multitude of deteriorating factors. Age related defects of innate immunity are observed not only in the macrophage/monocyte compartment, which is probably the main "culprit" of inflammaging, but also in other cells, i.e., NK cells, dendritic cells, and granulocytes, whereas the defects of adaptive immunity are observed in both the B-cell and the T-cell compartments. Aging of the immune system or "immunosenescence" is characterized by a time-dependent functional alteration of immunity leading to immunodeficiency [275, 276] that manifests in chronic inflammation [277], reduced resistance to infections [278], poor responses to vaccination [279], and increased incidence of autoimmunity and cancers. Similarly, the involvement of immune processes in clinical conditions, such as atherosclerosis, diabetes, and dementia, have been described [280, 281]. The impairment of the immune system exerts an influence on the increased morbidity and mortality observed in human subjects as they age [282].

There is also accumulating evidence indicating that aging-related changes in one tissue can lead to aging-specific deterioration of other tissues. Typical case are the inflammatory cytokines that can cause so called "contagious aging". In certain bystander effects senescent cells induce senescence in neighboring cells via gap junction-mediated cell-to-cell contacts and processes involving ROS [283]. The microenvironment contributes to the age-related functional defects of CD4 T cells, as assessed by using an adoptive transfer model in mice [284]. Likewise, impaired kidney function can increase the risk of heart disease in humans [285]. Conversely, lifespan-extending manipulations targeting one single tissue can delay the aging process in other tissues [286].

Defective intercellular communication underlying aging processes, including genetic, can be restored by nutritional or pharmacological interventions that may improve the cell–cell communication properties lost with aging [48]. Of special interest in this regard are the CR approaches to extend healthy lifespan [287] and the rejuvenation strategies based on the use of blood-borne systemic factors identified in parabiosis experiments [288, 289]. Moreover, the long-term administration of anti-inflammatory agents, such as aspirin, may increase longevity in mice and healthy aging in humans [290, 291]. Finally, it also appears possible to extend lifespan by manipulating the composition and functionality of the intestinal bacterial ecosystem of the human body [292]. The near future research will undoubtedly bring spectacular results in this field of human physiology that will also be translated to the clinical medicine.

### **3. Conclusion**

Although the stem cells are often considered "a fountain of youth" they are subjected to various aging and degenerative processes. Contrary to somatic cells, they have developed a plethora of mechanisms that prevent or delay aging and age-related pathology. Over recent decades we have witnessed an immense increase in advanced therapies. The cells used in therapeutic products must meet stringent standards of quality. The huge increase in stem cell based therapies especially demands that we use the most advanced analysis of stem cell grafts to ensure optimal performance.

The aging of stem cells is an important biological factor that contributes to the general aging of an organism. Therefore, senescence and the age related status of grafted stem cells have to be taken into account in every stem cell based therapy, as well as in tissue engineering procedure. Further research on the cellular mechanisms leading to the aging of stem cells will not only answer various burning questions related to current cell based therapies, but also pave the way to designing future counter-aging procedures.

### **Acknowledgements**

The work was supported by the Slovenian Research Agency (Grant No. P3-0371).

### **Author details**

that results in reduced production of gonadotropin-releasing hormone (GnRH) by neurons [274]. This GnRH decline can contribute to numerous aging-related changes such as bone fragility, muscle weakness, skin atrophy, and reduced neurogenesis. These findings suggest that the hypothalamus may modulate systemic aging by integrating NF-kB-driven inflammatory

Besides chronic inflammation, aged immune cells are prone to a multitude of deteriorating factors. Age related defects of innate immunity are observed not only in the macrophage/monocyte compartment, which is probably the main "culprit" of inflammaging, but also in other cells, i.e., NK cells, dendritic cells, and granulocytes, whereas the defects of adaptive immunity are observed in both the B-cell and the T-cell compartments. Aging of the immune system or "immunosenescence" is characterized by a time-dependent functional alteration of immunity leading to immunodeficiency [275, 276] that manifests in chronic inflammation [277], reduced resistance to infections [278], poor responses to vaccination [279], and increased incidence of autoimmunity and cancers. Similarly, the involvement of immune processes in clinical conditions, such as atherosclerosis, diabetes, and dementia, have been described [280, 281]. The impairment of the immune system exerts an influence on the increased morbidity and mortal-

There is also accumulating evidence indicating that aging-related changes in one tissue can lead to aging-specific deterioration of other tissues. Typical case are the inflammatory cytokines that can cause so called "contagious aging". In certain bystander effects senescent cells induce senescence in neighboring cells via gap junction-mediated cell-to-cell contacts and processes involving ROS [283]. The microenvironment contributes to the age-related functional defects of CD4 T cells, as assessed by using an adoptive transfer model in mice [284]. Likewise, impaired kidney function can increase the risk of heart disease in humans [285]. Conversely, lifespan-extending manipulations targeting one single tissue can delay the aging

Defective intercellular communication underlying aging processes, including genetic, can be restored by nutritional or pharmacological interventions that may improve the cell–cell communication properties lost with aging [48]. Of special interest in this regard are the CR approaches to extend healthy lifespan [287] and the rejuvenation strategies based on the use of blood-borne systemic factors identified in parabiosis experiments [288, 289]. Moreover, the long-term administration of anti-inflammatory agents, such as aspirin, may increase longevity in mice and healthy aging in humans [290, 291]. Finally, it also appears possible to extend lifespan by manipulating the composition and functionality of the intestinal bacterial ecosystem of the human body [292]. The near future research will undoubtedly bring spectacular results in this field of human physiology that will also be translated to the clinical medicine.

Although the stem cells are often considered "a fountain of youth" they are subjected to various aging and degenerative processes. Contrary to somatic cells, they have developed a

responses with GnRH-mediated neuroendocrine effects.

56 Stem Cells in Clinical Practice and Tissue Engineering

ity observed in human subjects as they age [282].

process in other tissues [286].

**3. Conclusion**

Primož Rožman\*, Katerina Jazbec and Mojca Jež

\*Address all correspondence to: primoz.rozman@ztm.si

Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia

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**Chapter 4**

**Provisional chapter**

**Monitoring of Chimerism Following Hematopoietic**

One of the most important events in the posthematopoietic stem cell transplantation is the immune system reconstitution—a process characterized by a considerable dynamic. During this period, patients are exposed to different life threatening complications. In this chapter, we consider chimerism levels in relation to the conditioning regimens and disease type. Furthermore, the predictive role of chimerism analysis as an important method in monitoring the early diagnosis of graft versus host disease (GVHD), minimal residual disease (MRD), graft failure or rejection, and disease relapse has been discussed.

In March 1969, Prof. Thomas and his team performed the first transplantation of hematopoietic stem cells (HSCs). Since then, the transplantation of bone marrow or peripheral stem cells has become a routine method for treatment of a number of malignant and nonmalignant hematologic diseases [1–6]. Allogeneic stem cell transplantation is effective in restoring normal hematopoiesis and is a preferred therapeutic method for malignant diseases of the blood, due to its graft-versus-leukemia (GVL) effect. This effect is mainly due to donor T cells that exhibit immunoreactivity against the minor histocompatibility antigens (mHags) of the recipient or epitopes specific for leukemic cells. The immune system recovery after allogeneic hematopoietic stem cells transplantation is of crucial importance during the postoperative period. This process lasts for months to years and depends mainly on the ability of the donor

**Monitoring of Chimerism Following Hematopoietic** 

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Stem Cell Transplantation**

**Stem Cell Transplantation**

Elissaveta Naumova

**Abstract**

**1. Introduction**

http://dx.doi.org/10.5772/67383

and Elissaveta Naumova

Tsvetelin Lukanov, Milena Ivanova-Shivarova and

Tsvetelin Lukanov, Milena Ivanova-Shivarova

Additional information is available at the end of the chapter

**Keywords:** chimerism, HSCT, STR, conditioning

hematopoietic stem cells to take over the recipient cells.

Additional information is available at the end of the chapter

**Provisional chapter**

### **Monitoring of Chimerism Following Hematopoietic Stem Cell Transplantation Monitoring of Chimerism Following Hematopoietic Stem Cell Transplantation**

Tsvetelin Lukanov, Milena Ivanova-Shivarova and Elissaveta Naumova Tsvetelin Lukanov, Milena Ivanova-Shivarova and Elissaveta Naumova Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67383

#### **Abstract**

One of the most important events in the posthematopoietic stem cell transplantation is the immune system reconstitution—a process characterized by a considerable dynamic. During this period, patients are exposed to different life threatening complications. In this chapter, we consider chimerism levels in relation to the conditioning regimens and disease type. Furthermore, the predictive role of chimerism analysis as an important method in monitoring the early diagnosis of graft versus host disease (GVHD), minimal residual disease (MRD), graft failure or rejection, and disease relapse has been discussed.

**Keywords:** chimerism, HSCT, STR, conditioning

#### **1. Introduction**

In March 1969, Prof. Thomas and his team performed the first transplantation of hematopoietic stem cells (HSCs). Since then, the transplantation of bone marrow or peripheral stem cells has become a routine method for treatment of a number of malignant and nonmalignant hematologic diseases [1–6]. Allogeneic stem cell transplantation is effective in restoring normal hematopoiesis and is a preferred therapeutic method for malignant diseases of the blood, due to its graft-versus-leukemia (GVL) effect. This effect is mainly due to donor T cells that exhibit immunoreactivity against the minor histocompatibility antigens (mHags) of the recipient or epitopes specific for leukemic cells. The immune system recovery after allogeneic hematopoietic stem cells transplantation is of crucial importance during the postoperative period. This process lasts for months to years and depends mainly on the ability of the donor hematopoietic stem cells to take over the recipient cells.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The determination of the genetic origin of hematopoiesis is referred to as chimerism analysis. The term "chimera" was introduced in medicine by Anderson and coworkers [7] in 1951, and in transplantology—by Ford [8] in 1956. It is used for people who have cell populations from different individuals of the same or a different type that arise occasionally during pregnancy or blood transfusions or are targeted—by transplantation of tissues, organs, and cells. The term chimerism refers to the coexistence of cells from two different organisms in a body.

them particularly depleted following treatment [13]. Conditioning regimens cause neutropenia, which are likely to last up to approximately 30 days depending on the source of stem cells [14]. Delayed recovery following immunodepletion is associated with a high degree of morbidity and mortality [13, 15, 16]. Lymphoid recovery is critically dependent on primary immune

NK cells [17–20]. Recovery of NK cells after HSCT depends on an expansion of the cytokineproducing CD56bright NK cell subset. Initial recovery of the T cell compartment relies on the peripheral expansion of memory T cells, driven by cytokines and the presence of alloreactive

which could be due to extrathymic clonal expansion [20–23]. The recovery of B cells takes

B cells [14]. Furthermore, immunodepletion from pretransplant conditioning causes enhanced senescence of the hematopoietic system coupled with an upregulation in the cyclindependent

With the development of less intensive conditioning regimens, the frontiers of hematopoietic stem cell transplantations have become even broader covering more diseases and more people becoming eligible for this treatment. The myeloablative regimens (MAs) are designed to fully eradicate the host immune system and facilitate the engraftment of donor cells. In contrast, reduced-intensity conditioning (RIC) and nonmyeloablative (NMA) regimens aim to suppress the immune system in view of preventing donor cell rejection rather than ablate it. Protocols using lower intensity conditioning regimens have been developed to treat hematological disorders in patients with medical comorbidities or elderly people who are not considered appropriate candidates for more intense and toxic conditioning. Thus, the duration of cytopenia is shorter and initial mixed donor chimerism (DC) is more likely to occur in those patients.

In 2009, Bacigalupo et al. published a report proposing a categorization of conditioning regimens into three categories—myeloablative conditioning, reduced-intensity conditioning, and nonmyeloablative conditioning. These categories were distinguished on the basis of cytopenia duration and the need of stem cell support [26]. The terminology reflects the early regimenrelated toxicity to the host bone marrow cells, but not the biological effect of the transplant.

The MA protocols include administration of high doses of total body irradiation (TBI) and/or alkylating agents, which do not allow autologous hematologic recovery and should benefit a faster donor cell engraftment. Since it is unlikely to fully eradicate a person's immune system, the term myeloablation should be considered as an operational definition, indicating a regimen usually causing an irreversible pancytopenia [26]. The myeloablative conditioning of patients results in higher transplant-related mortality (TRM) and higher frequency of GVHD,

RIC regimens are considered an intermediate category of regimens, which cannot be classified as either NMA or MA—they cause cytopenia that may be prolonged, but the dose of

T cells recover at similar rates in young and aged patients,

Monitoring of Chimerism Following Hematopoietic Stem Cell Transplantation

T cells, B cells, and

http://dx.doi.org/10.5772/67383

79

CD24++CD38++

system organs—the younger the patient, the faster the recovery of CD4+

longest time—up to 2 years and is preceded by expansion of transitional CD19<sup>+</sup>

kinase inhibitors p19Arf and p16ink4A mimicking some of the age-related effects [24, 25].

antigens [14]. Interestingly, CD8<sup>+</sup>


compared to other protocols.


Each stage of the transplant process potentially exposes the patient to complications and lifethreatening events; this is largely due to a lack of understanding of the mechanisms of engraftment, as well as the genetic differences that exist between donor and recipient. Additional factor for consideration is the sensitivity of the methods used. The most widely used one—PCR-STR, is thought to have 1–5% sensitivity. Factors, such as type and stage of disease, patient age, donor type, HLA compatibility, number of transplanted CD34+ cells, graft T cell depletion, and many others, influence the postSCT immune reconstitution, as well as all subsequent complications.

The recovery of hematopoiesis depends mainly on the possibility of the donor hematopoietic stem cells to generate progenitor cells and repopulate the bone marrow niches. It creates a dynamic donor-recipient chimera, the exploration of which—qualitatively or quantitatively, has become an important component of the posttransplant monitoring of the patients. Chimerism is an important indicator for relapse, graft rejection, minimal residual disease (MRD), and graft versus host disease (GVHD). The presence of persistent or emerging recipient cells could mean surviving leukemic cells that could lead to a recurrence of a malignant branch by inhibiting immunocompetent donor cells. Therefore, the accurate assessment of chimerism in the patient's blood or bone marrow provides important information about the engraftment process and aids in providing a more adequate treatment to the recipient. Initially, full donor hematopoiesis was thought to be essential for the survival of the graft after allogeneic stem cell transplantation [9], but in recent years it became clear that posttransplantation chimerism is a dynamic process, and patients with full donor chimerism (FDC) in a posttransplant period may subsequently develop mixed chimerism and vice versa—patients with mixed can develop complete chimerism.

### **2. Chimerism and different therapeutic strategies**

Patients undergoing allogeneic SCT are given chemotherapy alone or in combination with radiotherapy prior to reinfusion of blood or a bone marrow graft. In autologous transplantations, the conditioning aims to eradicate tumor cells, whereas in allogeneic transplantations, it aims to immunosuppress the recipient (for the purposes of preventing graft rejection), provide better control of possible GVHD, eradicate tumor cells (which correlates mainly with the intensity of the regimen used), and allow immune reconstitution. There are some exceptions, like recipients with severe aplastic anemia or those with severe combined immunodeficiency who do not require full immune system eradication.

Treatment with chemotherapy or radiation therapy results in severe depletion of all hematopoietic cells of the immune system. Both alkylating chemotherapeutic agents and irradiation target highly proliferative cells [10–12], including developing and naïve lymphocytes, making them particularly depleted following treatment [13]. Conditioning regimens cause neutropenia, which are likely to last up to approximately 30 days depending on the source of stem cells [14]. Delayed recovery following immunodepletion is associated with a high degree of morbidity and mortality [13, 15, 16]. Lymphoid recovery is critically dependent on primary immune system organs—the younger the patient, the faster the recovery of CD4+ T cells, B cells, and NK cells [17–20]. Recovery of NK cells after HSCT depends on an expansion of the cytokineproducing CD56bright NK cell subset. Initial recovery of the T cell compartment relies on the peripheral expansion of memory T cells, driven by cytokines and the presence of alloreactive antigens [14]. Interestingly, CD8<sup>+</sup> T cells recover at similar rates in young and aged patients, which could be due to extrathymic clonal expansion [20–23]. The recovery of B cells takes longest time—up to 2 years and is preceded by expansion of transitional CD19<sup>+</sup> CD24++CD38++ B cells [14]. Furthermore, immunodepletion from pretransplant conditioning causes enhanced senescence of the hematopoietic system coupled with an upregulation in the cyclindependent kinase inhibitors p19Arf and p16ink4A mimicking some of the age-related effects [24, 25].

With the development of less intensive conditioning regimens, the frontiers of hematopoietic stem cell transplantations have become even broader covering more diseases and more people becoming eligible for this treatment. The myeloablative regimens (MAs) are designed to fully eradicate the host immune system and facilitate the engraftment of donor cells. In contrast, reduced-intensity conditioning (RIC) and nonmyeloablative (NMA) regimens aim to suppress the immune system in view of preventing donor cell rejection rather than ablate it. Protocols using lower intensity conditioning regimens have been developed to treat hematological disorders in patients with medical comorbidities or elderly people who are not considered appropriate candidates for more intense and toxic conditioning. Thus, the duration of cytopenia is shorter and initial mixed donor chimerism (DC) is more likely to occur in those patients.

In 2009, Bacigalupo et al. published a report proposing a categorization of conditioning regimens into three categories—myeloablative conditioning, reduced-intensity conditioning, and nonmyeloablative conditioning. These categories were distinguished on the basis of cytopenia duration and the need of stem cell support [26]. The terminology reflects the early regimenrelated toxicity to the host bone marrow cells, but not the biological effect of the transplant.

#### - *Myeloablative conditioning regimens*

The determination of the genetic origin of hematopoiesis is referred to as chimerism analysis. The term "chimera" was introduced in medicine by Anderson and coworkers [7] in 1951, and in transplantology—by Ford [8] in 1956. It is used for people who have cell populations from different individuals of the same or a different type that arise occasionally during pregnancy or blood transfusions or are targeted—by transplantation of tissues, organs, and cells. The term chimerism refers to the coexistence of cells from two different organisms in a body.

Each stage of the transplant process potentially exposes the patient to complications and lifethreatening events; this is largely due to a lack of understanding of the mechanisms of engraftment, as well as the genetic differences that exist between donor and recipient. Additional factor for consideration is the sensitivity of the methods used. The most widely used one—PCR-STR, is thought to have 1–5% sensitivity. Factors, such as type and stage of disease, patient age, donor

others, influence the postSCT immune reconstitution, as well as all subsequent complications. The recovery of hematopoiesis depends mainly on the possibility of the donor hematopoietic stem cells to generate progenitor cells and repopulate the bone marrow niches. It creates a dynamic donor-recipient chimera, the exploration of which—qualitatively or quantitatively, has become an important component of the posttransplant monitoring of the patients. Chimerism is an important indicator for relapse, graft rejection, minimal residual disease (MRD), and graft versus host disease (GVHD). The presence of persistent or emerging recipient cells could mean surviving leukemic cells that could lead to a recurrence of a malignant branch by inhibiting immunocompetent donor cells. Therefore, the accurate assessment of chimerism in the patient's blood or bone marrow provides important information about the engraftment process and aids in providing a more adequate treatment to the recipient. Initially, full donor hematopoiesis was thought to be essential for the survival of the graft after allogeneic stem cell transplantation [9], but in recent years it became clear that posttransplantation chimerism is a dynamic process, and patients with full donor chimerism (FDC) in a posttransplant period may subsequently develop mixed chimerism and vice versa—patients with mixed can develop complete chimerism.

Patients undergoing allogeneic SCT are given chemotherapy alone or in combination with radiotherapy prior to reinfusion of blood or a bone marrow graft. In autologous transplantations, the conditioning aims to eradicate tumor cells, whereas in allogeneic transplantations, it aims to immunosuppress the recipient (for the purposes of preventing graft rejection), provide better control of possible GVHD, eradicate tumor cells (which correlates mainly with the intensity of the regimen used), and allow immune reconstitution. There are some exceptions, like recipients with severe aplastic anemia or those with severe combined immunodeficiency

Treatment with chemotherapy or radiation therapy results in severe depletion of all hematopoietic cells of the immune system. Both alkylating chemotherapeutic agents and irradiation target highly proliferative cells [10–12], including developing and naïve lymphocytes, making

cells, graft T cell depletion, and many

type, HLA compatibility, number of transplanted CD34+

78 Stem Cells in Clinical Practice and Tissue Engineering

**2. Chimerism and different therapeutic strategies**

who do not require full immune system eradication.

The MA protocols include administration of high doses of total body irradiation (TBI) and/or alkylating agents, which do not allow autologous hematologic recovery and should benefit a faster donor cell engraftment. Since it is unlikely to fully eradicate a person's immune system, the term myeloablation should be considered as an operational definition, indicating a regimen usually causing an irreversible pancytopenia [26]. The myeloablative conditioning of patients results in higher transplant-related mortality (TRM) and higher frequency of GVHD, compared to other protocols.

#### - *Reduced-intensity conditioning regimens*

RIC regimens are considered an intermediate category of regimens, which cannot be classified as either NMA or MA—they cause cytopenia that may be prolonged, but the dose of alkylating agents or TBI is reduced by at least 30%, as compared to MA, and they do require stem cell support [26].

merism at day 30 after HSCT. In such type of HSCT, cases of GVHD are more frequent, while relapses have been rarely observed. Chimerism analysis shows complete donor chimera [37]. In 50–100% of the patients transplanted with T cell-depleted bone marrow grafts, mixed chimera have been observed. In these transplant procedures very often donor-lymphocyte infu-

Monitoring of Chimerism Following Hematopoietic Stem Cell Transplantation

http://dx.doi.org/10.5772/67383

81

One of the main factors allowing increase in the number of HSCT is the development of nonmyeloablative conditioning and reduced-intensity conditioning regimens. The introduction of these protocols is related to the lower treatment-related mortality, allowing the application of HSCT as a treatment for elderly people and patients in a severe clinical condition. Another substantial advantage of these protocols is the better immune reconstitution due to the lower degree of thymus injury, despite the fact that studies showed similar outcomes for HSCT. This allows better regeneration of naïve T cells and proliferation of immune-competent cells of the recipient, which have survived after conditioning. The majority of RIC protocols are based on a combination of purine analogs, usually fludarabine with alkylating agents, such as busulfan or cyclophosphamide, and represent a different intensity of myelosuppression [42–44]. Another approach is to use low doses of TBI, alone or in combination with fludarabine [45]. This approach relies on the GVL effect and is associated with minimal toxicity. Several studies have shown the association between patient conditioning and posttransplant chimerism

Unlike HSCT with myeloablative conditioning, nonmyeloablative conditioning is related to a mixed chimerism [6]. Additionally, the probability to develop aGVHD is lower, while probability to develop cGVHD is comparable for both conditioning protocols. The immunobiology of allo-SCT after NMA conditioning differs from that after MA mainly by the release of less inflammatory cytokines. Tissue damage caused by myeloablation is translated into proinflammatory cytokines that are supposed to provide a milieu for the development of GVHD. As a result, aGVHD after NMA is delayed and may develop after day +100, remaining a significant cause of morbidity and mortality in NMA SCT patients. Mixed chimerism does

Relatively decreased aGVHD after RIC HSCT could be explained by a combination of pretransplant conditioning and initial mixed chimerism that could contribute to the development of tolerance [29, 45]. Some patients demonstrate late development of aGVHD that coincides with the transition from mixed to complete donor T cell chimera [46]. In patients who do not develop GVHD, donor lymphocyte infusion could be performed in order to achieve complete chimera and GVL effect. Therefore, in HSCT with nonmyeloablative conditioning, it is very important to monitor chimerism level in order to assess correctly the need of donor lymphocyte infusion and the effect of this treatment [37]. Lower levels of T and NK cells in the first 30 days following HSCT are associated with increased risk of graft rejection. Granulocyte and monocyte chimerism levels have limited relevance for graft rejection [46]. In contrast, high levels of donor T cells are predictive for development of aGVHD grade 2–4 [31, 46, 48]. Additionally, levels above 90% donor T and/or NK cells are associated with antitumour response and disease-free survival [46,

not mean grim prognosis but could be associated with increased risk of relapses.

**2.2. Investigation of chimerism in nonmyeloablative/reduced-intensity HSCT**

sion is performed, especially in CML patients.

levels [46, 47].


NMAs typically cause minimal cytopenia—they do not require stem cell support, but are immunosuppressive to the extent that, when followed by G-CSF mobilized peripheral blood stem cells (PBSC), they usually result in full donor cell engraftment. According to Bacigalupo et al., the NMA regimens are more "immune-ablative" than myelo-ablative [26]. The NMA conditioning results in lower TRM, as compared to MA but requires a larger amount of donor cells to facilitate a full replacement of the recipient's hematopoiesis [27, 28]. Acute GVHD after NMA is delayed and may develop after day +100, at a time when chronic GVHD is usually diagnosed after MA regimen. GVHD remains a significant cause of morbidity and mortality after the application of both MA and NMA [29].

Many studies address the importance of chimerism monitoring after allo-SCT, but the data are controversial [30–36]. Moreover, chimerism monitoring in patients with MA conditioning is considered to be less informative and unnecessary, since the aggressive pretransplant preparation is thought to result in irreversible pancytopenia and achievement of stable full donor chimerism [30, 37, 38]. The chimerism status seems to be even more complicated as new data show the vague borders between the postSCT effects of the different conditioning protocols [31, 39, 40].

The aggressive pretransplant therapy is more effective against the disease, but its use is limited to younger and healthier patients who are able to tolerate concomitant toxicity. MA conditioning aims to facilitate normal hematopoietic reconstitution with FDC by day +30, after allo-SCT. In unmanipulated grafts, chimerism analysis shows mostly FDC, as its early establishment may be an indication of GVHD. Mixed chimerism is more likely to be observed in patients who had received T cell-depleted graft. T cell depletion is used to reduce the frequency of GVHD—Mickelson et al. showed a significantly increased risk of developing acute or chronic GVHD in patients who reached levels of donor chimerism in T cells >90% (HR = 1.92, P = 0.08 and HR = 2.26, and P = 0.07, respectively) [31]. Moreover, speed and extent of donor chimerism have been shown to be influenced by additional factors including primary diagnosis and previous treatment [37].

#### **2.1. Investigation of chimerism in myeloablative HSCT**

From a historical point of view, ablative chemotherapy and total body irradiation were accepted as a treatment for elimination of malignant cells and targeting immunosuppression with view of facilitating donor cell engraftment. High dosage protocols aiming to achieve antileukemic effect are still in use for patients with aggressive malignant diseases. The main treatment includes cyclophosphamide and TBI or busulfan (BU), and more often a combination of busulfan and fludarabine at dosages eliminating all myeloid cells [41]. The aggressive pretransplant conditioning is very effective for the primary disease, but it is limited to younger patients who are able to tolerate treatment-related toxicity. Myeloablative conditioning schemes could facilitate reconstitution of hematopoiesis with a complete lymphocyte chimerism at day 30 after HSCT. In such type of HSCT, cases of GVHD are more frequent, while relapses have been rarely observed. Chimerism analysis shows complete donor chimera [37]. In 50–100% of the patients transplanted with T cell-depleted bone marrow grafts, mixed chimera have been observed. In these transplant procedures very often donor-lymphocyte infusion is performed, especially in CML patients.

#### **2.2. Investigation of chimerism in nonmyeloablative/reduced-intensity HSCT**

alkylating agents or TBI is reduced by at least 30%, as compared to MA, and they do require

NMAs typically cause minimal cytopenia—they do not require stem cell support, but are immunosuppressive to the extent that, when followed by G-CSF mobilized peripheral blood stem cells (PBSC), they usually result in full donor cell engraftment. According to Bacigalupo et al., the NMA regimens are more "immune-ablative" than myelo-ablative [26]. The NMA conditioning results in lower TRM, as compared to MA but requires a larger amount of donor cells to facilitate a full replacement of the recipient's hematopoiesis [27, 28]. Acute GVHD after NMA is delayed and may develop after day +100, at a time when chronic GVHD is usually diagnosed after MA regimen. GVHD remains a significant cause of morbidity and

Many studies address the importance of chimerism monitoring after allo-SCT, but the data are controversial [30–36]. Moreover, chimerism monitoring in patients with MA conditioning is considered to be less informative and unnecessary, since the aggressive pretransplant preparation is thought to result in irreversible pancytopenia and achievement of stable full donor chimerism [30, 37, 38]. The chimerism status seems to be even more complicated as new data show the vague borders between the postSCT effects of the different conditioning

The aggressive pretransplant therapy is more effective against the disease, but its use is limited to younger and healthier patients who are able to tolerate concomitant toxicity. MA conditioning aims to facilitate normal hematopoietic reconstitution with FDC by day +30, after allo-SCT. In unmanipulated grafts, chimerism analysis shows mostly FDC, as its early establishment may be an indication of GVHD. Mixed chimerism is more likely to be observed in patients who had received T cell-depleted graft. T cell depletion is used to reduce the frequency of GVHD—Mickelson et al. showed a significantly increased risk of developing acute or chronic GVHD in patients who reached levels of donor chimerism in T cells >90% (HR = 1.92, P = 0.08 and HR = 2.26, and P = 0.07, respectively) [31]. Moreover, speed and extent of donor chimerism have been shown to be influenced by additional factors including pri-

From a historical point of view, ablative chemotherapy and total body irradiation were accepted as a treatment for elimination of malignant cells and targeting immunosuppression with view of facilitating donor cell engraftment. High dosage protocols aiming to achieve antileukemic effect are still in use for patients with aggressive malignant diseases. The main treatment includes cyclophosphamide and TBI or busulfan (BU), and more often a combination of busulfan and fludarabine at dosages eliminating all myeloid cells [41]. The aggressive pretransplant conditioning is very effective for the primary disease, but it is limited to younger patients who are able to tolerate treatment-related toxicity. Myeloablative conditioning schemes could facilitate reconstitution of hematopoiesis with a complete lymphocyte chi-

stem cell support [26].

protocols [31, 39, 40].


80 Stem Cells in Clinical Practice and Tissue Engineering

mortality after the application of both MA and NMA [29].

mary diagnosis and previous treatment [37].

**2.1. Investigation of chimerism in myeloablative HSCT**

One of the main factors allowing increase in the number of HSCT is the development of nonmyeloablative conditioning and reduced-intensity conditioning regimens. The introduction of these protocols is related to the lower treatment-related mortality, allowing the application of HSCT as a treatment for elderly people and patients in a severe clinical condition. Another substantial advantage of these protocols is the better immune reconstitution due to the lower degree of thymus injury, despite the fact that studies showed similar outcomes for HSCT. This allows better regeneration of naïve T cells and proliferation of immune-competent cells of the recipient, which have survived after conditioning. The majority of RIC protocols are based on a combination of purine analogs, usually fludarabine with alkylating agents, such as busulfan or cyclophosphamide, and represent a different intensity of myelosuppression [42–44]. Another approach is to use low doses of TBI, alone or in combination with fludarabine [45]. This approach relies on the GVL effect and is associated with minimal toxicity. Several studies have shown the association between patient conditioning and posttransplant chimerism levels [46, 47].

Unlike HSCT with myeloablative conditioning, nonmyeloablative conditioning is related to a mixed chimerism [6]. Additionally, the probability to develop aGVHD is lower, while probability to develop cGVHD is comparable for both conditioning protocols. The immunobiology of allo-SCT after NMA conditioning differs from that after MA mainly by the release of less inflammatory cytokines. Tissue damage caused by myeloablation is translated into proinflammatory cytokines that are supposed to provide a milieu for the development of GVHD. As a result, aGVHD after NMA is delayed and may develop after day +100, remaining a significant cause of morbidity and mortality in NMA SCT patients. Mixed chimerism does not mean grim prognosis but could be associated with increased risk of relapses.

Relatively decreased aGVHD after RIC HSCT could be explained by a combination of pretransplant conditioning and initial mixed chimerism that could contribute to the development of tolerance [29, 45]. Some patients demonstrate late development of aGVHD that coincides with the transition from mixed to complete donor T cell chimera [46]. In patients who do not develop GVHD, donor lymphocyte infusion could be performed in order to achieve complete chimera and GVL effect. Therefore, in HSCT with nonmyeloablative conditioning, it is very important to monitor chimerism level in order to assess correctly the need of donor lymphocyte infusion and the effect of this treatment [37]. Lower levels of T and NK cells in the first 30 days following HSCT are associated with increased risk of graft rejection. Granulocyte and monocyte chimerism levels have limited relevance for graft rejection [46]. In contrast, high levels of donor T cells are predictive for development of aGVHD grade 2–4 [31, 46, 48]. Additionally, levels above 90% donor T and/or NK cells are associated with antitumour response and disease-free survival [46, 49]. Similar results have been reported by Mohty et al. (n = 102) [48]. In contrast, some studies do not reveal an association between the level of donor T lymphocytes and the development of GVHD [50, 51]. In their study, Lim et al. showed that rapid conversion to complete donor chimera is associated with decreased overall survival and increased TRM when compared with persistence of mixed chimerism. No association with relapse frequency was observed [52]. Mixed chimera is related to better survival—this observation could be explained by the persisting recipient cells, associated with resistance to infections and overall survival [52, 53]. Patients with low absolute count of dendritic cells, one month following HSCT, have a higher TRM (60% versus 12%, P < 0.02) and decreased overall survival (15% versus 45%, P < 0.002) [54]. There is also an association between decreased CD16<sup>+</sup> dendritic cells and increased infectionrelated mortality (50% versus 0%, P < 0.05). The possibility that the infection itself is likely to decrease the number of these cells cannot be excluded [54].

**4. Chimerism in malignant diseases**

GVHD [46, 62–64].

28 days [34, 65, 66].

late rejection.

value for MRD detection.

**5. Chimerism analysis: technical considerations**

Monitoring MRD and chimerism levels in patients with malignant diseases is very important. Allogeneic HSCT is an effective therapy in both low- and high-risk CLL. The analysis of 44 high-risk patients with 17p deletion and RIC transplantation showed a four-year cumulative frequency of disease progression of 34% [62]. Establishment of T cell mixed chimera 90 days following transplantation and chemorefractory disease are associated with increased risk of disease progression. Establishment of complete donor chimera and lack of MRD are associated with prolonged DFS. Limited data on the relapse outcome in CLL are available and some patients have positive response to DLL treatment and immunosuppression withdrawal. A better response has been observed in patients with 100% donor T cells. Many studies have shown that conversion from mixed to complete donor chimera predicts development of

Monitoring of Chimerism Following Hematopoietic Stem Cell Transplantation

http://dx.doi.org/10.5772/67383

83

In patients with acute leukemia (AML and ALL), mixed chimera may be associated with increased risk of graft rejection. A greater risk has been demonstrated in patients with high levels of residual cells and/or rapid increase of recipient cells [2, 4, 5]. Monitoring of the level of expression of WT1 and the kinetics of nonseparated CD34 chimerism allows early

WT1 expression under predefined cut off, relapse could be excluded within the following

Establishment of mixed chimera can be associated with many factors such as conditioning intensity, T cell-depleted grafts, CD34 cell dosage, techniques used for monitoring, and primary disease. Due to the dynamics in chimerism development, chimerism should be monitored at shorter intervals, especially in the first 1–2 months following transplantation when the risk of rejection is higher. In the early posttransplant period, the persistent mixed chimera could indicate graft rejection or early relapse. On the other hand, the increased number of recipient cells at a later stage after transplantation is associated with a relapse or

Due to its low sensitivity of about 1%, chimerism analysis could be used as a prognostic rather than an indirect indicator for MRD. With the introduction of RQ-PCR technique, the sensitivity of chimerism monitoring has improved to 0.1%, resulting in improvement of its prognostic

Expanding the curability of hematological diseases with stem cell transplantation, as well as introducing new conditioning protocols allows more patients to be eligible for this treatment. This raises the need for better posttransplant chimerism monitoring, since the different quantitative or semiquantitative methods have their advantages and disadvantages. While the significance of the absolute value is still under discussion, the relative changes in engraftment

levels and

detection of relapse in MDS and AML patients. In patients with stable CD34<sup>+</sup>

#### **2.3. Chimerism following donor lymphocyte infusion (DLI)**

Donor lymphocyte infusion is a new therapeutic strategy, applied mainly in cases of nonmyeloablative, T cell-depleted myeloablative HSCT, or haploidentical transplantations due to its GVL effect. These patients have delayed immune reconstitution, increasing the risk of disease relapse. Application of standard DLI is limited due to GVHD and aplasia. New protocols using G-CSF-stimulated blood progenitor cells, allodepleted donor T cells and mHag-specific CD8<sup>+</sup> cytotoxic T lymphocytes have been currently developed allowing wider application of this type of therapy. A limitation of the latter is estimating the right time for DLI. Lymphocyte infusions in the early relapses are associated with favorable outcome [55, 56]. Therefore, chimerism should be monitored regularly in order to detect recipient-specific hematopoiesis [6, 57, 58]. Infused lymphocytes are sensitized by surface antigens that are associated with leukemia or polymorphic minor histocompatibility antigens, which are expressed by the leukemic cells. This induces their transformation into cytotoxic lymphocytes killing cancer cells. DLI is most effective in patients with CML (approximately 75%), which could be explained by the antigen-presenting cells found in malignant clones. This therapy is not effective in patients with AML or multiple myeloma. The development of new therapeutic protocols has decreased aplasia and GVHD.

### **3. Chimerism in nonmalignant diseases**

Currently, a major part of inherited or acquired nonmalignant diseases, such as thalassemia, aplastic anemia, and immune deficiencies, can be successfully treated by HSCT. The aim of such type of transplantation is to achieve stable engraftment, enabling hematopoiesis, enzyme activity, or immune competence. Therefore, establishment of complete donor chimera is not necessary and the recipient can be conditioned by nonmyeloablative therapy. This results more often in the establishment of mixed chimera, increased risk of graft rejection, and decreased risk of GVHD. On the other hand, rapid development of complete chimera in NK and T cells is very important for the successful engraftment, although it increases the frequency of acute GVHD [59–61].

## **4. Chimerism in malignant diseases**

49]. Similar results have been reported by Mohty et al. (n = 102) [48]. In contrast, some studies do not reveal an association between the level of donor T lymphocytes and the development of GVHD [50, 51]. In their study, Lim et al. showed that rapid conversion to complete donor chimera is associated with decreased overall survival and increased TRM when compared with persistence of mixed chimerism. No association with relapse frequency was observed [52]. Mixed chimera is related to better survival—this observation could be explained by the persisting recipient cells, associated with resistance to infections and overall survival [52, 53]. Patients with low absolute count of dendritic cells, one month following HSCT, have a higher TRM (60% versus 12%, P < 0.02) and decreased overall survival (15% versus 45%, P < 0.002) [54].

related mortality (50% versus 0%, P < 0.05). The possibility that the infection itself is likely to

Donor lymphocyte infusion is a new therapeutic strategy, applied mainly in cases of nonmyeloablative, T cell-depleted myeloablative HSCT, or haploidentical transplantations due to its GVL effect. These patients have delayed immune reconstitution, increasing the risk of disease relapse. Application of standard DLI is limited due to GVHD and aplasia. New protocols using G-CSF-stimulated blood progenitor cells, allodepleted donor T cells and mHag-specific CD8<sup>+</sup> cytotoxic T lymphocytes have been currently developed allowing wider application of this type of therapy. A limitation of the latter is estimating the right time for DLI. Lymphocyte infusions in the early relapses are associated with favorable outcome [55, 56]. Therefore, chimerism should be monitored regularly in order to detect recipient-specific hematopoiesis [6, 57, 58]. Infused lymphocytes are sensitized by surface antigens that are associated with leukemia or polymorphic minor histocompatibility antigens, which are expressed by the leukemic cells. This induces their transformation into cytotoxic lymphocytes killing cancer cells. DLI is most effective in patients with CML (approximately 75%), which could be explained by the antigen-presenting cells found in malignant clones. This therapy is not effective in patients with AML or multiple myeloma. The development of new therapeutic protocols has decreased aplasia and GVHD.

Currently, a major part of inherited or acquired nonmalignant diseases, such as thalassemia, aplastic anemia, and immune deficiencies, can be successfully treated by HSCT. The aim of such type of transplantation is to achieve stable engraftment, enabling hematopoiesis, enzyme activity, or immune competence. Therefore, establishment of complete donor chimera is not necessary and the recipient can be conditioned by nonmyeloablative therapy. This results more often in the establishment of mixed chimera, increased risk of graft rejection, and decreased risk of GVHD. On the other hand, rapid development of complete chimera in NK and T cells is very important for the successful engraftment, although it increases the

dendritic cells and increased infection-

There is also an association between decreased CD16<sup>+</sup>

82 Stem Cells in Clinical Practice and Tissue Engineering

**3. Chimerism in nonmalignant diseases**

frequency of acute GVHD [59–61].

decrease the number of these cells cannot be excluded [54].

**2.3. Chimerism following donor lymphocyte infusion (DLI)**

Monitoring MRD and chimerism levels in patients with malignant diseases is very important. Allogeneic HSCT is an effective therapy in both low- and high-risk CLL. The analysis of 44 high-risk patients with 17p deletion and RIC transplantation showed a four-year cumulative frequency of disease progression of 34% [62]. Establishment of T cell mixed chimera 90 days following transplantation and chemorefractory disease are associated with increased risk of disease progression. Establishment of complete donor chimera and lack of MRD are associated with prolonged DFS. Limited data on the relapse outcome in CLL are available and some patients have positive response to DLL treatment and immunosuppression withdrawal. A better response has been observed in patients with 100% donor T cells. Many studies have shown that conversion from mixed to complete donor chimera predicts development of GVHD [46, 62–64].

In patients with acute leukemia (AML and ALL), mixed chimera may be associated with increased risk of graft rejection. A greater risk has been demonstrated in patients with high levels of residual cells and/or rapid increase of recipient cells [2, 4, 5]. Monitoring of the level of expression of WT1 and the kinetics of nonseparated CD34 chimerism allows early detection of relapse in MDS and AML patients. In patients with stable CD34<sup>+</sup> levels and WT1 expression under predefined cut off, relapse could be excluded within the following 28 days [34, 65, 66].

Establishment of mixed chimera can be associated with many factors such as conditioning intensity, T cell-depleted grafts, CD34 cell dosage, techniques used for monitoring, and primary disease. Due to the dynamics in chimerism development, chimerism should be monitored at shorter intervals, especially in the first 1–2 months following transplantation when the risk of rejection is higher. In the early posttransplant period, the persistent mixed chimera could indicate graft rejection or early relapse. On the other hand, the increased number of recipient cells at a later stage after transplantation is associated with a relapse or late rejection.

Due to its low sensitivity of about 1%, chimerism analysis could be used as a prognostic rather than an indirect indicator for MRD. With the introduction of RQ-PCR technique, the sensitivity of chimerism monitoring has improved to 0.1%, resulting in improvement of its prognostic value for MRD detection.

### **5. Chimerism analysis: technical considerations**

Expanding the curability of hematological diseases with stem cell transplantation, as well as introducing new conditioning protocols allows more patients to be eligible for this treatment. This raises the need for better posttransplant chimerism monitoring, since the different quantitative or semiquantitative methods have their advantages and disadvantages. While the significance of the absolute value is still under discussion, the relative changes in engraftment kinetics are a reliable sign for acceptance or rejection of the graft. This, in its turn, requires a determination of the factors intrinsic for the platform, which may influence the observed changes in chimerism result [67, 68]. Some of the most important characteristics of the methods for postSCT chimerism monitoring are their sensitivity, precision, and reproducibility [69–71].

Investigating microsatellites (STR) and minisatellites (VNTR) is considered the most sensitive and informative technique to study the levels of chimerism after HSCT. Micro (2–5 bp) and minisatellites (9–80 bp) are tandemly repeated blocks of noncoding DNA that are widespread throughout the human genome. The repeat-pattern of these blocks varies greatly among individuals in a population. There is a large number of STR (short tandem repeats) systems that have been mapped throughout the human genome, and they are found on almost every chromosome (**Table 1**).


The method is semiquantitative and has a moderate level of sensitivity—1–5%; neither gender nor HLA compatibility of donor and recipient can influence its informativity; it requires a very small number of cells, which makes it applicable in the first postSCT days.

mtDNA HV1 and HV2

**Chromosome STR markers RFLP-based VNTR markers Other PCR-based** 

17 D17S974, D17S976, and D17S1301 D17S79, D17S26 D17S5, YNZ22

19 D19S253, D19S433 LDLR (PM)

13 D13S308, D13S317, and D13S1492

Penta E, D15S659, and

18 MBP, D18S51, D18S535, D18S849, D18S853, and D18S1270

20 D20S85, D20S161, D20S470, D20S482, and

15 CYAR04 (P450), FES/FPS,

D15S822

D20S1082

21 Penta D, D21S11, D21S1437, and D21S2055 22 D22S683 and D22S1045

X HPRTB, ARA, STRX1, DXYS156,

GATA165B12, and GATA172D05

Y DYS19, DXYS156, DYS385 a/b, DYS388,

14 D14S306, D14S608, and D14S1434 D14S13

16 D16S537, D16S539, and D16S3253 D16S85

DXS101, DXS981, DXS6789, DXS6795, DXS6797, DXS6800, DXS6801, DXS6803, DXS6807,DXS6809, DXS7130, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS9895, DXS9898, DXS9905, DXS9908, DXS10011, GATA31E08,

DYS389 I/II, DYS390, DYS391, DYS392, DYS393, DYF406S1, DYS437, DYS438, DYS439, DYS444, DYS446, DYS447, DYS448, DYS449, DYS456, DYS458, DYS460, DYS461, DYS462, DYS463, DYS464 a/b/c/d, DYS481, DYS485, DYS495, DYS505, DYS508, DYS520, DYS522, DYS525, DYS531, DYS532, DYS533, DYS534, DYS540, DYS549, DYS556, DYS557, DYS570, DYS576, DYS578, DYS589, DYS594, DYS617, DYS635, DYS643, DYS724 (CD a/b), GATA-H4, and YCAII a/b

**Table 1.** Chromosome location of mini- and microsatellites [72].

**markers**

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Monitoring of Chimerism Following Hematopoietic Stem Cell Transplantation

Amelogenin

Amelogenin and Y-SNPs


**Table 1.** Chromosome location of mini- and microsatellites [72].

kinetics are a reliable sign for acceptance or rejection of the graft. This, in its turn, requires a determination of the factors intrinsic for the platform, which may influence the observed changes in chimerism result [67, 68]. Some of the most important characteristics of the methods for postSCT chimerism monitoring are their sensitivity, precision, and reproducibility [69–71].

Investigating microsatellites (STR) and minisatellites (VNTR) is considered the most sensitive and informative technique to study the levels of chimerism after HSCT. Micro (2–5 bp) and minisatellites (9–80 bp) are tandemly repeated blocks of noncoding DNA that are widespread throughout the human genome. The repeat-pattern of these blocks varies greatly among individuals in a population. There is a large number of STR (short tandem repeats) systems that have been mapped throughout the human genome, and they are found on almost every chromosome (**Table 1**).

**Chromosome STR markers RFLP-based VNTR markers Other PCR-based** 

D5S110

D10S28

D12S11

1 F13B, RENA4, D1S1171, D1S1627,

84 Stem Cells in Clinical Practice and Tissue Engineering

3 ACPP, D3S1349, D3S1352, D3S1358,

D1GATA113

and D3S4529 4 FABP, FGA (FIBRA), GABARB15, D4S2364,

D5S2500

8 LPL (LIPOL), D8S306,

and D8S1179 9 D9S52, D9S302, D9S304, D9S925, D9S1122, and D9S2157

and D10S2326

2 APOB, TPOX, D2S410,

D1S1656, D1S1677, D1S2142, and

D2S441, D2S436, D2S1242, D2S1338, D2S1360, D2S1772, and D2S1776

D3S1359, D3S1545, D3S1744, D3S3053,

D4S2366, D4S2368, and D4S2408

D8S320, D8S323, D8S344, D8S347, D8S639, D8S1132, D8S1115,

10 D10S89, D10S1248, D10S1435, D10S2325,

11 APOAI1, TH01 (TC11), UGB, D11S488, D11S554, and D11S4463

12 CD4, PLA2A1, VWA, D12S67, D12S391, D12S1090, and D12ATA63

5 CSF1PO, D5S373, D5S815, D5S818, and

6 F13A1, FOLP23, SE33 (ACTBP2), D6S366, D6S474, D6S477, D6S502, D6S965,

D6S1017, and D6S1043

7 D7S460, D7S809, D7S820, D7S821, D7S1517, D7S1520, and D7S3048

**markers**

D1S7 and D1S339 D1S80

D2S44 ApoB

D6S132 DQa

D7S21, D7S22, D7S467 D7S8 (PM)

D4S139 GC (PM) and GYPA

(PM)

HBGG (PM)

The method is semiquantitative and has a moderate level of sensitivity—1–5%; neither gender nor HLA compatibility of donor and recipient can influence its informativity; it requires a very small number of cells, which makes it applicable in the first postSCT days. Multiplex platforms use STR/VNTR (variable number tandem repeats) loci with identical amplification protocols and different allelic lengths, using fluorescently labeled primers. Due to its large size, VNTR more often showed discrepancies in the evaluation of chimerism between markers [73]. All loci are amplified together in a single PCR reaction and amplification products are electrophoretically separated on an automated DNA sequencer—fragment analysis. Further analysis is carried out by specialized software. Microsatellites are more widely used, since they are polymorphic, more sensitive, faster to work with, and cheaper. Despite their excellent performance in forensic science, their application in the study of chimerism has its limitations. The most important one results from the very design of the platform—the amount of DNA is indirectly evaluated based on measurement of fluorescence during electrophoresis. The various fluorophores differ in their efficiency to emit light, and the platform has no internal calibration for measurement. As a result, the measurement of one and the same absolute value of DNA will not be equally estimated in all loci. However, this fluorescence-based technology is considered the gold standard in the study of chimerism [58]. Another feature of the STR system is the presence of additional signals—stutter peaks, which are mainly n − 1 and to a lesser degree n + 1 signals [74, 75]. These artifacts are the result of slipped-strand mispairing during amplification. Their size depends on the size and type of the allele, and is in the range 2–13% of their respective base peaks. Stutter is less pronounced with larger repeat units (dinucleotides > tri- > tetra- > penta-). Longer repeat regions generate more stutter, and each successive stutter product is less intense. If stutter peaks match the corresponding donor or recipient peaks, this would affect the estimate of chimerism. This is especially important at low levels of the host residual cells, which would hamper the assessment of minimal residual disease. In heterozygous loci, the two alleles should be equal in amount, but due to stochastic effects during PCR amplification, an imbalance in the two detected alleles is established—this is especially true when the amount of DNA being amplified is limited. Under conditions of extreme imbalance, one allele may "drop-out".

The main objective in posttransplantation monitoring is early diagnosis of adverse events. Due to the dynamic nature of the development of chimerism, its monitoring should be carried out at short intervals, especially in the first 1–2 months after transplantation, when the risk of disease recurrence or graft failure/rejection is greatest. In the earliest posttransplantation period, the presence of persistent mixed chimerism or the disappearance of donor alleles are associated with both graft rejection and early relapse of the underlying disease. On the other hand, the increasing number of recipient cells in the later periods after the transplantation is associated with upcoming relapse or late allograft rejection. The mechanisms that direct hematopoietic recovery toward one or another course are not yet fully understood. The analysis of chimerism kinetics allows early differentiation between the lack of engraftment and its delay, as well as early detection of patients at high risk of developing GVHD or susceptible to a relapse. In this context, the study of chimerism is undoubtedly an important method for monitoring the outcome of HSCT.

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87

Department of Clinical Immunology and Stem Cell Bank, University Hospital Alexandrovska,

[1] Kvasnicka H, Wickenhauser C, Thiele J, Varus E, Hamm K, et al. Mixed chimerism of bone marrow vessels (endothelial cells, myofibroblasts) following allogeneic transplantation for chronic myelogenous leukemia. Leuk Lymphoma. 2003;**44**(2):321–328. DOI:

[2] Bader P, Hancock J, Kreyenberg H, Goulden N, Niethammer D, Oakhill A, et al. Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for post-transplant outcome in children with ALL. Leukemia. 2002;**16**(9):1668–

[3] Elmaagacli A, Peceney R, Steckel N, Trenschel R, Ottinger H, Grosse-Wilde H, et al. Outcome of transplantation of highly purified peripheral blood CD34+ cells with T cells add-back compared with unmanipulated bone marrow or peripheral blood stem cells from HLA-identical sibling donors in first chronic phase CML. Blood. 2003;**101**(2):446–

[4] Bader P, Beck J, Frey A, Schlegel P, Hebarth H, Handgretinger R, et al. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant.

Tsvetelin Lukanov\*, Milena Ivanova-Shivarova and Elissaveta Naumova

\*Address all correspondence to: ts\_lukanov@yahoo.com

10.1080/1042819021000035699

1672. DOI: 10.1038/sj.leu.2402552

453. DOI: 10.1182/blood-2002-05-1615

1998;**21**(5):487–495. DOI: 10.1038/sj.bmt.1701119

**Author details**

Sofia, Bulgaria

**References**

#### **6. Conclusion**

Different protocols have been used to condition patients prior to HSCT. They differ from each other in intensity of myeloablation. Myeloablative conditioning is very aggressive and leads to high toxicity and TRM, so its application is limited to younger patients and patients in good medical condition. The risk of TRM decreases with time, although the causes for this are not fully understood. Most likely this is due to advances in technology for HLA typing, better understanding of the role of HLA compatibility, particularly with regard to unrelated donors, and better medical services for patients. Various conditioning protocols are deemed to have varying degrees of ablation of the recipient immune system resulting in different profiles of posttransplantation recovery.

The levels of chimerism after HSCT depend on a number of factors, including the intensity of pretransplant conditioning, the use of T cell-depleted grafts, the number of transplanted stem cells, the sensitivity of the technique used for detection and the interval of study, and as well as the type of the disease.

The main objective in posttransplantation monitoring is early diagnosis of adverse events. Due to the dynamic nature of the development of chimerism, its monitoring should be carried out at short intervals, especially in the first 1–2 months after transplantation, when the risk of disease recurrence or graft failure/rejection is greatest. In the earliest posttransplantation period, the presence of persistent mixed chimerism or the disappearance of donor alleles are associated with both graft rejection and early relapse of the underlying disease. On the other hand, the increasing number of recipient cells in the later periods after the transplantation is associated with upcoming relapse or late allograft rejection. The mechanisms that direct hematopoietic recovery toward one or another course are not yet fully understood. The analysis of chimerism kinetics allows early differentiation between the lack of engraftment and its delay, as well as early detection of patients at high risk of developing GVHD or susceptible to a relapse. In this context, the study of chimerism is undoubtedly an important method for monitoring the outcome of HSCT.

### **Author details**

Multiplex platforms use STR/VNTR (variable number tandem repeats) loci with identical amplification protocols and different allelic lengths, using fluorescently labeled primers. Due to its large size, VNTR more often showed discrepancies in the evaluation of chimerism between markers [73]. All loci are amplified together in a single PCR reaction and amplification products are electrophoretically separated on an automated DNA sequencer—fragment analysis. Further analysis is carried out by specialized software. Microsatellites are more widely used, since they are polymorphic, more sensitive, faster to work with, and cheaper. Despite their excellent performance in forensic science, their application in the study of chimerism has its limitations. The most important one results from the very design of the platform—the amount of DNA is indirectly evaluated based on measurement of fluorescence during electrophoresis. The various fluorophores differ in their efficiency to emit light, and the platform has no internal calibration for measurement. As a result, the measurement of one and the same absolute value of DNA will not be equally estimated in all loci. However, this fluorescence-based technology is considered the gold standard in the study of chimerism [58]. Another feature of the STR system is the presence of additional signals—stutter peaks, which are mainly n − 1 and to a lesser degree n + 1 signals [74, 75]. These artifacts are the result of slipped-strand mispairing during amplification. Their size depends on the size and type of the allele, and is in the range 2–13% of their respective base peaks. Stutter is less pronounced with larger repeat units (dinucleotides > tri- > tetra- > penta-). Longer repeat regions generate more stutter, and each successive stutter product is less intense. If stutter peaks match the corresponding donor or recipient peaks, this would affect the estimate of chimerism. This is especially important at low levels of the host residual cells, which would hamper the assessment of minimal residual disease. In heterozygous loci, the two alleles should be equal in amount, but due to stochastic effects during PCR amplification, an imbalance in the two detected alleles is established—this is especially true when the amount of DNA being amplified is

limited. Under conditions of extreme imbalance, one allele may "drop-out".

Different protocols have been used to condition patients prior to HSCT. They differ from each other in intensity of myeloablation. Myeloablative conditioning is very aggressive and leads to high toxicity and TRM, so its application is limited to younger patients and patients in good medical condition. The risk of TRM decreases with time, although the causes for this are not fully understood. Most likely this is due to advances in technology for HLA typing, better understanding of the role of HLA compatibility, particularly with regard to unrelated donors, and better medical services for patients. Various conditioning protocols are deemed to have varying degrees of ablation of the recipient immune system resulting in different profiles of

The levels of chimerism after HSCT depend on a number of factors, including the intensity of pretransplant conditioning, the use of T cell-depleted grafts, the number of transplanted stem cells, the sensitivity of the technique used for detection and the interval of study, and as well

**6. Conclusion**

86 Stem Cells in Clinical Practice and Tissue Engineering

posttransplantation recovery.

as the type of the disease.

Tsvetelin Lukanov\*, Milena Ivanova-Shivarova and Elissaveta Naumova

\*Address all correspondence to: ts\_lukanov@yahoo.com

 Department of Clinical Immunology and Stem Cell Bank, University Hospital Alexandrovska, Sofia, Bulgaria

### **References**


[5] Bader P, Holle W, Klingebiel T, Handgretinger R, Benda N, Schlegel P, et al. Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft reject ion in children. Bone Marrow Transplant. 1997;**19**(7):697–702. DOI: 10.1038/sj.bmt.1700721

[19] Storek J, Witherspoon RP, Storb R. T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life. Bone Marrow

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[20] Dudakov J, van den Brink M. Greater than the sum of their parts: Combination strategies for immune regeneration following allogeneic hematopoietic stem cell transplantation. Best Pract Res Clin Haematol. 2011;**24**(3):467–476. DOI: 10.1016/j.beha.2011.05.003.

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bbmt.2013.02.015


**Section 3**

**Advances on Stem Cells in Medical Practice**

**Advances on Stem Cells in Medical Practice**

**Chapter 5**

**Provisional chapter**

**Human-Induced Pluripotent Stem Cell-Derived**

**Human-Induced Pluripotent Stem Cell-Derived** 

DOI: 10.5772/intechopen.71621

By combining tissue engineering techniques with human-induced pluripotent stem cell (hiPSC) technology, human-derived engineered cardiac tissues (ECTs) have been developed using several cell lineage compositions and 3-dimensional geometries. Although hiPSC ECTs are relatively immature compared with native adult heart tissues, they have promising potential as a platform technology for drug-screening and disease modeling, and as grafts for hiPSC-based regenerative heart therapy. This chapter provides the focused overview of the current status of cardiac tissue engineering technology and its

**Keywords:** iPS cell, engineered cardiac tissue, tissue engineering, drug screening,

With the progress of tissue engineering technology in the last decade, many kinds of engineered cardiac tissues (ECTs) have been developed and reported. These tissues possess striated myofibers which recapitulate unique contractile function of heart tissues. Takahashi, Yamanaka, and colleagues developed iPSCs from mice in 2006 and from human next year [1, 2]. Human iPS cells (hiPSCs) have the potential to differentiate into cardiomyocytes and other cardiac lineage cells. Recently, the efficiency of cardiac differentiation has rapidly improved, which makes it possible to robustly induce cardiac lineage cells [3–6]. By using cardiac cells derived from hiPSCs as a cell source for ECT generation, the potential of ECTs has expanded. The present chapter overviews the current status of ECT technology and its

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Engineered Cardiac Tissues**

**Engineered Cardiac Tissues**

http://dx.doi.org/10.5772/intechopen.71621

Bradley B. Keller

**Abstract**

**1. Introduction**

possible application.

possible application.

disease modeling, cardiac regeneration

Bradley B. Keller

Takeichiro Nakane, Hidetoshi Masumoto and

Takeichiro Nakane, Hidetoshi Masumoto and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

### **Human-Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues Engineered Cardiac Tissues**

**Human-Induced Pluripotent Stem Cell-Derived** 

DOI: 10.5772/intechopen.71621

Takeichiro Nakane, Hidetoshi Masumoto and Bradley B. Keller Bradley B. Keller Additional information is available at the end of the chapter

Takeichiro Nakane, Hidetoshi Masumoto and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71621

#### **Abstract**

By combining tissue engineering techniques with human-induced pluripotent stem cell (hiPSC) technology, human-derived engineered cardiac tissues (ECTs) have been developed using several cell lineage compositions and 3-dimensional geometries. Although hiPSC ECTs are relatively immature compared with native adult heart tissues, they have promising potential as a platform technology for drug-screening and disease modeling, and as grafts for hiPSC-based regenerative heart therapy. This chapter provides the focused overview of the current status of cardiac tissue engineering technology and its possible application.

**Keywords:** iPS cell, engineered cardiac tissue, tissue engineering, drug screening, disease modeling, cardiac regeneration

#### **1. Introduction**

With the progress of tissue engineering technology in the last decade, many kinds of engineered cardiac tissues (ECTs) have been developed and reported. These tissues possess striated myofibers which recapitulate unique contractile function of heart tissues. Takahashi, Yamanaka, and colleagues developed iPSCs from mice in 2006 and from human next year [1, 2]. Human iPS cells (hiPSCs) have the potential to differentiate into cardiomyocytes and other cardiac lineage cells. Recently, the efficiency of cardiac differentiation has rapidly improved, which makes it possible to robustly induce cardiac lineage cells [3–6]. By using cardiac cells derived from hiPSCs as a cell source for ECT generation, the potential of ECTs has expanded. The present chapter overviews the current status of ECT technology and its possible application.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

### **2. Methods for generating engineered cardiac tissues**

#### **2.1. Cell sheet technology**

Okano, Shimizu and colleagues have developed a culture surface grafted with temperatureresponsive polymer, poly(N-isopropyl acrylamide) (PIPAAm) [7]. This system enables confluent cells to detach themselves from the surface while maintaining a sheet structure simply by reducing the culture temperature. The cell sheets are fabricated without the use of exogenous matrices. It has endogenous matrix layer at the bottom side, and it secures biological attachment to the recipient heart surface within 30 min [8]. This scaffold-free tissue is a thin sheet, and three-dimensional thicker structure can be obtained by stacking several sheets. However, stacking more than three layers (approximately 80 μm thickness) triggers cell death at the center due to hypoxia [9]. At our lab, we overcame this limit by stacking cell sheets with gelatin hydrogel microspheres (GHM) [10]. Five-layered cell sheets with GHM displayed better cell viability *in vitro* compared with the control cell sheet without GHM. Furthermore, cell sheet modification with GHM improved the retention of grafted cell sheets on the rat heart epicardial surface after myocardial infarction. We succeeded in generating over 1 mm thick constructs from 15 sheets using this technology.

#### **2.2. Biomaterials**

Cardiomyocytes and other cells are embedded in biomaterials, such as collagen I, matrigel, and/or fibrin in a casting mold. Exogenous gel matrix promotes self-assembly of cells leading to form a tissue structure. The first successful cardiac tissue fabrication by this method was reported in the year 1997 [11]. Fixed anchors in these molds generate static strain, which enhances the cell alignment and contractile function. The geometry of mold controls the final tissue architecture, and a variety of tissue shapes have been formulated, including linear [12–16], circular [17], and mesh structures (**Figure 1**) [18–20]. By applying engineering manufacturing methods, it may be possible to automate the process of generating small-sized ECTs, leading to the development of high throughput *in vitro* analysis systems [21, 22]. In fact, recently, 3D bio-printing technology has been applied and provided the possibility of creating more complicated structures reproducibly by printing both scaffold matrix and living cells [23].

**2.4. Decellularized tissue**

**3. Maturation of the tissue**

both myocardial structure and function [13, 16, 19].

Scale bar: 10 mm. (Reproduced from Ref. [20] with permission).

**3.1. Cellular contents**

Deccllurization of a whole heart is achieved by the treatment with sodium dodecyl sulfate (SDS) and Triton X-100 under Langendorf perfusion [27]. The decellularized tissue maintains most of the tissue contents and function of the extracellular matrix. Hence, decellularized tissue provides a native scaffold which can support repopulation by mesoderm lineage cells. These decellularized tissues may be advantageous in the generation of *in vitro* cardiac tissues [28, 29].

**Figure 1.** Generation of a mesh ECT by biomaterial-based technique. (a) Schematic diagram for a mesh ECT generation. Cardiomyocytes (CM), endothelial cells (EC), and mural cells (MC) are induced from human iPS cells (h-iPSCs) using two different protocols. Cells and collagen-based matrix were combined and poured into custom polydimethylsiloxane (PDMS) molds at day 0 and cultured for 14 days. (b) Representative image of a PDMS mold (left) and a mesh ECT (right).

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A heart tissue is composed of multiple cell types including cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells. In human hearts, cardiomyocytes account for 25-50% of total cells, occupying around 70% of the whole volume [30]. Many reports have demonstrated that non-myocytes support the maturation of cardiomyocytes and play a significant role in

We induced cardiomyocytes (CM), endothelial cells (EC), and vascular mural cells (MC) from hiPSCs, and generated ECTs from several formulations of cardiomyocytes and non-myocytes,

#### **2.3. Prefabricated matrix**

Biomaterials provide a 'soft' environment for the cells to grow in. On the other hand, synthetic microporous ('spongy') scaffolds made from alginate, collagen, and gelatin or other stiffer materials, such as polystyrene, PLLA (Poly-L-Lactide Acid), PLGA (Poly(lactic-coglycolic acid)) and PCL (Polycaprolactone), have also been tested [24, 25]. The advantage of this method is that these rigid scaffolds contribute to engineering any desired structure and size with mechanical stability. However, the maturation of various mesodermal cells is matrix stiffness dependent, and therefore both the biomaterial gel and the supporting mold may impact cell maturation and survival [26].

Human-Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues http://dx.doi.org/10.5772/intechopen.71621 99

**Figure 1.** Generation of a mesh ECT by biomaterial-based technique. (a) Schematic diagram for a mesh ECT generation. Cardiomyocytes (CM), endothelial cells (EC), and mural cells (MC) are induced from human iPS cells (h-iPSCs) using two different protocols. Cells and collagen-based matrix were combined and poured into custom polydimethylsiloxane (PDMS) molds at day 0 and cultured for 14 days. (b) Representative image of a PDMS mold (left) and a mesh ECT (right). Scale bar: 10 mm. (Reproduced from Ref. [20] with permission).

#### **2.4. Decellularized tissue**

**2. Methods for generating engineered cardiac tissues**

constructs from 15 sheets using this technology.

Okano, Shimizu and colleagues have developed a culture surface grafted with temperatureresponsive polymer, poly(N-isopropyl acrylamide) (PIPAAm) [7]. This system enables confluent cells to detach themselves from the surface while maintaining a sheet structure simply by reducing the culture temperature. The cell sheets are fabricated without the use of exogenous matrices. It has endogenous matrix layer at the bottom side, and it secures biological attachment to the recipient heart surface within 30 min [8]. This scaffold-free tissue is a thin sheet, and three-dimensional thicker structure can be obtained by stacking several sheets. However, stacking more than three layers (approximately 80 μm thickness) triggers cell death at the center due to hypoxia [9]. At our lab, we overcame this limit by stacking cell sheets with gelatin hydrogel microspheres (GHM) [10]. Five-layered cell sheets with GHM displayed better cell viability *in vitro* compared with the control cell sheet without GHM. Furthermore, cell sheet modification with GHM improved the retention of grafted cell sheets on the rat heart epicardial surface after myocardial infarction. We succeeded in generating over 1 mm thick

Cardiomyocytes and other cells are embedded in biomaterials, such as collagen I, matrigel, and/or fibrin in a casting mold. Exogenous gel matrix promotes self-assembly of cells leading to form a tissue structure. The first successful cardiac tissue fabrication by this method was reported in the year 1997 [11]. Fixed anchors in these molds generate static strain, which enhances the cell alignment and contractile function. The geometry of mold controls the final tissue architecture, and a variety of tissue shapes have been formulated, including linear [12–16], circular [17], and mesh structures (**Figure 1**) [18–20]. By applying engineering manufacturing methods, it may be possible to automate the process of generating small-sized ECTs, leading to the development of high throughput *in vitro* analysis systems [21, 22]. In fact, recently, 3D bio-printing technology has been applied and provided the possibility of creating more com-

plicated structures reproducibly by printing both scaffold matrix and living cells [23].

Biomaterials provide a 'soft' environment for the cells to grow in. On the other hand, synthetic microporous ('spongy') scaffolds made from alginate, collagen, and gelatin or other stiffer materials, such as polystyrene, PLLA (Poly-L-Lactide Acid), PLGA (Poly(lactic-coglycolic acid)) and PCL (Polycaprolactone), have also been tested [24, 25]. The advantage of this method is that these rigid scaffolds contribute to engineering any desired structure and size with mechanical stability. However, the maturation of various mesodermal cells is matrix stiffness dependent, and therefore both the biomaterial gel and the supporting mold may

**2.1. Cell sheet technology**

98 Stem Cells in Clinical Practice and Tissue Engineering

**2.2. Biomaterials**

**2.3. Prefabricated matrix**

impact cell maturation and survival [26].

Deccllurization of a whole heart is achieved by the treatment with sodium dodecyl sulfate (SDS) and Triton X-100 under Langendorf perfusion [27]. The decellularized tissue maintains most of the tissue contents and function of the extracellular matrix. Hence, decellularized tissue provides a native scaffold which can support repopulation by mesoderm lineage cells. These decellularized tissues may be advantageous in the generation of *in vitro* cardiac tissues [28, 29].

#### **3. Maturation of the tissue**

#### **3.1. Cellular contents**

A heart tissue is composed of multiple cell types including cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells. In human hearts, cardiomyocytes account for 25-50% of total cells, occupying around 70% of the whole volume [30]. Many reports have demonstrated that non-myocytes support the maturation of cardiomyocytes and play a significant role in both myocardial structure and function [13, 16, 19].

We induced cardiomyocytes (CM), endothelial cells (EC), and vascular mural cells (MC) from hiPSCs, and generated ECTs from several formulations of cardiomyocytes and non-myocytes, such as CM+EC, CM+MC, and CM+EC+MC [16]. According to in vitro force measurement, CM+EC+MC ECTs showed most advanced electrophysiological properties. Furthermore, histological analysis revealed CM+EC+MC possessed more unidirectionally aligned myofiber with mature sarcomeric structures (**Figure 2**).

The mechanism for the improvement of tissue maturation as the result of a more complex lineage mixture is still unknown. Evidence is emerging that cardiomyocytes and fibroblasts are electrically coupled, which may modulate electrophysiologic function [31]. Direct interaction of different cell types or paracrine effects can be considered. It is reported that extracellular matrix derived from cardiac fibroblast supported the proliferation *in vitro* and indicated the usefulness of the coculture [32].

#### **3.2. Effects of extended culture duration on CM and ECT maturation and function**

Cardiomyocytes derived from pluripotent stem cells mature early in culture but are arrested at the late embryonic stage under 2-dimensional (2D) culture condition [33]. Meanwhile, it is widely recognized that cardiomyocytes in a 3-dimensional (3D) cardiac tissue acquire a more mature phenotype than those in 2D culture [25, 34].

In our study, we prolonged culture duration of mesh ECTs from 14 to 28days [20]. Longcultured constructs showed the tendency to augment the active contractile force along with the increase of beating frequency from 1.5Hz to 2.5Hz and maintained greater force compared to 14-day constructs. The shift from a negative to a neutral force-frequency relationship in 28-day constructs represents sustained functional maturation as well as more rapid force generation and relaxation cycle, and the ability to capture higher pacing frequency. Additional culture duration enhanced myofiber alignment along with the expression of several genes related to ion channels and gap junction (**Figure 3**).

#### **3.3. Impact of culture condition on ECT maturation and function**

In order to expand the dimension of ECTs, it is necessary to improve the distribution of oxygen and nutrient throughout tissues. Direct perfusion of culture medium reduces the gradients associated with diffusional mass transport between the construct surfaces and the inner phase and improves the microenvironmental conditions within ECTs. [35–37]. In addition to the increase of cellularity at the center of ECTs, perfusion system contributes to the maturation of cardiomyocytes and the tissue structure. It is reported that even the simple rocking dynamic culture yields engineered myocardium with near-adult functional output [38]. The system accelerates force generation and conduction velocity probably due to the activation of mTOR signaling.

**Figure 2.** Electromechanical properties and structural maturation of hiPSC-ECT**s**. (a) Schematic diagrams for generationg 3 types of ECTs containing cardiomyocytes (CM), endothelial cells (EC), and/or mural cells (MC) (upper) and the proportions of each cell type (lower) [n = 8 (CM+ EC), 7 (CM+ MC), and 12 (CM+ EC+ MC). (b–d) Results of contractile force measurements [n = 8 (CM+ EC), 7 (CM+ MC), and 12 (CM+ EC+ MC)]. (b) Maximum capture rate (left), relaxation time (center) and excitation threshold voltage (right) (c) Relationship between active force and pacing frequency (2Hz to 3.5Hz). (d) Young's modulus of ECTs. NS, not significant; \*P < 0.05,\*\*P < 0.01, \*\*\*P < 0.001. (e) Representative alignment analysis using cTnT-stained images after 2-dimensional (2D) and 3-dimensional (3D) culture for 3 types of ECTs (left). Calculated concentration parameter (κ ) (right) [n =3 (2D) and 5 (each 3D)]. Larger values of κ represent greater alignment to a single direction. cTnT, cardiac troponin T; Deg, degree. (f) Representative transmission electron microscopic images for each type of ECT cultured for 4 weeks. Arrows indicate myofibers. N, nucleus; Mt, mitochondria;

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I, I-band; A, A-band; Z, Z-line. (Reproduced from Ref. [16] with permission).

For generating ECTs, especially from biomaterial-based technique, materials from other sources, such as collagen derived from rat or matrigel, have been widely used with xenoserum containing culture medium. Tiburcy and colleagues modified their methods toward clinical application and developed a formulation for generating ECTs by using medical grade bovine collagen and maintaining them under serum-free defined condition [19]. This modification provided a proof-of-concept for a universally applicable technology for the engineering

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such as CM+EC, CM+MC, and CM+EC+MC [16]. According to in vitro force measurement, CM+EC+MC ECTs showed most advanced electrophysiological properties. Furthermore, histological analysis revealed CM+EC+MC possessed more unidirectionally aligned myofiber

The mechanism for the improvement of tissue maturation as the result of a more complex lineage mixture is still unknown. Evidence is emerging that cardiomyocytes and fibroblasts are electrically coupled, which may modulate electrophysiologic function [31]. Direct interaction of different cell types or paracrine effects can be considered. It is reported that extracellular matrix derived from cardiac fibroblast supported the proliferation *in vitro* and indicated the

Cardiomyocytes derived from pluripotent stem cells mature early in culture but are arrested at the late embryonic stage under 2-dimensional (2D) culture condition [33]. Meanwhile, it is widely recognized that cardiomyocytes in a 3-dimensional (3D) cardiac tissue acquire a more

In our study, we prolonged culture duration of mesh ECTs from 14 to 28days [20]. Longcultured constructs showed the tendency to augment the active contractile force along with the increase of beating frequency from 1.5Hz to 2.5Hz and maintained greater force compared to 14-day constructs. The shift from a negative to a neutral force-frequency relationship in 28-day constructs represents sustained functional maturation as well as more rapid force generation and relaxation cycle, and the ability to capture higher pacing frequency. Additional culture duration enhanced myofiber alignment along with the expression of several genes

In order to expand the dimension of ECTs, it is necessary to improve the distribution of oxygen and nutrient throughout tissues. Direct perfusion of culture medium reduces the gradients associated with diffusional mass transport between the construct surfaces and the inner phase and improves the microenvironmental conditions within ECTs. [35–37]. In addition to the increase of cellularity at the center of ECTs, perfusion system contributes to the maturation of cardiomyocytes and the tissue structure. It is reported that even the simple rocking dynamic culture yields engineered myocardium with near-adult functional output [38]. The system accelerates force generation and conduction velocity probably due to the activation of

For generating ECTs, especially from biomaterial-based technique, materials from other sources, such as collagen derived from rat or matrigel, have been widely used with xenoserum containing culture medium. Tiburcy and colleagues modified their methods toward clinical application and developed a formulation for generating ECTs by using medical grade bovine collagen and maintaining them under serum-free defined condition [19]. This modification provided a proof-of-concept for a universally applicable technology for the engineering

**3.2. Effects of extended culture duration on CM and ECT maturation and function**

with mature sarcomeric structures (**Figure 2**).

100 Stem Cells in Clinical Practice and Tissue Engineering

mature phenotype than those in 2D culture [25, 34].

related to ion channels and gap junction (**Figure 3**).

**3.3. Impact of culture condition on ECT maturation and function**

usefulness of the coculture [32].

mTOR signaling.

**Figure 2.** Electromechanical properties and structural maturation of hiPSC-ECT**s**. (a) Schematic diagrams for generationg 3 types of ECTs containing cardiomyocytes (CM), endothelial cells (EC), and/or mural cells (MC) (upper) and the proportions of each cell type (lower) [n = 8 (CM+ EC), 7 (CM+ MC), and 12 (CM+ EC+ MC). (b–d) Results of contractile force measurements [n = 8 (CM+ EC), 7 (CM+ MC), and 12 (CM+ EC+ MC)]. (b) Maximum capture rate (left), relaxation time (center) and excitation threshold voltage (right) (c) Relationship between active force and pacing frequency (2Hz to 3.5Hz). (d) Young's modulus of ECTs. NS, not significant; \*P < 0.05,\*\*P < 0.01, \*\*\*P < 0.001. (e) Representative alignment analysis using cTnT-stained images after 2-dimensional (2D) and 3-dimensional (3D) culture for 3 types of ECTs (left). Calculated concentration parameter (κ ) (right) [n =3 (2D) and 5 (each 3D)]. Larger values of κ represent greater alignment to a single direction. cTnT, cardiac troponin T; Deg, degree. (f) Representative transmission electron microscopic images for each type of ECT cultured for 4 weeks. Arrows indicate myofibers. N, nucleus; Mt, mitochondria; I, I-band; A, A-band; Z, Z-line. (Reproduced from Ref. [16] with permission).

on electrical stimulation at up to 6 Hz [41]. Biowires subjected to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca2+ handling properties compared to non-stimulated controls. These changes were in agreement with cardiomyocyte matu-

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Another group created a system which applied combined electromechanical stimulation mimicking isovolumic contraction and confirmed the improvement of functional properties over electrical or mechanical stimulation alone. In the report, the timing of the combined

Cardiotoxicity is one of the main causes of drug withdrawal from the market [43, 44]. To evaluate the safety and effectiveness of drugs, several kinds of pre-clinical studies are performed using non-human models. However, these models occasionally show incorrect results, such as pseudo-negative, due to the difference of electrophysiology in other species [45]. By using hiPSC technology, effects to humans can be examined from the early stage [46]. Moreover, hiPSC-derived ECTs are expected to be more specific and sensitive platforms

We have developed a 3D cardiac tissue model which reproduces Torsade de Pointes (TdP) showing both a typical polymorphic extracellular field potential and meandering spiral wave re-entry upon treatment with IKr channel blockers [47]. It is generated from hiPSC-derived cardiomyocytes and mesenchymal cells using the cell sheet technology. The appearance of TdP-like waveforms was significantly higher in this 3D model compared with 2D monolayer conditions indicating that the multilayered 3D structure is an essential factor for this arrhyth-

Many groups have developed various high throughput screening formats, such as strip format [48] and heart-on-chip [21, 49], and confirmed the similarity of hiPSC-derived engineered tissues with native heart tissues [14, 50–52]. Video optical recording system set

Cardiomyocytes from hiPSCs are at the premature stage, and even ECTs generated to date remain immature compared to native adult myocardium. Hence, further cues for maturation

iPS cells derived from a patient with a known genetic mutation for the disease can provide the disease model, which may offer a useful strategy for understanding the mechanism of the

as described above is necessary to imitate native tissues more precisely [51].

incubator is also useful to continuously monitor contractile abilities of

stimulation greatly affected the electrophysiological properties of ECTs [42].

ration and were dependent on the stimulation frequency.

mia along with the coexistence of non-cardiomyocytes.

disease and exploring a new treatment modality.

**4. Applications of ECTs**

**4.1. Drug screening**

for drug screening [14].

up in a usual CO2

**4.2. Disease modeling**

tissues [48].

**Figure 3.** Effects of extended culture duration on mesh ECT maturation. (a) Averaged normalized active force-time curves for mesh ECT cultured for either 14 (blue, D14) or 28 (red, D28) (n = 6) days under 2 Hz electrical stimulation. Contraction time (CT) or relaxation time (RT) represents the time of 90% increase or decrease of force respectively. Comparison of (b) contraction time 90% (n = 6; \*\*\*P < 0.01), (c) relaxation time 90% (\*\*\*P < 0.001) at 2 Hz pacing, (d) maximum capture rate (n = 6; \*\*\*P < 0.001), (e) force-frequency relationship (n = 6; \*P < 0.05 to \*\*\*P < 0.001 vs. D14), and (f) cardiomyocyte alignment concentration (κ), [n = 4 (D14) and n = 3 (D28); \*\*\*P < 0.001]. (g), (h) Q-PCR analysis (n = 3; \*P < 0.05 to \*\*\*P < 0.001). cTnT level was lower in D28, and other genes were normalized to the value of cTnT expression. (Reproduced from Ref. [20] with permission).

cardiac tissue. In addition, ECTs generated by this condition displayed advanced cardiomyocyte maturation compared to the conventional methods [19].

#### **3.4. Role of mechanical loading on ECT maturation and function.**

Several *in vitro* ECT studies revealed the impact of uniaxial mechanical loading on the alignment of cardiomyocytes and functional maturation. The simplest system for that is the two fixed anchors which generate static strain on the tissue between them. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy and proliferation rates versus unconditioned constructs [13]. We demonstrated that ECTs displayed a broad spectrum of altered gene expression in response to cyclic stretch, reflecting a complex regulation of proliferation, differentiation, and architectural alignment of cardiomyocytes and non-cardiomyocytes within ECTs [39].

Electrical stimulation is considered to be another important cue for further maturation of tissues [40]. Nunes and colleagues subjected their engineered tissues, named biowires, on electrical stimulation at up to 6 Hz [41]. Biowires subjected to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca2+ handling properties compared to non-stimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation frequency.

Another group created a system which applied combined electromechanical stimulation mimicking isovolumic contraction and confirmed the improvement of functional properties over electrical or mechanical stimulation alone. In the report, the timing of the combined stimulation greatly affected the electrophysiological properties of ECTs [42].

## **4. Applications of ECTs**

### **4.1. Drug screening**

Cardiotoxicity is one of the main causes of drug withdrawal from the market [43, 44]. To evaluate the safety and effectiveness of drugs, several kinds of pre-clinical studies are performed using non-human models. However, these models occasionally show incorrect results, such as pseudo-negative, due to the difference of electrophysiology in other species [45]. By using hiPSC technology, effects to humans can be examined from the early stage [46]. Moreover, hiPSC-derived ECTs are expected to be more specific and sensitive platforms for drug screening [14].

We have developed a 3D cardiac tissue model which reproduces Torsade de Pointes (TdP) showing both a typical polymorphic extracellular field potential and meandering spiral wave re-entry upon treatment with IKr channel blockers [47]. It is generated from hiPSC-derived cardiomyocytes and mesenchymal cells using the cell sheet technology. The appearance of TdP-like waveforms was significantly higher in this 3D model compared with 2D monolayer conditions indicating that the multilayered 3D structure is an essential factor for this arrhythmia along with the coexistence of non-cardiomyocytes.

Many groups have developed various high throughput screening formats, such as strip format [48] and heart-on-chip [21, 49], and confirmed the similarity of hiPSC-derived engineered tissues with native heart tissues [14, 50–52]. Video optical recording system set up in a usual CO2 incubator is also useful to continuously monitor contractile abilities of tissues [48].

Cardiomyocytes from hiPSCs are at the premature stage, and even ECTs generated to date remain immature compared to native adult myocardium. Hence, further cues for maturation as described above is necessary to imitate native tissues more precisely [51].

#### **4.2. Disease modeling**

cardiac tissue. In addition, ECTs generated by this condition displayed advanced cardiomyo-

**Figure 3.** Effects of extended culture duration on mesh ECT maturation. (a) Averaged normalized active force-time curves for mesh ECT cultured for either 14 (blue, D14) or 28 (red, D28) (n = 6) days under 2 Hz electrical stimulation. Contraction time (CT) or relaxation time (RT) represents the time of 90% increase or decrease of force respectively. Comparison of (b) contraction time 90% (n = 6; \*\*\*P < 0.01), (c) relaxation time 90% (\*\*\*P < 0.001) at 2 Hz pacing, (d) maximum capture rate (n = 6; \*\*\*P < 0.001), (e) force-frequency relationship (n = 6; \*P < 0.05 to \*\*\*P < 0.001 vs. D14), and (f) cardiomyocyte alignment concentration (κ), [n = 4 (D14) and n = 3 (D28); \*\*\*P < 0.001]. (g), (h) Q-PCR analysis (n = 3; \*P < 0.05 to \*\*\*P < 0.001). cTnT level was lower in D28, and other genes were normalized to the value of cTnT expression.

Several *in vitro* ECT studies revealed the impact of uniaxial mechanical loading on the alignment of cardiomyocytes and functional maturation. The simplest system for that is the two fixed anchors which generate static strain on the tissue between them. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy and proliferation rates versus unconditioned constructs [13]. We demonstrated that ECTs displayed a broad spectrum of altered gene expression in response to cyclic stretch, reflecting a complex regulation of proliferation, differentiation, and architectural alignment of cardiomyocytes and non-cardiomyocytes

Electrical stimulation is considered to be another important cue for further maturation of tissues [40]. Nunes and colleagues subjected their engineered tissues, named biowires,

cyte maturation compared to the conventional methods [19].

(Reproduced from Ref. [20] with permission).

102 Stem Cells in Clinical Practice and Tissue Engineering

within ECTs [39].

**3.4. Role of mechanical loading on ECT maturation and function.**

iPS cells derived from a patient with a known genetic mutation for the disease can provide the disease model, which may offer a useful strategy for understanding the mechanism of the disease and exploring a new treatment modality.

Cardio-facio-cutaneous syndrome (CFCS) is one of the RASopathies, and cardiac abnormalities are the most common findings among CFCS, including hypertrophic cardiomyopathy (HCM) in approximately 40% of patients. Nearly 75% of patients with CFCS exhibit mutations in *BRAF*, which encodes a serine/threonine kinase and a direct effector of Ras. Cashman and colleagues created 3D human engineered cardiac tissue model of HCM using human cardiomyocytes yielded by directed differentiation of iPSCs established from a patient with CFCS carrying an activated BRAF mutation [53].

phenotype [19]. Tissues responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release, which are classical hallmarks of heart failure. Notably, the pathological phenotype could be partially or fully prevented by β1- or α1-adrenoreceptor blockade, demonstrating the applicability of ECTs in the *in vitro* simulation of heart failure and its prevention by pharmacological

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HiPSCs are now one of the most promising cell sources for cardiac regenerative cell therapy [54–56]. There are major methods of cell delivery, including intracoronary or intramuscular injection of dispersed cells and epicardial transplantation of engineered tissues [57]. It is possible to deliver a large number of differentiated cells with organized architecture by ECT implantation. The grafted tissues survive and support the heart wall, which overcomes the

A variety of studies has revealed the efficacy of ECT implantation for myocardial structural and functional recovery in injured hearts of several animal models [6, 10, 58, 59]. We implanted ECTs in an athymic nude rat myocardial infarction model. ECTs with vascular cells displayed the invasion of vasculature from the host heart to the tissue and their perfusion. Survived ECTs replaced the ventricular wall in the injured area and prevented the scar formation after myocardial infarction and improved cardiac function (**Figure 4**). ECTs survived during 4-week follow-up period [16, 20]. However, further work is required to identify the underlying mechanism for the functional recovery. Meanwhile, the first transplantation of cardiac progenitor patch derived from human embryonic stem cells in a severe heart failure patient was performed in France, offering

In this chapter, we have reviewed several aspects of current cardiac tissue engineering technologies and presented the possible applications of these tissues for *in vitro* drug toxicity testing, human disease modeling, and paradigms for myocardial recovery with muscle replacement following injury. This rapidly evolving new field is now incorporating manufacturing process to expand the scalability and reduce the cost of generating these novel engi-

means.

**4.3. Transplantation therapy**

an encouraging result [60].

neered *in vitro* myocardial tissues.

**Conflict of interest**

None.

**5. Conclusion**

problem of poor retention rate following cell injection [34].

Chronic catecholamine overstimulation contributes to heart failure progression. Overstimulation of ECTs with norepinephrine provides a simulation of a human heart failure

**Figure 4.** Therapeutic effects of hiPSC-mesh ECT implantation in a rat myocardial infarction model. (a) Schematic timeline of surgery. A mesh ECT matured *in vitro* for 14 days (or sham suture) is implanted in a nude rat (week 0) 1 week after the induction of myocardial infarction by ligating left anterior descending artery (LAD). Echocardiogram (Echo) is performed prior to LAD ligation at week-1 (W-1), prior to implantation at week 0 (W0), then week 2 (W2) and week 4 (W4). (b) Grafted mesh ECT on the heart surface covering infarction site. (c) Representative Masson's trichrome staining images of sham treated (left) and mesh ECT implanted (right) rat hearts at W4. Scale bar: 2 mm. Red dotted line indicates engrafted area. (d) Comparison of scar area (% of LV area) at W4 (n=5, \*P<0.05 Implant versus Sham). (**e-g**) Results of echocardiogram [n=5 (Implant, red solid line) and 5 (Sham, blue dotted line)]. **(**e**)** Left ventricular end diastolic area (LVAd; mm<sup>2</sup> ), **(**f**)** ejection fraction, EF (%), and **(**g**)** cardiac index, CI (mL/min/kg) (\*P<0.05 Implant versus Sham at W4). (Reproduced from Ref. [20] with permission).

phenotype [19]. Tissues responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release, which are classical hallmarks of heart failure. Notably, the pathological phenotype could be partially or fully prevented by β1- or α1-adrenoreceptor blockade, demonstrating the applicability of ECTs in the *in vitro* simulation of heart failure and its prevention by pharmacological means.

#### **4.3. Transplantation therapy**

Cardio-facio-cutaneous syndrome (CFCS) is one of the RASopathies, and cardiac abnormalities are the most common findings among CFCS, including hypertrophic cardiomyopathy (HCM) in approximately 40% of patients. Nearly 75% of patients with CFCS exhibit mutations in *BRAF*, which encodes a serine/threonine kinase and a direct effector of Ras. Cashman and colleagues created 3D human engineered cardiac tissue model of HCM using human cardiomyocytes yielded by directed differentiation of iPSCs established from a patient with

Chronic catecholamine overstimulation contributes to heart failure progression. Overstimulation of ECTs with norepinephrine provides a simulation of a human heart failure

**Figure 4.** Therapeutic effects of hiPSC-mesh ECT implantation in a rat myocardial infarction model. (a) Schematic timeline of surgery. A mesh ECT matured *in vitro* for 14 days (or sham suture) is implanted in a nude rat (week 0) 1 week after the induction of myocardial infarction by ligating left anterior descending artery (LAD). Echocardiogram (Echo) is performed prior to LAD ligation at week-1 (W-1), prior to implantation at week 0 (W0), then week 2 (W2) and week 4 (W4). (b) Grafted mesh ECT on the heart surface covering infarction site. (c) Representative Masson's trichrome staining images of sham treated (left) and mesh ECT implanted (right) rat hearts at W4. Scale bar: 2 mm. Red dotted line indicates engrafted area. (d) Comparison of scar area (% of LV area) at W4 (n=5, \*P<0.05 Implant versus Sham). (**e-g**) Results of echocardiogram [n=5 (Implant, red solid line) and 5 (Sham, blue dotted line)]. **(**e**)** Left ventricular end diastolic area

), **(**f**)** ejection fraction, EF (%), and **(**g**)** cardiac index, CI (mL/min/kg) (\*P<0.05 Implant versus Sham at W4).

CFCS carrying an activated BRAF mutation [53].

104 Stem Cells in Clinical Practice and Tissue Engineering

(LVAd; mm<sup>2</sup>

(Reproduced from Ref. [20] with permission).

HiPSCs are now one of the most promising cell sources for cardiac regenerative cell therapy [54–56]. There are major methods of cell delivery, including intracoronary or intramuscular injection of dispersed cells and epicardial transplantation of engineered tissues [57]. It is possible to deliver a large number of differentiated cells with organized architecture by ECT implantation. The grafted tissues survive and support the heart wall, which overcomes the problem of poor retention rate following cell injection [34].

A variety of studies has revealed the efficacy of ECT implantation for myocardial structural and functional recovery in injured hearts of several animal models [6, 10, 58, 59]. We implanted ECTs in an athymic nude rat myocardial infarction model. ECTs with vascular cells displayed the invasion of vasculature from the host heart to the tissue and their perfusion. Survived ECTs replaced the ventricular wall in the injured area and prevented the scar formation after myocardial infarction and improved cardiac function (**Figure 4**). ECTs survived during 4-week follow-up period [16, 20]. However, further work is required to identify the underlying mechanism for the functional recovery. Meanwhile, the first transplantation of cardiac progenitor patch derived from human embryonic stem cells in a severe heart failure patient was performed in France, offering an encouraging result [60].

### **5. Conclusion**

In this chapter, we have reviewed several aspects of current cardiac tissue engineering technologies and presented the possible applications of these tissues for *in vitro* drug toxicity testing, human disease modeling, and paradigms for myocardial recovery with muscle replacement following injury. This rapidly evolving new field is now incorporating manufacturing process to expand the scalability and reduce the cost of generating these novel engineered *in vitro* myocardial tissues.

### **Conflict of interest**

None.

### **Author details**

Takeichiro Nakane1,2,3, Hidetoshi Masumoto1,2,3\* and Bradley B. Keller<sup>3</sup>

\*Address all correspondence to: masumoto@kuhp.kyoto-u.ac.jp

1 Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan

[10] Matsuo T, Masumoto H, Tajima S, et al. Efficient long-term survival of cell grafts after myocardial infarction with thick viable cardiac tissue entirely from pluripotent stem

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2 Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

3 Kosair Charities Pediatric Heart Research Program, Cardiovascular Innovation Institute, University of Louisville, Louisville, Kentucky, The United States of America

### **References**


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**Author details**

106 Stem Cells in Clinical Practice and Tissue Engineering

Kyoto, Japan

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3 Kosair Charities Pediatric Heart Research Program, Cardiovascular Innovation Institute,

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**Chapter 6**

**Provisional chapter**

**Stem Cells in Treatment of Coronary Heart Disease and**

Cardiovascular and coronary heart diseases involve molecular and tissue level damage of blood vessels and heart. Coronary Heart Disease and heart failure are the leading cause of mortality worldwide. Stem cell transplantation is emerging as a new treatment option. Stem cells are capable to reach and settle down at damaged cardiac tissue. This stem cell option also repairs the myocardial infarction area in heart or vascular territories and ultimately reduces the infarct-related mortality. Non-invasive cardiovascular imaging monitors the real-time status of cardiovascular remodeling or differentiated stem cell autografting. Cardiac magnetic resonance imaging (MRI) and bioluminescence are robust non-invasive monitoring techniques to visualize cardiovascular structure changes due to myocardial dysfunction or restorative myocardial recovery. The present chapter highlights the sources, types, delivery methods of stem cells in cardiovascular treatment, advantages and current limitations of stem cell monitoring, scopes of ultra-high field cardiac 900 MHz MRI and bioluminescence methods applied in stem cell transplantation, to translate stem cell molecular events into clinical success and evaluation of rejuvenation rate with future perspectives. *In conclusion*, right choice of stem cells, pluripotent stem cell delivery, transplantation and real-time monitoring of stem cell trafficking enhances

**Stem Cells in Treatment of Coronary Heart Disease** 

**and Its Monitoring: Tissue Engineering and Clinical** 

DOI: 10.5772/intechopen.70229

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Cardiovascular diseases (CVD) and coronary heart disease (CHD) are worldwide leading causes of present mortality as high as 32.8% [1]. Last 5-year American Heart Association

the stem cell therapeutic efficacy in cardiac engraftment and differentiation.

**Keywords:** stem cell delivery, transplantation, magnetic resonance imaging, coronary disease,

**Its Monitoring: Tissue Engineering and Clinical**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70229

cardiac tissue engineering

**1. Introduction**

**Evaluation**

**Evaluation**

Rakesh Sharma

**Abstract**

Rakesh Sharma


**Provisional chapter**

### **Stem Cells in Treatment of Coronary Heart Disease and Its Monitoring: Tissue Engineering and Clinical Evaluation and Its Monitoring: Tissue Engineering and Clinical Evaluation**

**Stem Cells in Treatment of Coronary Heart Disease** 

DOI: 10.5772/intechopen.70229

Rakesh Sharma Additional information is available at the end of the chapter

Rakesh Sharma

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[53] Cashman TJ, Josowitz R, Johnson BV, et al. Human engineered cardiac tissues created using induced pluripotent stem cells reveal functional characteristics of BRAF-mediated

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[57] Lalit PA, Hei DJ, Raval AN, et al. Induced pluripotent stem cells for post-myocardial infarction repair: Remarkable opportunities and challenges. Circulation Research. 2014;

[58] Kawamura M, Miyagawa S, Miki K, et al. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine isch-

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[60] Menasché P, Vanneaux V, Hagège A, et al. Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: First clinical case report: Figure 1.

Analysis of contractile force. Stem Cell Reports. 2016;**7**:29-42

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110 Stem Cells in Clinical Practice and Tissue Engineering

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**114**:1328-1345

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70229

#### **Abstract**

Cardiovascular and coronary heart diseases involve molecular and tissue level damage of blood vessels and heart. Coronary Heart Disease and heart failure are the leading cause of mortality worldwide. Stem cell transplantation is emerging as a new treatment option. Stem cells are capable to reach and settle down at damaged cardiac tissue. This stem cell option also repairs the myocardial infarction area in heart or vascular territories and ultimately reduces the infarct-related mortality. Non-invasive cardiovascular imaging monitors the real-time status of cardiovascular remodeling or differentiated stem cell autografting. Cardiac magnetic resonance imaging (MRI) and bioluminescence are robust non-invasive monitoring techniques to visualize cardiovascular structure changes due to myocardial dysfunction or restorative myocardial recovery. The present chapter highlights the sources, types, delivery methods of stem cells in cardiovascular treatment, advantages and current limitations of stem cell monitoring, scopes of ultra-high field cardiac 900 MHz MRI and bioluminescence methods applied in stem cell transplantation, to translate stem cell molecular events into clinical success and evaluation of rejuvenation rate with future perspectives. *In conclusion*, right choice of stem cells, pluripotent stem cell delivery, transplantation and real-time monitoring of stem cell trafficking enhances the stem cell therapeutic efficacy in cardiac engraftment and differentiation.

**Keywords:** stem cell delivery, transplantation, magnetic resonance imaging, coronary disease, cardiac tissue engineering

#### **1. Introduction**

Cardiovascular diseases (CVD) and coronary heart disease (CHD) are worldwide leading causes of present mortality as high as 32.8% [1]. Last 5-year American Heart Association

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

(AHA) data show high prevalence of heart failure as high as 50% and mortality as high as 32.8%. Coronary artery bypass grafting (CABG) was routine interventional and surgical treatment to bring low morality of coronary heart disease and now stem cell therapy is a new option. Still, major life threat is myocardial necrosis of myocardial tissue that cannot restore the original function of myocardium. Currently, stem cell research has opened vista in transplantation therapy, and its feasibility and effectiveness are well proven in animal experiments as well as in small-scale clinical trials [2–15].

**1.2. What should be the goal of stem cell therapy?**

repair and recovery.

**1.3. Potential mechanisms**

Stem cells are used as autograft (self-renewing, undifferentiated clonigenic) transplantation in myocardial repair or regeneration to bring tissue functionality back to normal for long-term survival in patients with permanent myocardial damage. Some stem cells are used as allograft (multipotent) daughter cells that give rise to multiple progenies [18]. The result of this asymmetric replication of stem cells is that after each division of stem cells, some progeny enters into the differentiation phase. Bone marrow cells and embryonic stem cells have differentiation plasticity and capacity. Goal is achieved by stimulating blood stem cells to cardiomyocytes providing a continuous supply of cardiac stem cells by trans-differentiation. Other exciting option is 'therapeutic cloning' means transplanted stem cells reprogram into induced pluripotent stem (iPS) cells at target organ. Still many issues are: (1) How many number of optimized stem cells needed when performing cell transplantation therapy?; (2) How survival time of transplanted cells can be best monitored?; (3) How do transplanted stem cells undergo differentiation into cardiomyocytes, smooth muscle cells, or endothelial cells?; (4) Do transplanted stem cells produce electrochemical coupling closer to normal myocardial tissue and normal cardiac cells and their functions really recover?; (5) What is the mechanism of cell transplantation in the treatment of myocardial perfusion and cardiac function after a short enhancement (myocardial cell regeneration or paracrine or other)? In nutshell, regeneration and healing of damaged cardiac tissue by myocardial repair is critical in survival. For interested readers, myocardial repair refers to the restoration of tissue architecture is shown in **Figure 1** and its remodeling of metabolic functions after injury is described in **Figure 7** in detail. Regeneration is defined as 100% myocardial

Stem Cells in Treatment of Coronary Heart Disease and Its Monitoring: Tissue Engineering...

http://dx.doi.org/10.5772/intechopen.70229

113

Currently, cardiac stem cell therapy researchers are exploring new differentiation and surface protein expression markers. Main focus is to design stem cell therapy supports using different 3D extracellular matrices, polymeric scaffolds. Most intriguing advances are in innovative new methods of safe stem cell delivery with subsequent repair monitoring and follow up of stem cell therapy [19]. If incomplete repair, it leaves myocardial scar or fibrosis of collagen or necrosis after inflammation. After repair, myocardial recovery involves the proliferation of stem cells and interaction with native tissue cells to fill up myocardial mass and extra cellular matrix (remodeling) with its improved cellular paracrine function. The improved function is monitored by non-invasive MRI and bioluminescent techniques. Moreover, total success depends on choice of supporting engineered stem cell delivery method and monitoring the extent of repair or regenerating cardiac territories with its visualization by physiochemical

Basic mechanism is 'myocardial revascularization and regeneration' to combat little or no blood supply left to slowly dying heart after myocardial ischemia or infarct or hypoxia. If sufficient oxygen diffusion from endocardium and collateral vessels provide sufficient oxygen

methods during follow up of post-stem cell therapy benefits.

The present chapter is divided into six sections. Section 1 introduces the evolution of stem cell therapy and its mechanism in regeneration and heart restoration. Section 2 introduces different stem cells and their purpose in repair and remodeling myocardium. Section 3 defines transplantation. Section 4 describes different modes of stem cell delivery. Section 5 highlights the purpose of rapid noninvasive real-time monitoring the myocardial repair and evaluation of heart territories. Section 6 reviews different clinical trials, available current nanotechnology and tissue engineering tools, and new approaches with future perspectives. A sketch of metabolic regulation during rejuvenation is presented for exploring new thoughts on secretory molecules regulating remodeling stem cells to explain regeneration of heart with possibility of better regeneration outcome. The chapter is written for interested physicians, surgeons, tissue engineers, scientists, and entrepreneurs.

#### **1.1. Evolution of stem cell therapy: regeneration and healing**

History records two types of bone marrow cells (BMC): hematopoietic stem cells [red blood cells (RBC), white blood cells (WBC), lymphocytes, macrophages] and bone marrow stroma mesenchymal stem cells (osteogenic cells for bone formation, chondrogenic cells for cartilage formation, adipogenic cells for fat tissue, and myoblast cells for heart regeneration). Stroma mesenchymal cells are sources of stem cells. Now, stem cell treatment is emerging in heart regeneration and restoration by using intracoronary, intramyocardial, and epicardial injections.

Stem cell transplantation therapy was reported useful first time in recovering myocardial viability after myocardial infarction in ischemic heart disease [16]. Later, autologous intracoronary delivery of mononuclear bone marrow cells 5–9 days after percutaneous transluminal coronary angioplasty [(PTCA); performed within 12 hours of myocardial infarction) was successful in 10 patients. Patients showed improved wall motion [2]. These clinical trials showed angiogenesis, decreased perfusion defects, and improved ejection fraction by endocardial injection of bone marrow cells (BMC) directly in hibernating myocardium useful in heart failure patients. Later 'Myoblast Autologous Grafting in Ischemic Cardiomyopathy MAGICcell-5-combination cytokine clinical trial' using intracoronary blood stem cells or induced pluripotent stem cells along with granulocyte-colony-stimulating factor therapy recorded improved angiogenesis and cardiac function [17]. Currently, induced pluripotent stem cells bearing specific membrane surface marker proteins are emerging as potential engineered cells useful for constructing 3D matrices in cardiac repair.

#### **1.2. What should be the goal of stem cell therapy?**

(AHA) data show high prevalence of heart failure as high as 50% and mortality as high as 32.8%. Coronary artery bypass grafting (CABG) was routine interventional and surgical treatment to bring low morality of coronary heart disease and now stem cell therapy is a new option. Still, major life threat is myocardial necrosis of myocardial tissue that cannot restore the original function of myocardium. Currently, stem cell research has opened vista in transplantation therapy, and its feasibility and effectiveness are well proven in animal experiments

The present chapter is divided into six sections. Section 1 introduces the evolution of stem cell therapy and its mechanism in regeneration and heart restoration. Section 2 introduces different stem cells and their purpose in repair and remodeling myocardium. Section 3 defines transplantation. Section 4 describes different modes of stem cell delivery. Section 5 highlights the purpose of rapid noninvasive real-time monitoring the myocardial repair and evaluation of heart territories. Section 6 reviews different clinical trials, available current nanotechnology and tissue engineering tools, and new approaches with future perspectives. A sketch of metabolic regulation during rejuvenation is presented for exploring new thoughts on secretory molecules regulating remodeling stem cells to explain regeneration of heart with possibility of better regeneration outcome. The chapter is written for interested physicians, surgeons, tis-

History records two types of bone marrow cells (BMC): hematopoietic stem cells [red blood cells (RBC), white blood cells (WBC), lymphocytes, macrophages] and bone marrow stroma mesenchymal stem cells (osteogenic cells for bone formation, chondrogenic cells for cartilage formation, adipogenic cells for fat tissue, and myoblast cells for heart regeneration). Stroma mesenchymal cells are sources of stem cells. Now, stem cell treatment is emerging in heart regeneration and restoration by using intracoronary, intramyocardial, and epicardial

Stem cell transplantation therapy was reported useful first time in recovering myocardial viability after myocardial infarction in ischemic heart disease [16]. Later, autologous intracoronary delivery of mononuclear bone marrow cells 5–9 days after percutaneous transluminal coronary angioplasty [(PTCA); performed within 12 hours of myocardial infarction) was successful in 10 patients. Patients showed improved wall motion [2]. These clinical trials showed angiogenesis, decreased perfusion defects, and improved ejection fraction by endocardial injection of bone marrow cells (BMC) directly in hibernating myocardium useful in heart failure patients. Later 'Myoblast Autologous Grafting in Ischemic Cardiomyopathy MAGICcell-5-combination cytokine clinical trial' using intracoronary blood stem cells or induced pluripotent stem cells along with granulocyte-colony-stimulating factor therapy recorded improved angiogenesis and cardiac function [17]. Currently, induced pluripotent stem cells bearing specific membrane surface marker proteins are emerging as potential engineered

as well as in small-scale clinical trials [2–15].

112 Stem Cells in Clinical Practice and Tissue Engineering

sue engineers, scientists, and entrepreneurs.

injections.

**1.1. Evolution of stem cell therapy: regeneration and healing**

cells useful for constructing 3D matrices in cardiac repair.

Stem cells are used as autograft (self-renewing, undifferentiated clonigenic) transplantation in myocardial repair or regeneration to bring tissue functionality back to normal for long-term survival in patients with permanent myocardial damage. Some stem cells are used as allograft (multipotent) daughter cells that give rise to multiple progenies [18]. The result of this asymmetric replication of stem cells is that after each division of stem cells, some progeny enters into the differentiation phase. Bone marrow cells and embryonic stem cells have differentiation plasticity and capacity. Goal is achieved by stimulating blood stem cells to cardiomyocytes providing a continuous supply of cardiac stem cells by trans-differentiation. Other exciting option is 'therapeutic cloning' means transplanted stem cells reprogram into induced pluripotent stem (iPS) cells at target organ. Still many issues are: (1) How many number of optimized stem cells needed when performing cell transplantation therapy?; (2) How survival time of transplanted cells can be best monitored?; (3) How do transplanted stem cells undergo differentiation into cardiomyocytes, smooth muscle cells, or endothelial cells?; (4) Do transplanted stem cells produce electrochemical coupling closer to normal myocardial tissue and normal cardiac cells and their functions really recover?; (5) What is the mechanism of cell transplantation in the treatment of myocardial perfusion and cardiac function after a short enhancement (myocardial cell regeneration or paracrine or other)? In nutshell, regeneration and healing of damaged cardiac tissue by myocardial repair is critical in survival. For interested readers, myocardial repair refers to the restoration of tissue architecture is shown in **Figure 1** and its remodeling of metabolic functions after injury is described in **Figure 7** in detail. Regeneration is defined as 100% myocardial repair and recovery.

Currently, cardiac stem cell therapy researchers are exploring new differentiation and surface protein expression markers. Main focus is to design stem cell therapy supports using different 3D extracellular matrices, polymeric scaffolds. Most intriguing advances are in innovative new methods of safe stem cell delivery with subsequent repair monitoring and follow up of stem cell therapy [19]. If incomplete repair, it leaves myocardial scar or fibrosis of collagen or necrosis after inflammation. After repair, myocardial recovery involves the proliferation of stem cells and interaction with native tissue cells to fill up myocardial mass and extra cellular matrix (remodeling) with its improved cellular paracrine function. The improved function is monitored by non-invasive MRI and bioluminescent techniques. Moreover, total success depends on choice of supporting engineered stem cell delivery method and monitoring the extent of repair or regenerating cardiac territories with its visualization by physiochemical methods during follow up of post-stem cell therapy benefits.

#### **1.3. Potential mechanisms**

Basic mechanism is 'myocardial revascularization and regeneration' to combat little or no blood supply left to slowly dying heart after myocardial ischemia or infarct or hypoxia. If sufficient oxygen diffusion from endocardium and collateral vessels provide sufficient oxygen

**Figure 1.** Different sources of adult stem cells are shown in heart regeneration in panel A and different conditions of myocardial injury are shown for the need of heart regeneration in panel B.

to preserve progenitor cells, cardiac repair is done by progenitor cell migration from healthy adjacent myocardium or from the blood circulation.

During regeneration, in fact, initially myoblasts, hemingioblasts, multipotent BMCs and adipocytes transform into cardiac specific progenitor cells. These resident stem cells, circulating hematopoietic cells, progenitor cells and BMCs collectively repair the dying heart by establishing revascularization and regeneration of heart as shown in **Figure 1**. These progenitor cells mainly differentiate into endothelial phenotype and cardiac phenotype to produce paracrine factors for perivascular incorporation and fusion to develop into myocytes and coronary vessels as shown in **Figure 2**. In this process, it requires specific transcription factors. In nutshell, bone marrow mononuclear stem cells, mesenchymal stem cells, endothelial stem cells, and hematopoietic stem cells undergo local neovascularization, neoangiogenesis and paracrine function to have positive effect on endogenous cell angiogenesis and energy metabolism by secretary molecules to inhibit myocyte apoptosis [20] as shown in **Figure 2**. As a result, heart left ventricle ejection fraction, arteriole, ventricular walls, enddiastolic and end-systolic ejection volumes, perfusion rate, contractility are improved with oxygen sufficiency. Revascularization and differentiation are mainly triggered by cycline dependent myocyte membrane surface proteins and remodeling factors as described in detail (see **Figure 7** in section 6).

#### **1.4. Cardiovascular tissue has progenitor differentiating cells to replenish dead or dying cells**

Stem cells can be mobilized from bone marrow, fat tissue, or blood, and then cultured to produce large numbers of pluripotent stem cells to transplant into the area of heart injury. It can be explained by the concept of 'cardiac chimerism' that explains the role of putative stem cells and progenitor cells present in transplanted heart during regeneration from circulating stem cells. For example, human circulating endothelial progenitor cells from bone cells are rich in membrane surface proteins such as CD34, CD31, KDR, and c-kit positive myocardial

**Figure 2.** Panel on top: Different mechanisms are shown for differentiation of stem cells to improve revascularization and cardiac regeneration after stem cell therapy. Panel on bottom: Somatic nuclear transfer mechanism is shown for

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differentiated cells. Induced pluripotent cells have potential of cardiac repair and used in treatment.

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to preserve progenitor cells, cardiac repair is done by progenitor cell migration from healthy

**Figure 1.** Different sources of adult stem cells are shown in heart regeneration in panel A and different conditions of

During regeneration, in fact, initially myoblasts, hemingioblasts, multipotent BMCs and adipocytes transform into cardiac specific progenitor cells. These resident stem cells, circulating hematopoietic cells, progenitor cells and BMCs collectively repair the dying heart by establishing revascularization and regeneration of heart as shown in **Figure 1**. These progenitor cells mainly differentiate into endothelial phenotype and cardiac phenotype to produce paracrine factors for perivascular incorporation and fusion to develop into myocytes and coronary vessels as shown in **Figure 2**. In this process, it requires specific transcription factors. In nutshell, bone marrow mononuclear stem cells, mesenchymal stem cells, endothelial stem cells, and hematopoietic stem cells undergo local neovascularization, neoangiogenesis and paracrine function to have positive effect on endogenous cell angiogenesis and energy metabolism by secretary molecules to inhibit myocyte apoptosis [20] as shown in **Figure 2**. As a result, heart left ventricle ejection fraction, arteriole, ventricular walls, enddiastolic and end-systolic ejection volumes, perfusion rate, contractility are improved with oxygen sufficiency. Revascularization and differentiation are mainly triggered by cycline dependent myocyte membrane surface proteins and remodeling factors as described in detail

**1.4. Cardiovascular tissue has progenitor differentiating cells to replenish dead or** 

Stem cells can be mobilized from bone marrow, fat tissue, or blood, and then cultured to produce large numbers of pluripotent stem cells to transplant into the area of heart injury. It can be explained by the concept of 'cardiac chimerism' that explains the role of putative stem cells and progenitor cells present in transplanted heart during regeneration from circulating stem cells. For example, human circulating endothelial progenitor cells from bone cells are rich in membrane surface proteins such as CD34, CD31, KDR, and c-kit positive myocardial

adjacent myocardium or from the blood circulation.

114 Stem Cells in Clinical Practice and Tissue Engineering

myocardial injury are shown for the need of heart regeneration in panel B.

(see **Figure 7** in section 6).

**dying cells**

**Figure 2.** Panel on top: Different mechanisms are shown for differentiation of stem cells to improve revascularization and cardiac regeneration after stem cell therapy. Panel on bottom: Somatic nuclear transfer mechanism is shown for differentiated cells. Induced pluripotent cells have potential of cardiac repair and used in treatment.

differentiation proteins visible in myocardial cell biopsy or cultures [21]. Bone-derived endocardial progenitor cells also do cardiac repair of functional myocardium by declining angiogenic activity. Bearzi et al. reported chimeric heart containing human myocardium with myocytes, coronary arterioles, and capillaries formed in mice injected with human cardiac stem cells [22]. It also supported the view of human stem cell therapy of cardiomyopathy [22]. The following description introduces readers with stem cell types, sources, stem cell engineering, and clinical application in heart repair.

egg rejuvenates the DNA of adult donor cells means restores telomere length without DNA loss during advancing age. This hypothetical idea poses ethical questions. On the other side of coin, iPS cells may treat or correct harmful mutations or diseases such as sickle cell

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• **Cardiac stem cells are** composed of four types including: resident stem cells, circulating hematopoietic cells, circulating progenitor cells, bone marrow cells. These all cells have significant role in cardiac regeneration after myocardial infarction. Urbanek et al. 2005 reported high number of activated stem cells (myocytes, smooth muscle cells, and embryonic cells) formed after cell regeneration in acute myocardial infarcts over chronic infarcts. Poor cell regeneration caused predisposition to chronic congestive heart failure [19]. Answer lies in telomere attrition, leading to decreased telomerase levels in chronic infarct and higher telomerase activity in acute infarcts. Telomerase enzyme is a marker showing growth potential of myocytes, endothelial cells, and smooth muscle cell lineages. Telomerase protects the DNA at the end of a chromosome during mitosis. Autologous transplantation raises hope of increasing telomerase activity to correct end stage

• **Mesenchymal stem cells** are nonhematopoietic cells in adult bone marrow and adipose tissues. These differentiate or modify *in vitro* to adopt phenotypic characters of cardiomyocytes and vascular cells by mesenchymal stem cell allogeneic therapy or cardiac repair by

• **Allogeneic stem cells** are "off the shelf" mesenchymal stem cell products from bone marrow of healthy donor. These are useful in therapy phase I trials as they target the myocardial injury site due to the presence of several stromal cell-derived factor-1 (SDF-1), major histocompatibility antigen class 2 molecules, and phenotypes CD145+, CD166+, and CD45− protein markers. These cells can differentiate into bone, tendon, fat, and muscle tissues. These cells also secrete immunosuppressive cytokines. Moreover, these cells can be administered by intravenous route. These stem cells also target and differentiate into cardiac

**3. Ideal stem cell transplantation to treat cardiovascular diseases**

In stem cell transplantation methods, ideally adult stem cells, embryonic stem cells (ESC), or induced pluripotent stem cells (iPSC) are locally fixed at dying myocardial tissue sites. However, major challenge is to monitor them timely and confirm the real-time improvement in dying or recovering myocardial tissue physiology efficiently to treat the ischemic heart disease. In other words, capability of MR imaging and monitoring heart metabolism visualize the anterior wall in acute myocardial infarction patients to detect improved myocardial perfusion and myocardial recovery status. Real-time cell imaging also confirms the efficacy of injected bone marrow stem cells (BMC cells) in the recovery of myocardial fragility and viability without any increase in left ventricular ejection fraction (LVEF) [27]. In initial experimental study, the success of embryonic stem cell transplantation in rat myocardial ischemia model showed

hemoglobin.

cardiomyopathy.

paracrine function [4].

myocytes and blood vessels [26].

### **2. Stem cell types and regeneration**

Human body has continuously dividing tissues, stable tissues, or permanent tissues. Hematopoietic cells in bone marrow continuously divide and readily regenerate. These regenerating matured cells are short-lived and continuously replenished by stem cells to maintain a constant equilibrium between replicating and dying mature cells, for example, skin and Gastrointestinal tract (GIT). Stable tissues with least replicating cells are heart, liver, kidney cells, endothelial cells, fibroblasts, and smooth muscle cells. Permanent cells are neuron and cardiac muscle cells. They can replicate but cannot terminally differentiate.

#### **2.1. Sources of stem cells**


egg rejuvenates the DNA of adult donor cells means restores telomere length without DNA loss during advancing age. This hypothetical idea poses ethical questions. On the other side of coin, iPS cells may treat or correct harmful mutations or diseases such as sickle cell hemoglobin.

differentiation proteins visible in myocardial cell biopsy or cultures [21]. Bone-derived endocardial progenitor cells also do cardiac repair of functional myocardium by declining angiogenic activity. Bearzi et al. reported chimeric heart containing human myocardium with myocytes, coronary arterioles, and capillaries formed in mice injected with human cardiac stem cells [22]. It also supported the view of human stem cell therapy of cardiomyopathy [22]. The following description introduces readers with stem cell types, sources, stem cell

Human body has continuously dividing tissues, stable tissues, or permanent tissues. Hematopoietic cells in bone marrow continuously divide and readily regenerate. These regenerating matured cells are short-lived and continuously replenished by stem cells to maintain a constant equilibrium between replicating and dying mature cells, for example, skin and Gastrointestinal tract (GIT). Stable tissues with least replicating cells are heart, liver, kidney cells, endothelial cells, fibroblasts, and smooth muscle cells. Permanent cells are neuron and

• **Embryonic stem cells** originate from endoderm of embryo after fertilization. Endoderm cells produce 220 kinds of specialized cells during mammalian development by irreversible differentiation process [23]. Later, embryonic precursor cells differentiate into adult muscle

• **iPS cells** are formed from regular adult cells by a "cocktail" of inducers or transcription factors so called "induced" pluripotent stem cells (iPS). These transform into the embryolike state, without eggs or embryos. The iPS cells are pluripotent and make any type of tissue in human body because iPS cells can resemble genetically and immunologically matched with the recipient body. Now, transplantation of these cells into the desired organ offers regenerative therapy of that tissue. However, turning back the biological clock of adult cells to an embryonic state is myth or miraculous escape from aging "immortal divinity". Interested readers may read comprehensive review on pluripotent cells [23, 24]. Yamanaka, 2007 introduced a combination of genes into adult cells changed their behavior as embryonic stem cell, hence called them 'pluripotent' stem cells. In fact, four gene transacting factors Oct3/4, Sox2, c-Myc, and Klf4 in adult myocyte cells possibly transformed them pluripotent stem cells [24]. Yu et al. 2007 reported the delivery of trans-acting factors Oct4, NANOG, Sox2, and LIN28 sufficient to reprogram a human somatic fibroblast cell into pluripotent cell bearing same telomerase and surface markers as embryonic cells [25]. Now, cellular programming by somatic nuclear transfer or cloning enables iPS cells behaving like embryonic cells [25]. Cloning develops embryo by the injection of new DNA material from an adult stem cell to an egg cell whose DNA is removed. This enucleated oocyte is the best source of pluripotent stem cells [24] as shown in **Figure 2**. The said engineered

cardiac muscle cells. They can replicate but cannot terminally differentiate.

or bone marrow cells, fat stem cells or multipotent cells.

engineering, and clinical application in heart repair.

**2. Stem cell types and regeneration**

116 Stem Cells in Clinical Practice and Tissue Engineering

**2.1. Sources of stem cells**


### **3. Ideal stem cell transplantation to treat cardiovascular diseases**

In stem cell transplantation methods, ideally adult stem cells, embryonic stem cells (ESC), or induced pluripotent stem cells (iPSC) are locally fixed at dying myocardial tissue sites. However, major challenge is to monitor them timely and confirm the real-time improvement in dying or recovering myocardial tissue physiology efficiently to treat the ischemic heart disease. In other words, capability of MR imaging and monitoring heart metabolism visualize the anterior wall in acute myocardial infarction patients to detect improved myocardial perfusion and myocardial recovery status. Real-time cell imaging also confirms the efficacy of injected bone marrow stem cells (BMC cells) in the recovery of myocardial fragility and viability without any increase in left ventricular ejection fraction (LVEF) [27]. In initial experimental study, the success of embryonic stem cell transplantation in rat myocardial ischemia model showed significant recovery as reduced left ventricular expansion and reduced area of myocardial infarction after 3–6 weeks. However, in this recovery process, stem cell transcription factors such as Oct3/4, Sox2, Klf4, and c-Myc transformed the embryonic stem cells into induced pluripotent stem cells or iPSCs [28]. These pluripotent cells form regenerative myocardial tissue, smooth muscle, or endothelial vascular cells *in situ* to repair myocardial infarction in heart or increased ventricular wall thickness and electrical stability [29]. Recently, different clinical centers claim their success differently to transplant pluripotent stem cells in remodeling myocardial muscle or endothelial vascular cells [10, 28–35]. In fact, stem cell treatment centers follow the strategy that pluripotent stem cells may be stable rather than terminally differentiated as meta-analysis of randomized controlled clinical trials on stem cell therapy also indicated clearly that intracoronary adult bone marrow stem cells improve left ventricular function and reduce the risk of recurrent heart failure soon after acute myocardial infarction (AMI) [36]. **Table 1** shows the major stem cell types commonly used in medical practice using autograft or allograft transplantation in myocardial repair. Mainly adult stem cells, embryonic stem cells (ESC), or induced pluripotent stem cells (iPSC) are choice.


**Table 1.** Potential applications of different stem cell types for cardiomyocytes in heart transplantation for myocardial repair.

> with central lumen placed at a desired position (see **Figure 3**). It allows intracoronary cells to "home-in" or retention of stem cells by extravasation of BMC to the infarcted area in the presence of chemokines and adhesion molecules, SDF-1, and beta-2-integrin factors induced

> **Figure 3.** Different delivery sites of stem cell injections are shown in panel A. Yag laser with three needles is shown for BM Laser Repair procedure to deliver stem cells and rejuvenation molecules in panel B. NOGA Myostar catheter is shown for delivery of stem cells in left ventricle in panel C. The evaluation of heart recovery as improved anterolateral

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Coronary infusion of cells is performed four to six times, with 3-minute sequential balloon inflations followed by 3-minute rest periods, to create a "stop flow" situation for maximal retention period to come into contact with the microcirculation of the infarct-related artery. It maximizes the migration and retention of cells into the infarct and peri-infarct tissues for successful transplantation. After transplantation, baseline and post procedure LV angiograms are monitored for 24 hours, with cardiac markers checked at every 6 and 12 hours. Injection of stem cells into a contralateral artery may increase retention in ischemic area if there are well-formed collaterals. Imaging studies further confirm the success of contralateral stem cell injections to increase the retention of cells in occluded artery territories. The crucial issues are: retention of cells, improved ejection fraction, improved regional wall LV function, microvascular plugging, biodistribution, homing to myocardium, proapoptotic factors in the ischemic

by ischemic cell injury [32, 35–37].

wall after stem cell therapy by MRI is shown in panel D.

myocardium, CD34+ cells [35, 38].

### **4. Delivery protocols of stem cell therapy**

Each protocol differs in cell retention and regeneration rates depending upon method and site of injection, i.e., intracoronary, intramyocardial, transendocardial, or via coronary sinus delivery (see **Figure 3**), time of delivery, inflammatory response. Other factor is timing of administration rapid or slow injection rates. The early administration of cells facilitates better retention of stem cells or rejuvenating homing signals evidenced in TIME trial [6], while a long delay may cause scar formation in the LateTIME trial [7] as highlighted in **Tables 2** and **3**.

#### **4.1. Intracoronary stem cell therapy**

It is done by cell transplantation through transcoronary passage of cells at infarct site along with a standard percutaneous transluminal coronary angioplasty (PTCA) procedure or coronary artery bypass grafting (CABG) procedure, with the use of an over-the-wire balloon Stem Cells in Treatment of Coronary Heart Disease and Its Monitoring: Tissue Engineering... http://dx.doi.org/10.5772/intechopen.70229 119

significant recovery as reduced left ventricular expansion and reduced area of myocardial infarction after 3–6 weeks. However, in this recovery process, stem cell transcription factors such as Oct3/4, Sox2, Klf4, and c-Myc transformed the embryonic stem cells into induced pluripotent stem cells or iPSCs [28]. These pluripotent cells form regenerative myocardial tissue, smooth muscle, or endothelial vascular cells *in situ* to repair myocardial infarction in heart or increased ventricular wall thickness and electrical stability [29]. Recently, different clinical centers claim their success differently to transplant pluripotent stem cells in remodeling myocardial muscle or endothelial vascular cells [10, 28–35]. In fact, stem cell treatment centers follow the strategy that pluripotent stem cells may be stable rather than terminally differentiated as meta-analysis of randomized controlled clinical trials on stem cell therapy also indicated clearly that intracoronary adult bone marrow stem cells improve left ventricular function and reduce the risk of recurrent heart failure soon after acute myocardial infarction (AMI) [36]. **Table 1** shows the major stem cell types commonly used in medical practice using autograft or allograft transplantation in myocardial repair. Mainly adult stem cells, embryonic stem

**Fate of stem cells Autologous origin of adult** 

Embryonic stem cells Fetal cardiomyocytes Mesenchymal stem cells Endothelial progenitor cells

Resident cardiac stem cells Fetal cardiomyocytes Endothelial progenitor cells Induced pluripotent stem

Adipose derived stem cells Umblical cord derived cells Endothelial progenitor cells Multipotent adult

**stem cells**

**Fate of stem cells**

progenitor cells

cells

Each protocol differs in cell retention and regeneration rates depending upon method and site of injection, i.e., intracoronary, intramyocardial, transendocardial, or via coronary sinus delivery (see **Figure 3**), time of delivery, inflammatory response. Other factor is timing of administration rapid or slow injection rates. The early administration of cells facilitates better retention of stem cells or rejuvenating homing signals evidenced in TIME trial [6], while a long delay may cause scar formation in the LateTIME trial [7] as highlighted in **Tables 2** and **3**.

**Table 1.** Potential applications of different stem cell types for cardiomyocytes in heart transplantation for myocardial

It is done by cell transplantation through transcoronary passage of cells at infarct site along with a standard percutaneous transluminal coronary angioplasty (PTCA) procedure or coronary artery bypass grafting (CABG) procedure, with the use of an over-the-wire balloon

cells (ESC), or induced pluripotent stem cells (iPSC) are choice.

**4. Delivery protocols of stem cell therapy**

Skeletal myoblast cells Bone marrow mononuclear CD34+

**4.1. Intracoronary stem cell therapy**

**Allogenic origin of stem** 

118 Stem Cells in Clinical Practice and Tissue Engineering

**cells**

repair.

**Figure 3.** Different delivery sites of stem cell injections are shown in panel A. Yag laser with three needles is shown for BM Laser Repair procedure to deliver stem cells and rejuvenation molecules in panel B. NOGA Myostar catheter is shown for delivery of stem cells in left ventricle in panel C. The evaluation of heart recovery as improved anterolateral wall after stem cell therapy by MRI is shown in panel D.

with central lumen placed at a desired position (see **Figure 3**). It allows intracoronary cells to "home-in" or retention of stem cells by extravasation of BMC to the infarcted area in the presence of chemokines and adhesion molecules, SDF-1, and beta-2-integrin factors induced by ischemic cell injury [32, 35–37].

Coronary infusion of cells is performed four to six times, with 3-minute sequential balloon inflations followed by 3-minute rest periods, to create a "stop flow" situation for maximal retention period to come into contact with the microcirculation of the infarct-related artery. It maximizes the migration and retention of cells into the infarct and peri-infarct tissues for successful transplantation. After transplantation, baseline and post procedure LV angiograms are monitored for 24 hours, with cardiac markers checked at every 6 and 12 hours. Injection of stem cells into a contralateral artery may increase retention in ischemic area if there are well-formed collaterals. Imaging studies further confirm the success of contralateral stem cell injections to increase the retention of cells in occluded artery territories. The crucial issues are: retention of cells, improved ejection fraction, improved regional wall LV function, microvascular plugging, biodistribution, homing to myocardium, proapoptotic factors in the ischemic myocardium, CD34+ cells [35, 38].


**Table 2.** Randomized control trials showing administration of pluripotent stem cells with primary outcome of improved cardiac mass by monitoring improvement in left ventricle ejection function by imaging.

*How success in heart recovery after heart transplantation is assessed?* After heart recovery, improvement in cardiac functions is the success key. Important cardiac parameters are improved ejection fraction (LVEF), improved contractile function, improved regional wall thickness reduction or improved LV function, Ejection Diastolic and Ejection Systolic Volumes, improved perfusion along with decreased adverse perfusion defects, all these events within less than a week as shown in **Table 2** and **Figure 3** (see panel D). In support, several randomized trials clearly shown that administration of intracoronary autologous bone marrow nuclear cells in patients soon after myocardial infarction improved the ejection fraction within 5 days [8, 39] shown in **Table 2** and illustrated in **Figure 3** (see panel D). Other randomized

**Table 3.** A chronology of clinical trials using different stem cell delivery and engineered constructs.

**Clinical trial Administration Engineered tissue construct used Reference**

Bolli et al. 2011 SCIPIO Trial Intracoronary Pluripotent cells [5]

Zhao et al. 2013 Intracoronary Pluripotent cells [90] Kurbonov et al. 2013 Intracoronary Engineered stem cells [91] Forcillo et al. 2013 Via CABG+i.m. Stem cells [92] Assmann et al. 2013 Via CABG+epicardial Engineered stem cells [93] Nasseri et al. 2014 i.m Stem cells [94] Brickwedel et al. 2014 Via CABG Engineered stem cells [95]

Hao et al. 2015 Intracoronary Stem cells [97] Chang et al. 2015 Intracoronary Stem cells [98] Gao et al. 2015 Intracoronary Stem cell engineering [99] Fiarresga et al. 2015 Intracoronary Stem cell engineering [100] Helseth et al. 2015 Intracoronary Stem cell engineering [101] Eirin et al. 2015 Intrarenal Pluripotent cells [102] Lee et al. 2015 Intracoronary Engineered stem cells [103] Tseliou et al. 2016 Intracoronary Stem cell engineering [104] Hasan et al. 2016 Intracoronary Stem cell engineering [105] Xiao et al. 2017 Intracoronary Stem cell engineering [106] Gao et al. 2017 Intracoronary Pluripotent cells in 3D scaffold [107]

Intracoronary No change in LVEF in 4 months followup

Intracoronary Pluripotent cells [13]

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Intracoronary Pluripotent cells [7]

Intracoronary Pluripotent cells [89]

Engineered stem cells [96]

[12]

121

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Hirsch et al. 2011 HEBE clinical trial

Roncalli et al. 2011 BONAMI Trial

Trial

Trial

Traverse et al. 2011 LateTIME

Makkar et al. 2012 CADUCEUS

Hong et al. 2014 Intracoronary + retrograde

coronary sinus


**Study name Published n Days Primary outcome Imaging modality**

51–60% in 3 months

months

in 1 year

months

Autologous BMNCs CABG + SC 5 1y >5 days old MI Improved perfusion Autologous BMNCs PTCA + SC 13 3m 5–9 days post MI Better perfusion, wall

BMNCs+EPCs PTCA + SC 23 4m <3 days post MI Better LVEF, EDV, perfusion,

BMCs+AC133 CABG + SC 12 3–9 m 0–3 m post MI Better EF, better perfusion BMNCs EMM + SC 8 3 m severe IHD Improved perfusion, angina,

Autogous BMNCs EMM + SC 14 2 m CHF Improved LVEF, perfusion,

Myoblasts CABG + SC 10 11 m CHF Improved EVEF, contractile

**Table 2.** Randomized control trials showing administration of pluripotent stem cells with primary outcome of improved

CABG + SC 12 12 m Old MI + ischemic CAD improved LVDF, regional

5 6 m IHD improved LVEF, wall thicker

30 6 m <5 days post MI Improved LVEF, contractile

34 3–6 m 10 days post MI Better perfusion, high EDV,

10 6 m >48 hours AMI(old) Higher stent restenosis in

in large infarcts after 6 months(18 m follow up)

4 months but regional contractility improved & infarct size less

months but global LVEF same after 4 months







motion, less infarct size

% contractile function

contractile function

contractile function

function

function

G-CSF group

contractility

at the injection site

ESV, wall movement, LVEF


TOPCARE-AMI 2002 59 4–5.5 -Global LVEF improved

BOOST 2004 60 5–6.3 -Global LVEF improved

REPAIR-AMI 2006 187 3–6 -LVEF improved 2.5% in 4

ASTAMI 2006 97 6–7 -No change in global LVEF

LEUVEN-AMI 2006 66 1 -No change in LVEF in

FINCELL 2008 77 3 -LVEF improved 5% in 6

HEBE 2010 200 3–8 -LVEF improved 6% in 6

B. Other clinical stem cell trials using different stem cell types

IM SC injection at LVAD site

PTCA

18 days post PTCA

IC infusion + PTCA

cardiac mass by monitoring improvement in left ventricle ejection function by imaging.

Autologous skeletal myoblasts

Autologous skeletal myoblasts

Autologous blood SCs + inj G-CSF

Autologous BMCs IC infusion +

Autologous BMSCs IC infusion +

A. Proven stem cell treatment and evaluation

120 Stem Cells in Clinical Practice and Tissue Engineering

**Table 3.** A chronology of clinical trials using different stem cell delivery and engineered constructs.

*How success in heart recovery after heart transplantation is assessed?* After heart recovery, improvement in cardiac functions is the success key. Important cardiac parameters are improved ejection fraction (LVEF), improved contractile function, improved regional wall thickness reduction or improved LV function, Ejection Diastolic and Ejection Systolic Volumes, improved perfusion along with decreased adverse perfusion defects, all these events within less than a week as shown in **Table 2** and **Figure 3** (see panel D). In support, several randomized trials clearly shown that administration of intracoronary autologous bone marrow nuclear cells in patients soon after myocardial infarction improved the ejection fraction within 5 days [8, 39] shown in **Table 2** and illustrated in **Figure 3** (see panel D). Other randomized trials showed clear evidence of improved regional wall LV function [9]. The Repair-AMI trial showed a significant decrease in major adverse events [10]. However, several clinical trial and pilot studies have failed to demonstrate that bone marrow nuclear cells really improve LV function in the setting of acute myocardial infarction because of empirical calibration or lack of preclinical results[7, 11–13, 31, 40]. Other critical issue is successful cardiac recovery or revived myocardial function rapidly and fast as much as possible. In the previous studies, most of the autologous bone marrow mononuclear cell implantations were performed within week following ST elevation myocardial infarction event. Specific mention here is the evidence of most favorable cardiac recovery effect on LV function obtained on the fifth day after delivery of stem cells in small cohort of patients in the Repair-AMI trial [10] as shown in **Figure 3**.

low cardiac output, by an 8Fr MYOSTAR™ catheter (Biologics Delivery Systems) with nitinol tubing and retractable needle set up at a depth of 4.5–6 mm inside cardiac tissue and placed at an appropriate angle 45° under fluoroscopy observation as shown in **Figure 3**. Volumes of approximated 0.3 cc of stem cells are injected by manually advancing the needle initially

Stem Cells in Treatment of Coronary Heart Disease and Its Monitoring: Tissue Engineering...

MRI). Without motion, still patient is kept under observation. Later, patient is monitored for 18–24 hours attached with cardiac life support device and recovery, and myocardial viability is monitored by continuous real-time LV angiography. First time, Federal Drug Agency (FDA) approved the protocol of autologous BMC stem cells as milestone showing salvaged hibernating myocardium with improved angiogenesis, 75% decreased perfusion defects, and improved 20–29% ejection fraction [17]. Now, improved protocols in clinical trials are in practice throughout the US and Europe as shown in **Tables 2** and clinical trials in recent 5 years

It is other approach to deliver potentially therapeutic stem cells in coronary sinus. A double lumen catheter attached with a larger proximal and a smaller distal balloons is used for delivery of cells in distal lumen. The stem cells are injected and their transport is confirmed angiographically in the mid- to distal interventricular vein that runs parallel to the left anterior

This approach depends upon the intravenous access site as shown in **Figure 3** panel A. The cells get trapped in the lungs, liver, and spleen, so that only a small number may enter in coronary circulation, and myocardial homing is minimal [15]. Myocardial homing depends on the expression of adhesion molecules, cytokines, and homing receptors. In following sections, growing interest of real-time noninvasive monitoring of pre- and post-cardiac recovery of myocardium tissue by advanced 900 MHz MRI methods in preclinical studies and real-time stem cell behavior are discussed. 900 MHz MRI facility is available only in laboratory at our

place in the light of less known facts, limitations and challenges to use this facility.

**5. Need of noninvasive in vivo monitoring stem cells in preclinical** 

Molecular events by imaging methods offer excellent opportunity to visualize and track stem cell behavior in vivo to evaluate their efficacy of cardiac cell recovery or therapy in preclinical studies. Monitoring the settled home-in rejuvenated stem cells functioning well at cardiac infarct site is based on the fact that active myocardial metabolite protons and water relaxation dynamics is characteristic while 31P MR peaks predict the settled stem cell physiology [45]. Ultrasound imaging, positron emission tomography/single photon emission computed

in areas of thinned myocardium (<0.5 mm2

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123

at several different space volumes of 1 cm<sup>3</sup>

**4.3. Retrograde coronary sinus injection**

descending artery, as shown in **Figure 3** panel A.

shown in **Table 3**.

**4.4. Intravenous delivery**

**studies**

In the light of above, it is very important that timing of 'appropriate stem cell conditioned delivery' in right manner soon after myocardial infarction may have an influence on stem cell treatment as highlighted in **Figure 3** (see panel C). This timing and delivery issue has debated over the stem cell choice, delivery mode of stem cells, and timing of stem cell implantation after acute myocardial infarction. Two factors need attention here for successful implantation and its action on recovery of myocardium: (1) Release rate of circulating progenitor mononuclear cells from bone marrow within hours of acute myocardial infarction [27, 41, 42]; (2) Release of enormous hematopoietic stem cells, endothelial progenitor stem cells, mesenchymal stem cells, and a very small number of embryonic-like pluripotent cells with cardiorejuvenating properties [43]. Moreover, other factors are also determinant in the success of cardiac rejuvenation such as inadequate cell count, improper processing, and timing of stem cell administration.

Important concern in regard to negative findings is timing of stem cell administration. The National Heart Lung and Blood Institute sponsored Cardiovascular Cell Therapy Research Network reported two prospective clinical trials, TIME [6] and LateTIME [7]. The TIME trial was proposed to compare the effects of bone marrow mononuclear source cells delivered at 3–7 days in patients with predominantly ST elevation myocardial infarction. The LateTIME trial proved the hypothesis that delayed delivery of autologous bone marrow cells at 2–3 weeks following acute myocardial infarction may improve global LV systolic function. LateTIME trial calibrated the cell count and processing issues but did not show any detectable improvement in LV function over a period of 2 years [44]. For interested readers, intramyocardial stem cell therapy protocol is described in following section.

#### **4.2. Intramyocardial stem cell therapy protocol**

In open heart surgery, direct visualization of the heart is a preferred method as an endocardial approach during supervised intramyocardial injection of stem cells. Using endocardial approach for intramyocardial stem cell therapy, a transmyocardial injection of stem cells is guided by LV electromechanical mapping with NOGA™ software (Biologics Delivery Systems, Diamond Bar, CA) to deliver stem cells in target infarct area [15] as shown in **Figure 3**. For instance, in routine stem cells are injected into nonviable myocardium soon after an observed low cardiac output, by an 8Fr MYOSTAR™ catheter (Biologics Delivery Systems) with nitinol tubing and retractable needle set up at a depth of 4.5–6 mm inside cardiac tissue and placed at an appropriate angle 45° under fluoroscopy observation as shown in **Figure 3**. Volumes of approximated 0.3 cc of stem cells are injected by manually advancing the needle initially at several different space volumes of 1 cm<sup>3</sup> in areas of thinned myocardium (<0.5 mm2 by MRI). Without motion, still patient is kept under observation. Later, patient is monitored for 18–24 hours attached with cardiac life support device and recovery, and myocardial viability is monitored by continuous real-time LV angiography. First time, Federal Drug Agency (FDA) approved the protocol of autologous BMC stem cells as milestone showing salvaged hibernating myocardium with improved angiogenesis, 75% decreased perfusion defects, and improved 20–29% ejection fraction [17]. Now, improved protocols in clinical trials are in practice throughout the US and Europe as shown in **Tables 2** and clinical trials in recent 5 years shown in **Table 3**.

#### **4.3. Retrograde coronary sinus injection**

It is other approach to deliver potentially therapeutic stem cells in coronary sinus. A double lumen catheter attached with a larger proximal and a smaller distal balloons is used for delivery of cells in distal lumen. The stem cells are injected and their transport is confirmed angiographically in the mid- to distal interventricular vein that runs parallel to the left anterior descending artery, as shown in **Figure 3** panel A.

#### **4.4. Intravenous delivery**

trials showed clear evidence of improved regional wall LV function [9]. The Repair-AMI trial showed a significant decrease in major adverse events [10]. However, several clinical trial and pilot studies have failed to demonstrate that bone marrow nuclear cells really improve LV function in the setting of acute myocardial infarction because of empirical calibration or lack of preclinical results[7, 11–13, 31, 40]. Other critical issue is successful cardiac recovery or revived myocardial function rapidly and fast as much as possible. In the previous studies, most of the autologous bone marrow mononuclear cell implantations were performed within week following ST elevation myocardial infarction event. Specific mention here is the evidence of most favorable cardiac recovery effect on LV function obtained on the fifth day after delivery of stem cells in small cohort of patients in the Repair-AMI trial [10] as shown

In the light of above, it is very important that timing of 'appropriate stem cell conditioned delivery' in right manner soon after myocardial infarction may have an influence on stem cell treatment as highlighted in **Figure 3** (see panel C). This timing and delivery issue has debated over the stem cell choice, delivery mode of stem cells, and timing of stem cell implantation after acute myocardial infarction. Two factors need attention here for successful implantation and its action on recovery of myocardium: (1) Release rate of circulating progenitor mononuclear cells from bone marrow within hours of acute myocardial infarction [27, 41, 42]; (2) Release of enormous hematopoietic stem cells, endothelial progenitor stem cells, mesenchymal stem cells, and a very small number of embryonic-like pluripotent cells with cardiorejuvenating properties [43]. Moreover, other factors are also determinant in the success of cardiac rejuvenation such as inadequate cell count, improper processing, and timing of stem

Important concern in regard to negative findings is timing of stem cell administration. The National Heart Lung and Blood Institute sponsored Cardiovascular Cell Therapy Research Network reported two prospective clinical trials, TIME [6] and LateTIME [7]. The TIME trial was proposed to compare the effects of bone marrow mononuclear source cells delivered at 3–7 days in patients with predominantly ST elevation myocardial infarction. The LateTIME trial proved the hypothesis that delayed delivery of autologous bone marrow cells at 2–3 weeks following acute myocardial infarction may improve global LV systolic function. LateTIME trial calibrated the cell count and processing issues but did not show any detectable improvement in LV function over a period of 2 years [44]. For interested readers, intramyocar-

In open heart surgery, direct visualization of the heart is a preferred method as an endocardial approach during supervised intramyocardial injection of stem cells. Using endocardial approach for intramyocardial stem cell therapy, a transmyocardial injection of stem cells is guided by LV electromechanical mapping with NOGA™ software (Biologics Delivery Systems, Diamond Bar, CA) to deliver stem cells in target infarct area [15] as shown in **Figure 3**. For instance, in routine stem cells are injected into nonviable myocardium soon after an observed

dial stem cell therapy protocol is described in following section.

**4.2. Intramyocardial stem cell therapy protocol**

in **Figure 3**.

122 Stem Cells in Clinical Practice and Tissue Engineering

cell administration.

This approach depends upon the intravenous access site as shown in **Figure 3** panel A. The cells get trapped in the lungs, liver, and spleen, so that only a small number may enter in coronary circulation, and myocardial homing is minimal [15]. Myocardial homing depends on the expression of adhesion molecules, cytokines, and homing receptors. In following sections, growing interest of real-time noninvasive monitoring of pre- and post-cardiac recovery of myocardium tissue by advanced 900 MHz MRI methods in preclinical studies and real-time stem cell behavior are discussed. 900 MHz MRI facility is available only in laboratory at our place in the light of less known facts, limitations and challenges to use this facility.

### **5. Need of noninvasive in vivo monitoring stem cells in preclinical studies**

Molecular events by imaging methods offer excellent opportunity to visualize and track stem cell behavior in vivo to evaluate their efficacy of cardiac cell recovery or therapy in preclinical studies. Monitoring the settled home-in rejuvenated stem cells functioning well at cardiac infarct site is based on the fact that active myocardial metabolite protons and water relaxation dynamics is characteristic while 31P MR peaks predict the settled stem cell physiology [45]. Ultrasound imaging, positron emission tomography/single photon emission computed tomography (PET/ SPECT), magnetic resonance imaging (MRI), optical imaging, and CT imaging are routine molecular imaging techniques. Magnetic resonance of odd-numbered protons in cardiac tissue molecules with resonant radiofrequency in high magnetic field generates the physiological MR cardiac MRI fingerprint as most promising in clinical transformation to provide the structural-functional information of resettled cardiac mass with superior resolution and high sensitivity relatively safer and without radiation [45, 46].

Iron oxide nanoparticles produce strong transverse relaxation constant/dephased transverse relaxation constant (T2/T2\*) ratio as negative contrast effect due to dephasing effect [54–56].

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Super paramagnetic iron oxide nanoparticles (SPION) are family of paramagnetic/ superparamagnetic contrast agents. It consists of a ferrite (maghemite or magnetite) core and a polymer coating. Depending on the diameter size (including both metal core and polymer coat), the nanoparticles can be divided into the SPION (diameter size 60–150 nm), USPION (diameter size 10–40 nm), and MION (diameter size 10–30 nm) categories [57]. Ferucarbotran (Resovist®) and ferumoxides (Endorem or Feridex®) are MRI enhancement contrast agents approved by FDA for clinical diagnosis of liver tumors and metastatic lymph nodes. High concentrations of ferromagnetic material can shorten both the T1/T2 constants as well as the effect of T2\*, resulting in a significant reduction in MR relaxation with higher biological safety of stem cells

Most of these labeled stem cells do not internalize SPIONs and they need endocytosis polymer boosters. Positively charged polymer transfection agents (TAs) or polycations enhance the endocytosis across negatively charged membrane surface. So, they can be coated on the surface of magnetic iron oxide particles to boost SPION endocytosis or stem cells nonspecifically uptake these SPION particles through the negatively charged membrane surface. At present, composites of SPION and polycation TAs are the most commonly used methods to

• Negative charge on stem cell membrane surface does not permit ferric oxide particles to attach them with the stem cells. To accomplish it, iron oxide particle surface modifications can enhance cellular endocytosis. In this direction, several surface modification approaches of polycation binding, incubation with hematopoietic cells, monoclonal antibody-antigen binding, receptor binding, magnetoelectric perforation and others are used in but these are

• Positive charged polycation TAs macromolecules such as polylysine or protamine sulfate are used in making SPION/TA composites with strong positive/negative interaction or cationic polymer material coating [63]. These SPIO/TA composites easily adhere to the surface of stem cell membranes and persuade the phagocytosis of iron oxide particles without aggregation of SPION particles [51]. Ferumoxides (Feridex) with USPIO (MION-46 L) and added polycationic TAs have been in use to raise the concentration of intracellular SPION particles [32]. After 4–48-hour incubation with 25 μg Fe/mL TA-(USPION), target stem cells demonstrated a significant reduction in T2 signal intensities due to dephasing effect [52]. Ferumoxides mixed with protamine sulfate (50:3) μg/ml offers an

• Overnight incubation of the human mesenchymal stem cells with hematopoietic CD34+

cells and specific mammalian cells, increases the iron content in the stem cells 1.47–17.31 pg/

stem

**5.2. Iron oxide particle stem cell labeling**

**5.3. Positively charged polymer transfection agents**

enhance the endocytosis of iron oxide particles [49, 60–62].

[48, 58–60].

still in infancy.

optimized protocol [53].

cell [64].

Other major challenges are visualizing myocardial functionality and real-time monitoring the status of transplanted stem cell behavior within native tissue as true representative of altered or improved visible myocardial territories or metabolic recovery. For this purpose, the smart imaging contrast agents or contrast labeling of stem cells offer to visualize the behavior of transplanted stem cells in tissue in situ. Different techniques of cell MRI, bioluminescence, chemiluminescence, myofibril scanning, and DNA end-labeling are routine methods to track myocardial functionality, viability, and fragility [47, 49]. Recently, nanoparticle-labeled stem cells have been developed to achieve dephasing susceptibility contrast and monitoring the stem cell behavior, physiological changes and molecular events by 900 MHz MR imaging stem cells [50].

#### **5.1. MRI contrast labeling of stem cells: source of contrast in images**

Tracking of transplanted stem cells and their behavior in native tissue is done using stem cell MRI contrast agents such as gadolinium (Gd) chelating agents (Gd-DTPA) and manganese chloride (MnCl2 ) [51–53]. Mostly, these image contrast agents provide longitudinal relaxation constant (T1) enhanced positive contrast effects of stem cell originated proton nuclear magnetic resonance (NMR) relaxation as shown in **Figure 4**. Recently, several stem cell specific iron oxide paramagnetic/super paramagnetic contrast agents emerged as negative contrast agents.

**Figure 4.** The metal (M) is encapsulated in chelator coat (Gd-DTPA in dextran coated SPIO). The water accessibility (τm), rotational tumbling time (τ<sup>f</sup> ), electron spin state T1e of superparamagnetic metal, chemical exchange rate (τex), and MR frequency (δ) are shown as source of contrast. On right: Distinct longitudinal relaxation T1 constants as graphs at different contrast agent concentrations are shown in panel A and respective T1 images are shown in panel B. Reproduced from [52] with permission.

Iron oxide nanoparticles produce strong transverse relaxation constant/dephased transverse relaxation constant (T2/T2\*) ratio as negative contrast effect due to dephasing effect [54–56].

#### **5.2. Iron oxide particle stem cell labeling**

tomography (PET/ SPECT), magnetic resonance imaging (MRI), optical imaging, and CT imaging are routine molecular imaging techniques. Magnetic resonance of odd-numbered protons in cardiac tissue molecules with resonant radiofrequency in high magnetic field generates the physiological MR cardiac MRI fingerprint as most promising in clinical transformation to provide the structural-functional information of resettled cardiac mass with superior

Other major challenges are visualizing myocardial functionality and real-time monitoring the status of transplanted stem cell behavior within native tissue as true representative of altered or improved visible myocardial territories or metabolic recovery. For this purpose, the smart imaging contrast agents or contrast labeling of stem cells offer to visualize the behavior of transplanted stem cells in tissue in situ. Different techniques of cell MRI, bioluminescence, chemiluminescence, myofibril scanning, and DNA end-labeling are routine methods to track myocardial functionality, viability, and fragility [47, 49]. Recently, nanoparticle-labeled stem cells have been developed to achieve dephasing susceptibility contrast and monitoring the stem cell behavior, physiological changes and molecular events by 900 MHz MR imaging stem cells [50].

Tracking of transplanted stem cells and their behavior in native tissue is done using stem cell MRI contrast agents such as gadolinium (Gd) chelating agents (Gd-DTPA) and manganese

constant (T1) enhanced positive contrast effects of stem cell originated proton nuclear magnetic resonance (NMR) relaxation as shown in **Figure 4**. Recently, several stem cell specific iron oxide paramagnetic/super paramagnetic contrast agents emerged as negative contrast agents.

**Figure 4.** The metal (M) is encapsulated in chelator coat (Gd-DTPA in dextran coated SPIO). The water accessibility (τm),

(δ) are shown as source of contrast. On right: Distinct longitudinal relaxation T1 constants as graphs at different contrast agent concentrations are shown in panel A and respective T1 images are shown in panel B. Reproduced from [52] with permission.

), electron spin state T1e of superparamagnetic metal, chemical exchange rate (τex), and MR frequency

) [51–53]. Mostly, these image contrast agents provide longitudinal relaxation

resolution and high sensitivity relatively safer and without radiation [45, 46].

**5.1. MRI contrast labeling of stem cells: source of contrast in images**

chloride (MnCl2

124 Stem Cells in Clinical Practice and Tissue Engineering

rotational tumbling time (τ<sup>f</sup>

Super paramagnetic iron oxide nanoparticles (SPION) are family of paramagnetic/ superparamagnetic contrast agents. It consists of a ferrite (maghemite or magnetite) core and a polymer coating. Depending on the diameter size (including both metal core and polymer coat), the nanoparticles can be divided into the SPION (diameter size 60–150 nm), USPION (diameter size 10–40 nm), and MION (diameter size 10–30 nm) categories [57]. Ferucarbotran (Resovist®) and ferumoxides (Endorem or Feridex®) are MRI enhancement contrast agents approved by FDA for clinical diagnosis of liver tumors and metastatic lymph nodes. High concentrations of ferromagnetic material can shorten both the T1/T2 constants as well as the effect of T2\*, resulting in a significant reduction in MR relaxation with higher biological safety of stem cells [48, 58–60].

#### **5.3. Positively charged polymer transfection agents**

Most of these labeled stem cells do not internalize SPIONs and they need endocytosis polymer boosters. Positively charged polymer transfection agents (TAs) or polycations enhance the endocytosis across negatively charged membrane surface. So, they can be coated on the surface of magnetic iron oxide particles to boost SPION endocytosis or stem cells nonspecifically uptake these SPION particles through the negatively charged membrane surface. At present, composites of SPION and polycation TAs are the most commonly used methods to enhance the endocytosis of iron oxide particles [49, 60–62].


• Monoclonal antibodies of pancreatic cancer specific antigen (PAP2a) fused with dextranmodified SPIONs show antigen-antibody reaction to target the iron oxide particles in pancreatic cancer cells and promote the receptor-mediated SPIO endocytosis [65].

**5.7. Limitations of tracking SPION labeling stem cells**

popular in the field of stem cell tracking because of their high sensitivity.

**5.8. Reporter gene labeling in stem cells**

of angle proportional to degree of distortion.

SPION particle cell tracking method for cell labeling has some shortcomings. The MRI signal in preclinical or clinical studies is usually generated from surrounding tissue areas of noninterest cardiopulmonary junction [56]. The paramagnetic material usually accumulates in hemorrhagic infarction. So, hemoglobin shows false low signal intensity on T2\*-weighted image [70]. In case of death and rupture of transplanted cells, targeted SPIO nanoparticles can be trapped in surrounding tissue cells or reticuloendothelial cells. Subsequently, SPION are redistributed, deposited, or differentiated in extracellular environment to generate false positive signal. In author's opinion, direct iron oxide labeling is only suitable for short-term stem cell tracking in vivo or in vitro experiments. Other reason of false negative signal can be partial volume effects or low concentrations of cells in one imaging voxel. After every cell division, intracellular iron content remains half. So, every cell division evidences gradual reduction in cell detection sensitivity. The said fact was reported as MRI nonvisible heart cells after 6 weeks post-transplant stem cell administrated to the heart [71]. Despite these limitations and shortcomings, paramagnetic/superparamagnetic iron oxide particles are still highly

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Reporter gene labeling is other method based on fusion of an MRI reporter gene to a target gene in stem cells. In transfection of a target stem cell, genes are incorporated into the cellular DNA via transgenic methods. These products of reporter genes are expressed in living stem cells and produces reporter gene expression as indirectly MRI visible in vivo. Transgenic gene labeling methods are highly valuable in long-term studies of labeled stem cell survival, proliferation, and differentiation in vivo. The MRI reporter gene expression can make two products of its expression in stem cells: (1) Intracellular enzymes including β-galactosidase, cytosine deaminase, creatinine kinase, tyrosinase, and arginine kinase [72]; (2) Ferritin or transferrin receptors [73]. Recently, a MRI reporter gene (a ferritin receptor) has emerged as a choice of robust contrast. Excessive expression of ferritin can increase iron uptake. Inside cells, redistribution of intracellular iron enhances transverse relaxation rates and reduces T2 relaxation constants. Recently, adenovirus-ferritin reporter gene injection into murine corpus striatum

generated robust contrast on T2 and T2\*-weighted imaging within 5–39 days [74].

Clearly MRI reporter gene imaging is still a choice, but it cannot rule out the potential damage to cell proliferation and differentiation. Still open issues are the sources and safety of cells, issues relating to gene mutation and sensitivity [75]. Now, MR microimaging technology has advanced with available 900 MHz magnetic fields to visualize cardiovascular myofibrillar territories up to 30 micrometer resolution using SPION and SPOIT nanoparticle-enhanced relaxation susceptibility signal intensities of revived cardiac muscles enough to decipher the insight of stem cells as shown in **Figure 5**. Author developed mice beating heart microimages using antibody-coated nanoparticles to visualize cardiac muscle orientation angles as fingerprints of cardiac revival and rejuvenation [76]. It can be easily noticed that dying heart left ventricle wall clearly shows the damage sites with clear muscle mass with altered orientation

• Iron oxide particle surfaces can be modified by specific target receptors such as vascular cell adhesion molecule-1 and membrane mucin A5. These nanoparticles target specific tissues or organs, but the presence of specific target receptors limits the application of modified nanoparticles in cell tracking [66, 67].

#### **5.4. Magnetoelectric perforation method**

It increases the efficiency of nanoparticle endocytosis. Toxicity testing of mesenchymal stem cells, neural stem cells and adipose cells in vitro all indicate safer use of magnetoelectric perforation because of less cell incubation time and effective safer SPION contrast agent to target cells approved by the FDA [68, 69]. Still, stem cell transplantation biological safety considerations need attention and further research.

#### **5.5. Biosafety of iron oxide particle labeling on stem cells**

Cell labeling with iron oxide requires intensive toxicity evaluation tests for every protocol and characterization of cell type before translating them in clinical application. Feridex®, Resovist®, and Endorem® are FDA-approved agents. These agents in stem cells are cleared by the reticuloendothelial system. Peripheral blood mononuclear cells labeled in vitro with Ferumoxide® upon administrated these cells through intravenous injection in organs showed localized T2\*-weighted images and R2\* maps of cell migration at the tissue inflammation damage areas [65]. However, extensive experiments are needed to verify the bio-safety of paramagnetic SPION contrast agents.

#### **5.6. Sensitivity of in vivo MRI detection of labeled stem cells**

The intracellular iron distribution in stem cells influences greatly the MRI detection signal from labeled stem cells. Several inherent factors are determinant of image quality such as MRI sequence selection, spatial resolution, magnetic field intensity, and surrounding stem cell or cardiac tissue heterogeneity to affect the molecule sensitive signal. Known factors are: (1) Higher intracellular iron content in cells shortens the relaxation time; (2) The T<sup>2</sup> \*-weighted image is highly sensitive for iron oxide particle labeling load; (3) Field inhomogeneity and surrounding tissues; and (4) MRI sensitivity can reach 3000 times that of T<sup>1</sup> weighting or 60 times that of T<sup>2</sup> weighting due to iron oxide-induced dephasing effect [54]. To nullify the ironinduced MRI signal sensitivity, specific techniques are chosen. T<sup>2</sup> \* sequence or steady-state free precession (SSFP) is a choice to detect SPION-labeled cells. However, the T<sup>2</sup> \* sequence gets artifact by intracellular magnetic field inhomogeneity and interference of the surrounding normal tissues at high magnetic field. Fast 3D gradient echo (GE) sequences balance this effect of T<sup>2</sup> \* sensitivity, spatial resolution within imaging time. At the present time, the best choice is **g**radient **e**cho **a**cquisition for **s**uperparamagnetic particles with **p**ositive contrast (GRASP), to create a positive contrast of SPION free from T<sup>2</sup> \* artifacts and high sensitive and specific hyperintense signal of cell tracking even for smaller imaging voxel size in the high field MRI [55].

#### **5.7. Limitations of tracking SPION labeling stem cells**

• Monoclonal antibodies of pancreatic cancer specific antigen (PAP2a) fused with dextranmodified SPIONs show antigen-antibody reaction to target the iron oxide particles in pan-

• Iron oxide particle surfaces can be modified by specific target receptors such as vascular cell adhesion molecule-1 and membrane mucin A5. These nanoparticles target specific tissues or organs, but the presence of specific target receptors limits the application of modi-

It increases the efficiency of nanoparticle endocytosis. Toxicity testing of mesenchymal stem cells, neural stem cells and adipose cells in vitro all indicate safer use of magnetoelectric perforation because of less cell incubation time and effective safer SPION contrast agent to target cells approved by the FDA [68, 69]. Still, stem cell transplantation biological safety consider-

Cell labeling with iron oxide requires intensive toxicity evaluation tests for every protocol and characterization of cell type before translating them in clinical application. Feridex®, Resovist®, and Endorem® are FDA-approved agents. These agents in stem cells are cleared by the reticuloendothelial system. Peripheral blood mononuclear cells labeled in vitro with Ferumoxide® upon administrated these cells through intravenous injection in organs showed localized T2\*-weighted images and R2\* maps of cell migration at the tissue inflammation damage areas [65]. However, extensive experiments are needed to verify the bio-safety of paramagnetic SPION contrast agents.

The intracellular iron distribution in stem cells influences greatly the MRI detection signal from labeled stem cells. Several inherent factors are determinant of image quality such as MRI sequence selection, spatial resolution, magnetic field intensity, and surrounding stem cell or cardiac tissue heterogeneity to affect the molecule sensitive signal. Known factors are: (1)

image is highly sensitive for iron oxide particle labeling load; (3) Field inhomogeneity and

gets artifact by intracellular magnetic field inhomogeneity and interference of the surrounding normal tissues at high magnetic field. Fast 3D gradient echo (GE) sequences balance this effect

intense signal of cell tracking even for smaller imaging voxel size in the high field MRI [55].

\* sensitivity, spatial resolution within imaging time. At the present time, the best choice is **g**radient **e**cho **a**cquisition for **s**uperparamagnetic particles with **p**ositive contrast (GRASP), to

weighting due to iron oxide-induced dephasing effect [54]. To nullify the iron-

\*-weighted

\* sequence

weighting or 60

\* sequence or steady-state

\* artifacts and high sensitive and specific hyper-

Higher intracellular iron content in cells shortens the relaxation time; (2) The T<sup>2</sup>

free precession (SSFP) is a choice to detect SPION-labeled cells. However, the T<sup>2</sup>

surrounding tissues; and (4) MRI sensitivity can reach 3000 times that of T<sup>1</sup>

induced MRI signal sensitivity, specific techniques are chosen. T<sup>2</sup>

create a positive contrast of SPION free from T<sup>2</sup>

creatic cancer cells and promote the receptor-mediated SPIO endocytosis [65].

fied nanoparticles in cell tracking [66, 67].

**5.4. Magnetoelectric perforation method**

126 Stem Cells in Clinical Practice and Tissue Engineering

ations need attention and further research.

times that of T<sup>2</sup>

of T<sup>2</sup>

**5.5. Biosafety of iron oxide particle labeling on stem cells**

**5.6. Sensitivity of in vivo MRI detection of labeled stem cells**

SPION particle cell tracking method for cell labeling has some shortcomings. The MRI signal in preclinical or clinical studies is usually generated from surrounding tissue areas of noninterest cardiopulmonary junction [56]. The paramagnetic material usually accumulates in hemorrhagic infarction. So, hemoglobin shows false low signal intensity on T2\*-weighted image [70]. In case of death and rupture of transplanted cells, targeted SPIO nanoparticles can be trapped in surrounding tissue cells or reticuloendothelial cells. Subsequently, SPION are redistributed, deposited, or differentiated in extracellular environment to generate false positive signal. In author's opinion, direct iron oxide labeling is only suitable for short-term stem cell tracking in vivo or in vitro experiments. Other reason of false negative signal can be partial volume effects or low concentrations of cells in one imaging voxel. After every cell division, intracellular iron content remains half. So, every cell division evidences gradual reduction in cell detection sensitivity. The said fact was reported as MRI nonvisible heart cells after 6 weeks post-transplant stem cell administrated to the heart [71]. Despite these limitations and shortcomings, paramagnetic/superparamagnetic iron oxide particles are still highly popular in the field of stem cell tracking because of their high sensitivity.

#### **5.8. Reporter gene labeling in stem cells**

Reporter gene labeling is other method based on fusion of an MRI reporter gene to a target gene in stem cells. In transfection of a target stem cell, genes are incorporated into the cellular DNA via transgenic methods. These products of reporter genes are expressed in living stem cells and produces reporter gene expression as indirectly MRI visible in vivo. Transgenic gene labeling methods are highly valuable in long-term studies of labeled stem cell survival, proliferation, and differentiation in vivo. The MRI reporter gene expression can make two products of its expression in stem cells: (1) Intracellular enzymes including β-galactosidase, cytosine deaminase, creatinine kinase, tyrosinase, and arginine kinase [72]; (2) Ferritin or transferrin receptors [73]. Recently, a MRI reporter gene (a ferritin receptor) has emerged as a choice of robust contrast. Excessive expression of ferritin can increase iron uptake. Inside cells, redistribution of intracellular iron enhances transverse relaxation rates and reduces T2 relaxation constants. Recently, adenovirus-ferritin reporter gene injection into murine corpus striatum generated robust contrast on T2 and T2\*-weighted imaging within 5–39 days [74].

Clearly MRI reporter gene imaging is still a choice, but it cannot rule out the potential damage to cell proliferation and differentiation. Still open issues are the sources and safety of cells, issues relating to gene mutation and sensitivity [75]. Now, MR microimaging technology has advanced with available 900 MHz magnetic fields to visualize cardiovascular myofibrillar territories up to 30 micrometer resolution using SPION and SPOIT nanoparticle-enhanced relaxation susceptibility signal intensities of revived cardiac muscles enough to decipher the insight of stem cells as shown in **Figure 5**. Author developed mice beating heart microimages using antibody-coated nanoparticles to visualize cardiac muscle orientation angles as fingerprints of cardiac revival and rejuvenation [76]. It can be easily noticed that dying heart left ventricle wall clearly shows the damage sites with clear muscle mass with altered orientation of angle proportional to degree of distortion.

valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease to define the effectiveness and underlying mechanisms of cardiac cell therapy. The light-emitting capability of BLI illustrates the insights of cardiac regeneration [80]. Recently, survival kinetics of induced pluripotent stem cell and engraftment of viable cells was monitored by BLI imaging by visualizing the retention of bioluminescent agents in adult stem cells as shown in **Figure 6** to monitor stem cells [34]. Efforts are still continuing for regenerating the heart and using myocardial stem cells in cardiovascular system in treatment of heart disease or remodeling [81]. Now, new trend of noninvasive in vivo MR imaging with spectroscopy is emerging to visualize cardiac muscle metabolites [81] and products of gene expression or imaging reporter gene induced inhomogeneity signal

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Future research may focus on conversion of adult cells into iPS cells, and conversion of these iPS cells to relevant cell types to treat individual diseases. In near future, multimodal single platform bioluminescent/NIR/FRET optical cum MRI/CT/PET microimaging techniques will emerge to track the pluripotent stem cell sensitive superior detection methods by monitoring the distribution of molecular events in differentiating myocardial progenitor cells in less time. It remains to see in coming years if differentiating stem cells remain safe and stem cells are not affected by radionuclide, chelators, contrast agents, and electromagnetic radiations used to image these cells. To expose these stems cells in different preservative media solutions for storage without any effect on their capability to remodeling is also a challenge in tissue engineering art. Before transplantation and regrafting, it needs thorough investigation of perfect autograft and metabolic compatibility, myocardial contractility to remain viable longer. Larger double blinded placebo-controlled clinical trials are needed on trans-aortic or trans-septal approach to reach different zones of endocardial necrosis. In cases of intramyocardial or epicardial necrosis, epicardial approach should be compared with endocardial one. Brachial can be an alternate option for patients who have peripheral vascular disease with difficult femoral approach*.* Safer delivery of stem cells to the heart opens vista of transplantation of stem cells as tissue-engineered constructs. Throughout life, every person experiences many injuries and recovers with time spontaneously by wound healing, organ recovery, or repair mechanisms without even realizing the past injuries in the first place. In this repair and wound healing process, proliferation of existing stem cells makes an individual capable of repairing or restoring the injured tissue(s). In fact, these pluripotent stem cells contain the genetic fingerprint or molecule metabolic blueprint as memory of tissue origin how a particular tissue cell was assembled from biomolecules and functionalized into physiochemical units of organs constructed to begin with from embryonic progenitor cells. If these pluripotent stem cells are maintained artificially in physiological cultures, rejuvenation potential of stem cells maintains all properties of biotransforming and differentiating into organ cells. This potential offers an excellent opportunity of clinical applications. In fact, these restorative potentials in stem cells are possible due to simultaneous multiple functions of stem cells, such as self-renewal, multipotency, and paracrine functions. Of mention, paracrine secretion releases colony-stimulating factors,

peaks from regenerating stem cells [82].

**6. Future perspectives**

**Figure 5.** Monitoring cardiac cells by 900 MHz MR microimaging on left panels. Axial image shows details of muscle fibers in ventricle wall (shown as arrows). Cardiac muscle fibers are shown on right panels with superparamagnetic iron oxide troponin nanoparticles (shown in circle) to indicate angles of muscle fiber orientation on right panels. On top right, change in muscle fiber orientation angle is shown before and after infarction (shown as vector directions).

#### **5.9. Regenerating stem cell** *in vivo* **dual optical imaging**

Cellular engraftment may be monitored by reporter gene construct (fluc-mrfp-ttk) visualization by optical methods such as bioluminescence (BLI), chemiluminescence combined with MRI, PET, fusion multimodal imaging (FMI), near infra-red (NIR), and radionuclide methods [77]. The dual-modality imaging has unique strength to monitor cell delivery, survival status, graft morphology, and impact on post-MI remodeling on same platform in less time [78]. Recently, application of BLI for tracking transplanted stem cells was reviewed on the association of stem cell viability with the therapeutic efficacy of stem cell evaluated in preclinical disease models of vascular disease [79]. Reporter gene technology with BLI provides

**Figure 6.** Noninvasive bioluminescence (BLI) monitoring of cardiac differentiation in the experimental model of acute myocardial infarction shows BLI images showing decrease in RLuc by CMV promoter and increase in PLuc by cardiacspecific cTnI promoter in adipose tissue–derived progenitor cells after myocardial implantation. BLI can finely quantify cardiac regeneration degree relative to the number of surviving cells under ischemic conditions. See Ref. [80].

valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease to define the effectiveness and underlying mechanisms of cardiac cell therapy. The light-emitting capability of BLI illustrates the insights of cardiac regeneration [80]. Recently, survival kinetics of induced pluripotent stem cell and engraftment of viable cells was monitored by BLI imaging by visualizing the retention of bioluminescent agents in adult stem cells as shown in **Figure 6** to monitor stem cells [34]. Efforts are still continuing for regenerating the heart and using myocardial stem cells in cardiovascular system in treatment of heart disease or remodeling [81]. Now, new trend of noninvasive in vivo MR imaging with spectroscopy is emerging to visualize cardiac muscle metabolites [81] and products of gene expression or imaging reporter gene induced inhomogeneity signal peaks from regenerating stem cells [82].

### **6. Future perspectives**

**5.9. Regenerating stem cell** *in vivo* **dual optical imaging**

128 Stem Cells in Clinical Practice and Tissue Engineering

Cellular engraftment may be monitored by reporter gene construct (fluc-mrfp-ttk) visualization by optical methods such as bioluminescence (BLI), chemiluminescence combined with MRI, PET, fusion multimodal imaging (FMI), near infra-red (NIR), and radionuclide methods [77]. The dual-modality imaging has unique strength to monitor cell delivery, survival status, graft morphology, and impact on post-MI remodeling on same platform in less time [78]. Recently, application of BLI for tracking transplanted stem cells was reviewed on the association of stem cell viability with the therapeutic efficacy of stem cell evaluated in preclinical disease models of vascular disease [79]. Reporter gene technology with BLI provides

**Figure 6.** Noninvasive bioluminescence (BLI) monitoring of cardiac differentiation in the experimental model of acute myocardial infarction shows BLI images showing decrease in RLuc by CMV promoter and increase in PLuc by cardiacspecific cTnI promoter in adipose tissue–derived progenitor cells after myocardial implantation. BLI can finely quantify cardiac regeneration degree relative to the number of surviving cells under ischemic conditions. See Ref. [80].

**Figure 5.** Monitoring cardiac cells by 900 MHz MR microimaging on left panels. Axial image shows details of muscle fibers in ventricle wall (shown as arrows). Cardiac muscle fibers are shown on right panels with superparamagnetic iron oxide troponin nanoparticles (shown in circle) to indicate angles of muscle fiber orientation on right panels. On top right,

change in muscle fiber orientation angle is shown before and after infarction (shown as vector directions).

Future research may focus on conversion of adult cells into iPS cells, and conversion of these iPS cells to relevant cell types to treat individual diseases. In near future, multimodal single platform bioluminescent/NIR/FRET optical cum MRI/CT/PET microimaging techniques will emerge to track the pluripotent stem cell sensitive superior detection methods by monitoring the distribution of molecular events in differentiating myocardial progenitor cells in less time. It remains to see in coming years if differentiating stem cells remain safe and stem cells are not affected by radionuclide, chelators, contrast agents, and electromagnetic radiations used to image these cells. To expose these stems cells in different preservative media solutions for storage without any effect on their capability to remodeling is also a challenge in tissue engineering art. Before transplantation and regrafting, it needs thorough investigation of perfect autograft and metabolic compatibility, myocardial contractility to remain viable longer. Larger double blinded placebo-controlled clinical trials are needed on trans-aortic or trans-septal approach to reach different zones of endocardial necrosis. In cases of intramyocardial or epicardial necrosis, epicardial approach should be compared with endocardial one. Brachial can be an alternate option for patients who have peripheral vascular disease with difficult femoral approach*.* Safer delivery of stem cells to the heart opens vista of transplantation of stem cells as tissue-engineered constructs.

Throughout life, every person experiences many injuries and recovers with time spontaneously by wound healing, organ recovery, or repair mechanisms without even realizing the past injuries in the first place. In this repair and wound healing process, proliferation of existing stem cells makes an individual capable of repairing or restoring the injured tissue(s). In fact, these pluripotent stem cells contain the genetic fingerprint or molecule metabolic blueprint as memory of tissue origin how a particular tissue cell was assembled from biomolecules and functionalized into physiochemical units of organs constructed to begin with from embryonic progenitor cells. If these pluripotent stem cells are maintained artificially in physiological cultures, rejuvenation potential of stem cells maintains all properties of biotransforming and differentiating into organ cells. This potential offers an excellent opportunity of clinical applications. In fact, these restorative potentials in stem cells are possible due to simultaneous multiple functions of stem cells, such as self-renewal, multipotency, and paracrine functions. Of mention, paracrine secretion releases colony-stimulating factors, growth factors, regulatory energy molecules, and stimulatory cytokines from a number of retained stem cells during regenerative processes at tissue sites as shown in recent clinical trials in **Table 3**. These secretary molecules lead to further mobilization of endogenous progenitor cells. We do not understand the complete sequence of underlying mechanisms of stem cell during regeneration and cardiac healing, even though everyone experiences the benefits of cardiac rejuvenation even without complete knowledge of origin of electrophysiology of heart, cardiomyocyte functions, and mechanism of molecular events.

The embryonic stem cells have excellent capacity to differentiate into virtually any type of tissue cells [83]. Presently, investigators and government agencies have intensified the detailed search for a similar cell lineage or stem cell rejuvenate database in adults [84]. However, many challenges remain to understand how these adult stem cells over-ride the complex tasks (failed heart in to beating heart again) to take up residence quickly when placed in just the right place to gain control and restore or correct the necessary cardiomyocyte shape to assume paracrine functions to perform their multiple plasticity functions in a complex different cellular environments (rejuvenation). Other major challenge is perfect retention of these cells after implantation via intracoronary, intramyocardial, and retrograde coronary sinus approach. In fact, a significant percent of stem cells leaves the heart soon after implantation and stem cell administration before they stick at damage site [84]. So, the clinical ramifications may be significant but they are limited. One fact is clear that remarkable universal nature of stem cells offers the exciting possibility of a universal stem cell transformation capability into any tissue cell or organ that can circulate throughout the body and reside wherever needed to promote regeneration or repair of local tissue if retention of stem cells is good. These stem cells have multiple functions and behave proangiogenic and proparacaine, thereby stem cells may consume or produce potentially detrimental substances as indicated in recent clinical trials shown in **Figure 7**, while stem cells may also survive in nontarget organs [85].

From clinical practice standpoint, the major hurdles to the clinical application and translational research in regard to adult stem cells are the limited small number of stem cells isolated from any adult tissue with successful propagation and harvesting of multipotent adult stem cells [86]. Other hurdle is the development of perfect "stem cell cocktails" to optimize the proliferation and of adult stem cells and differentiation in timely manner [30]. These hurdles indicate the urgent attention on supervised expansion of adult stem cells in cultures uniformly keeping stem cell intrinsic properties intact may be the answer to stable retention [87]. Although extensive cultures of human adult cells may suddenly change the intrinsic properties of stem cells in vivo [88], putting them unfit rendering them with no restoring capability to repair or reverse the injured or diseased tissue in prospective heart failure patients.

Author offers his opinion that cardiac stem cell therapy in future will have an acceptable wide spectrum of preclinical and double blinded placebo-controlled clinical trials on trans-aortic or trans-septal approaches solving the issue of epicardial or endocardial necrosis in cardiovascular regenerative medicine as shown in **Table 3** with emphasis on intracoronary and retrograde coronary methods or possibly combined with 3D scaffold biomatrices delivery. In development of engineered and constructed scaffold, intensive investigations will introduce new rejuvenator secretory molecules in remodeling and metabolic regulation to provide insight of right choice and optimization for best cardiac repair. Researchers may explore

more options of differentiated stem cell remodeling in addition to the engineered constructs, rejuvenative molecules and regenerative metabolic pathways highlighted in clinical trials shown in **Figure 7**. What secretory molecules and metabolic regulatory events are common in

**Figure 7.** Different secretory molecules are shown either synthesized or released from damaged myocardium from altered cardiomyocyte metabolic pathways (shown in left panel). The process of rejuvenation in stem cells is shown to correct the metabolic events (see at top on right) to lead repair, rejuvenation and restoration of cardiomyocyte viability

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with improved functions by remodeling in metabolic steps (shown in bottom at right).

growth factors, regulatory energy molecules, and stimulatory cytokines from a number of retained stem cells during regenerative processes at tissue sites as shown in recent clinical trials in **Table 3**. These secretary molecules lead to further mobilization of endogenous progenitor cells. We do not understand the complete sequence of underlying mechanisms of stem cell during regeneration and cardiac healing, even though everyone experiences the benefits of cardiac rejuvenation even without complete knowledge of origin of electrophysiology of

The embryonic stem cells have excellent capacity to differentiate into virtually any type of tissue cells [83]. Presently, investigators and government agencies have intensified the detailed search for a similar cell lineage or stem cell rejuvenate database in adults [84]. However, many challenges remain to understand how these adult stem cells over-ride the complex tasks (failed heart in to beating heart again) to take up residence quickly when placed in just the right place to gain control and restore or correct the necessary cardiomyocyte shape to assume paracrine functions to perform their multiple plasticity functions in a complex different cellular environments (rejuvenation). Other major challenge is perfect retention of these cells after implantation via intracoronary, intramyocardial, and retrograde coronary sinus approach. In fact, a significant percent of stem cells leaves the heart soon after implantation and stem cell administration before they stick at damage site [84]. So, the clinical ramifications may be significant but they are limited. One fact is clear that remarkable universal nature of stem cells offers the exciting possibility of a universal stem cell transformation capability into any tissue cell or organ that can circulate throughout the body and reside wherever needed to promote regeneration or repair of local tissue if retention of stem cells is good. These stem cells have multiple functions and behave proangiogenic and proparacaine, thereby stem cells may consume or produce potentially detrimental substances as indicated in recent clinical tri-

als shown in **Figure 7**, while stem cells may also survive in nontarget organs [85].

to repair or reverse the injured or diseased tissue in prospective heart failure patients.

Author offers his opinion that cardiac stem cell therapy in future will have an acceptable wide spectrum of preclinical and double blinded placebo-controlled clinical trials on trans-aortic or trans-septal approaches solving the issue of epicardial or endocardial necrosis in cardiovascular regenerative medicine as shown in **Table 3** with emphasis on intracoronary and retrograde coronary methods or possibly combined with 3D scaffold biomatrices delivery. In development of engineered and constructed scaffold, intensive investigations will introduce new rejuvenator secretory molecules in remodeling and metabolic regulation to provide insight of right choice and optimization for best cardiac repair. Researchers may explore

From clinical practice standpoint, the major hurdles to the clinical application and translational research in regard to adult stem cells are the limited small number of stem cells isolated from any adult tissue with successful propagation and harvesting of multipotent adult stem cells [86]. Other hurdle is the development of perfect "stem cell cocktails" to optimize the proliferation and of adult stem cells and differentiation in timely manner [30]. These hurdles indicate the urgent attention on supervised expansion of adult stem cells in cultures uniformly keeping stem cell intrinsic properties intact may be the answer to stable retention [87]. Although extensive cultures of human adult cells may suddenly change the intrinsic properties of stem cells in vivo [88], putting them unfit rendering them with no restoring capability

heart, cardiomyocyte functions, and mechanism of molecular events.

130 Stem Cells in Clinical Practice and Tissue Engineering

**Figure 7.** Different secretory molecules are shown either synthesized or released from damaged myocardium from altered cardiomyocyte metabolic pathways (shown in left panel). The process of rejuvenation in stem cells is shown to correct the metabolic events (see at top on right) to lead repair, rejuvenation and restoration of cardiomyocyte viability with improved functions by remodeling in metabolic steps (shown in bottom at right).

more options of differentiated stem cell remodeling in addition to the engineered constructs, rejuvenative molecules and regenerative metabolic pathways highlighted in clinical trials shown in **Figure 7**. What secretory molecules and metabolic regulatory events are common in differentiated stem cell remodeling?. During remodeling, substrates are transported across the extracellular membrane into the cytosol and are metabolized in various ways. For oxidation, the respective metabolic intermediates [e.g., pyruvate or acylcoenzyme A (CoA)] are transported across the inner mitochondrial membrane by specific transport systems. Once inside the mitochondrion, substrates are oxidized or carboxylated (anaplerosis) and fed into the Krebs cycle for the generation of reducing equivalents [reduced nicotinamide adenine dinucleotide (NADH)2 ; reduced flavin adenine dinucleotide (FADH)] and GTP. The reducing equivalents are used by the electron transport chain to generate a proton gradient, which in turn is used for the production of ATP. This principal functionality can be recovered in various ways during reverting heart failure (HF), thereby regaining ATP production or improving cellular function in other many ways. Researchers may explore more molecular options of remodeling in addition to the molecules and regenerative metabolic pathways shown in **Figure 7**.

imaging or restorative monitoring reporter of rejuvenating stem cell genes to evaluate success of transplantation and cardiac repair. On the other side of coin, researchers are continuously developing new real-time physiological cum functional MRSI options to explore new stem cell molecular probes and smart MRS imaging sequences with improved MRI sensitive specific stem cell differentiation and rejuvenating detection by targeting energy metabolites, myocardial viability, and vital physiochemical molecules. Noninvasive monitoring is necessary and bioluminescence or other radionuclide methods may be alarming because the potential biological damage caused by radionuclide exposed reporter genes and bioluminescence induced immune responses is concern in differentiating stem cells. Seriously, all these issues need research to minimize artifacts within safe limits. With the help of stem cell imaging and monitoring, transplantation of stem cells sooner or later will be optimized for the effective

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The author acknowledges his training at NSF sponsored NHMFL, Florida State University Users' scholar grant, during his experiments in stem cell research Lab and 21 Tesla MR micro-

[1] Go AS, Lloyd-Jones DM, et al. Heart disease and stroke statistics—2012 update: A report

[2] Assmus B, Schächinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation.

[3] Kang HJ, Kim HS, Zhang SY, et al. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: The

MAGIC cell randomised clinical trial. Lancet. 2004;**363**(9411):751-756. [PubMed]

from the American Heart Association. Circulation. 2012;**125**(1):e2-e220

long-lasting therapy of myocardial infarction and heart failure on some day.

**Acknowledgements**

imager facility.

**Author details**

Rakesh Sharma¹,²\*

**References**

\*Address all correspondence to: rksz2009@gmail.com 1 Innovations and Solutions Inc., Tallahassee, FL, USA

2002;**106**(24):3009-3017. [PubMed]

2 Florida State University Research Foundation, Tallahassee, FL, USA

Mainly two types of cardiovascular tissue biomaterials synthetic (polymer, ceramic, or metals) and biologic (cell-based, extracellular matrix-based, whole tissue) and hybrid biomaterials will be available. Advanced therapy medicinal products (ATMP), cardiomyocytes, ECM hydrogels and scaffolds, urinary bladder matrix (UBM) scaffolds, glycosaminoglycans (GAGs), collagen, fibronectin and laminin matrix, endothelial cells (ECs) and vascular smooth muscle cells(VSMCs), poly-tetra fluoro ethylene (PTFE), cardiac patches, cell-seeded matrices, and pericytes seeding with biodegradable tissue engineering-based graft (PLLA/PCL, designed to be degraded in 3–5 years) are currently used methods for dynamic seeding with total BMCs or selected bone marrow aspirate mononuclear cells (BMMNCs). Electrospinning is routine to design scaffolds. For heart regeneration, cell delivery vehicle is implanted using smooth muscle cells (SMC), fibroblasts, endothelial progenitor cells, embryonic CD 34 stem cells, BM cells, tissue-engineered vascular grafts are becoming promised biomaterials. Vascular CorMatrix® patch, vascular grafts made of PG/PGA, PCL/PLA polymers offer clinical use [107–109]. The stem cell treatment will have a universal role in reversing the aging process, although a natural phenomenon. Naturally with the aging process, there is a continuous decline in stem cell number and their viability or physiochemical cardiac capability with time. Due to these facts, aging and heart diseases are interlinked and advancing age promotes organ diseases. Therefore, restorative repairing capability of stem cells may provide a renewable life, and a "fountain of youth" as evidenced by jelly fish rejuvenation.

### **7. Conclusion**

The in vivo imaging techniques are useful in dynamic monitoring of cardiac stem cell therapy following myocardial infarction. Choice of stem cells and mode of delivery are very crucial in getting successful stem cell therapy positive outcome. Cardiovascular remodeling evaluation by MRI has merits because it is safe, sensitive, lacks radiation, provides good resolution, generates a real-time events' blueprint or first-hand information of myocardial viability with functional information of cardiac territories and their physiochemical changes in cardiac functions during stem cell rejuvenating process and after myocardial repair. Present time, ultrahigh magnetic field CMR possibly has preclinical prospects as in vivo noninvasive molecular imaging or restorative monitoring reporter of rejuvenating stem cell genes to evaluate success of transplantation and cardiac repair. On the other side of coin, researchers are continuously developing new real-time physiological cum functional MRSI options to explore new stem cell molecular probes and smart MRS imaging sequences with improved MRI sensitive specific stem cell differentiation and rejuvenating detection by targeting energy metabolites, myocardial viability, and vital physiochemical molecules. Noninvasive monitoring is necessary and bioluminescence or other radionuclide methods may be alarming because the potential biological damage caused by radionuclide exposed reporter genes and bioluminescence induced immune responses is concern in differentiating stem cells. Seriously, all these issues need research to minimize artifacts within safe limits. With the help of stem cell imaging and monitoring, transplantation of stem cells sooner or later will be optimized for the effective long-lasting therapy of myocardial infarction and heart failure on some day.

### **Acknowledgements**

differentiated stem cell remodeling?. During remodeling, substrates are transported across the extracellular membrane into the cytosol and are metabolized in various ways. For oxidation, the respective metabolic intermediates [e.g., pyruvate or acylcoenzyme A (CoA)] are transported across the inner mitochondrial membrane by specific transport systems. Once inside the mitochondrion, substrates are oxidized or carboxylated (anaplerosis) and fed into the Krebs cycle for the generation of reducing equivalents [reduced nicotinamide adenine dinucleotide

are used by the electron transport chain to generate a proton gradient, which in turn is used for the production of ATP. This principal functionality can be recovered in various ways during reverting heart failure (HF), thereby regaining ATP production or improving cellular function in other many ways. Researchers may explore more molecular options of remodeling in addi-

Mainly two types of cardiovascular tissue biomaterials synthetic (polymer, ceramic, or metals) and biologic (cell-based, extracellular matrix-based, whole tissue) and hybrid biomaterials will be available. Advanced therapy medicinal products (ATMP), cardiomyocytes, ECM hydrogels and scaffolds, urinary bladder matrix (UBM) scaffolds, glycosaminoglycans (GAGs), collagen, fibronectin and laminin matrix, endothelial cells (ECs) and vascular smooth muscle cells(VSMCs), poly-tetra fluoro ethylene (PTFE), cardiac patches, cell-seeded matrices, and pericytes seeding with biodegradable tissue engineering-based graft (PLLA/PCL, designed to be degraded in 3–5 years) are currently used methods for dynamic seeding with total BMCs or selected bone marrow aspirate mononuclear cells (BMMNCs). Electrospinning is routine to design scaffolds. For heart regeneration, cell delivery vehicle is implanted using smooth muscle cells (SMC), fibroblasts, endothelial progenitor cells, embryonic CD 34 stem cells, BM cells, tissue-engineered vascular grafts are becoming promised biomaterials. Vascular CorMatrix® patch, vascular grafts made of PG/PGA, PCL/PLA polymers offer clinical use [107–109]. The stem cell treatment will have a universal role in reversing the aging process, although a natural phenomenon. Naturally with the aging process, there is a continuous decline in stem cell number and their viability or physiochemical cardiac capability with time. Due to these facts, aging and heart diseases are interlinked and advancing age promotes organ diseases. Therefore, restorative repairing capability of stem cells may provide a renew-

tion to the molecules and regenerative metabolic pathways shown in **Figure 7**.

able life, and a "fountain of youth" as evidenced by jelly fish rejuvenation.

The in vivo imaging techniques are useful in dynamic monitoring of cardiac stem cell therapy following myocardial infarction. Choice of stem cells and mode of delivery are very crucial in getting successful stem cell therapy positive outcome. Cardiovascular remodeling evaluation by MRI has merits because it is safe, sensitive, lacks radiation, provides good resolution, generates a real-time events' blueprint or first-hand information of myocardial viability with functional information of cardiac territories and their physiochemical changes in cardiac functions during stem cell rejuvenating process and after myocardial repair. Present time, ultrahigh magnetic field CMR possibly has preclinical prospects as in vivo noninvasive molecular

; reduced flavin adenine dinucleotide (FADH)] and GTP. The reducing equivalents

(NADH)2

132 Stem Cells in Clinical Practice and Tissue Engineering

**7. Conclusion**

The author acknowledges his training at NSF sponsored NHMFL, Florida State University Users' scholar grant, during his experiments in stem cell research Lab and 21 Tesla MR microimager facility.

### **Author details**

Rakesh Sharma¹,²\*


### **References**


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**Chapter 7**

**Provisional chapter**

**Impact of the Donor KIR Genotype on the Clinical**

**Outcome of Hematopoietic Stem Cell Unrelated** 

**Impact of the Donor KIR Genotype on the Clinical** 

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Keywords:** UDHSCT, NK cells, donor KIR B status, AML, GvL effect

In recent years, the anti-leukemic potential of Natural Killer (NK) cells and their role in hematologic malignancies and in Hematopoietic Stem Cell Transplants (HSCT) has attracted greater interest and a recent study by Cooley S. et al. showed a better clinical outcome when patients with Acute Myeloid Leukemia received a transplant from unrelated Group B KIR haplotypes donors. As a consequence of these results, an algorithm called "KIR B-content score" was formulated. The aim of our research is a retrospective analysis of HSC unrelated transplants performed in our center to analyze the effect of the donor KIR B status on the clinical-outcome. Our results showed a better overall survival-rate in the AML recipients, HLA mismatched with the donor, when the donor KIR B content status is Best/Better (37% vs 18% at three years P=0,028). Moreover, we observed that AML recipients, whose Donors KIR B status was Best/Better, had more incidence of aGvHD grade I and II than patients whose Donors KIR B status was Neutral (70% vs 26%) and also a lower rate of relapse (36% vs 58%) and a better Disease Free Survival-rate (58% vs 38% at three years P=0,1) because of

**Outcome of Hematopoietic Stem Cell Unrelated**

**Transplants: A Single Center Experience**

**Transplants: A Single Center Experience**

Rosalba Bavetta, Serena Mistretta, Maria Igea Vega,

Floriana Bruno, Emanuela Collura, Giovanna Regina,

Francesco Ingrassia, Valentina Cappuzzo,

Maria Igea Vega, Paola Bruna Affaticati,

Francesco Ingrassia, Valentina Cappuzzo,

Valentina Randazzo, Alessandro Indovina, Felicia Farina and Raimondo Marcenò

Valentina Randazzo, Alessandro Indovina, Felicia Farina and Raimondo Marcenò

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Paola Bruna Affaticati, Maria Blando,

Emanuela Collura, Giovanna Regina,

Rosalba Bavetta, Serena Mistretta,

Maria Blando, Floriana Bruno,

http://dx.doi.org/10.5772/67047

a better GvL effect.

**Abstract**


**Provisional chapter**

### **Impact of the Donor KIR Genotype on the Clinical Outcome of Hematopoietic Stem Cell Unrelated Transplants: A Single Center Experience Outcome of Hematopoietic Stem Cell Unrelated Transplants: A Single Center Experience**

**Impact of the Donor KIR Genotype on the Clinical** 

Francesco Ingrassia, Valentina Cappuzzo, Rosalba Bavetta, Serena Mistretta, Maria Igea Vega, Paola Bruna Affaticati, Maria Blando, Floriana Bruno, Emanuela Collura, Giovanna Regina, Valentina Randazzo, Alessandro Indovina, Felicia Farina and Raimondo Marcenò Rosalba Bavetta, Serena Mistretta, Maria Igea Vega, Paola Bruna Affaticati, Maria Blando, Floriana Bruno, Emanuela Collura, Giovanna Regina, Valentina Randazzo, Alessandro Indovina, Felicia Farina and Raimondo Marcenò

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Francesco Ingrassia, Valentina Cappuzzo,

http://dx.doi.org/10.5772/67047

#### **Abstract**

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[104] Tseliou E, Kanazawa H, Dawkins J et al. Widespread myocardial delivery of heartderived stem cells by nonoclusive triple-vessel intracoronary infusion in porcine ischemic cardiomyopathy: Superior attenuation of adverse remodeling documented by

[105] Hasan A, Waters R, Roula B, Dana R, Yara S, Alexandre T, Paul A. Engineered biomaterials to enhance stem cell-based cardiac tissue engineering and therapy. Macromolecular

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ease. Stem Cell Research & Therapy. 2015;**6**:7

Yonsei Medical Journal. 2015;**56**:1522-1529

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Translating Regenerative Medicine to the Clinic; 2016:39-69

Bioscience. 2016;**16**(7):958-977

142 Stem Cells in Clinical Practice and Tissue Engineering

In recent years, the anti-leukemic potential of Natural Killer (NK) cells and their role in hematologic malignancies and in Hematopoietic Stem Cell Transplants (HSCT) has attracted greater interest and a recent study by Cooley S. et al. showed a better clinical outcome when patients with Acute Myeloid Leukemia received a transplant from unrelated Group B KIR haplotypes donors. As a consequence of these results, an algorithm called "KIR B-content score" was formulated. The aim of our research is a retrospective analysis of HSC unrelated transplants performed in our center to analyze the effect of the donor KIR B status on the clinical-outcome. Our results showed a better overall survival-rate in the AML recipients, HLA mismatched with the donor, when the donor KIR B content status is Best/Better (37% vs 18% at three years P=0,028). Moreover, we observed that AML recipients, whose Donors KIR B status was Best/Better, had more incidence of aGvHD grade I and II than patients whose Donors KIR B status was Neutral (70% vs 26%) and also a lower rate of relapse (36% vs 58%) and a better Disease Free Survival-rate (58% vs 38% at three years P=0,1) because of a better GvL effect.

**Keywords:** UDHSCT, NK cells, donor KIR B status, AML, GvL effect

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

### **1. Introduction**

#### **1.1. Hematopoietic stem cell transplantation (HSCT)**

Hematopoietic stem cell transplantation (HSCT) has emerged as one of the most useful therapeutic strategies in the treatment of hematologic malignancies (acute or chronic myeloid or lymphoid leukemia) or hereditary (Thalassemia major) for which conventional therapies offer little or no chance of cure. HSCT consists of the replacement of the diseased or nonfunctional bone marrow with healthy stem cells capable of regenerating all blood cells and restoring the normal hematologic and immunologic functions. The source of hematopoietic stem cells (HSC) can be bone marrow (BMT), peripheral blood after appropriate stimulation (PBHSCT) or cord blood (CBHSCT). The transplant can be autologous (the patient's own HSC transplant after suitable treatment) or allogeneic (HSC transplant from a healthy donor). In the latter case, it is essential to find a donor with genetic characteristics similar to those of the receiver because one of the most important variables that influence the success of HSCT is the compatibility of the genes of the HLA system. The Class I and II HLA matching between donor and recipient is crucial to prevent alloreactivity and, consequently, rejection, graft failure and, above all, the graft versus host disease (GVHD), because the transplanted cells from the donor are immunologically competent and can attack cells and tissues of the recipient [1–3].

**1.2. Natural killer cells and KIRs**

frequent event in tumor or virus-infected cells.

KIRs and their ligands are summarized in **Table 1**.

**Figure 1.** "Missing-self hypothesis" explains the mechanism of action of NK cells.

The human NK cells are a part of the immune system with an important role in the host's defense against infections from pathogens and in "immune surveillance" against cancer cells. Their function depends on several families of activating and inhibitory receptors including KIR (killer-cell immunoglobulin-like receptors). The "missing-self" concept [7, 8], put forward by Karré and colleagues in the 1980s, formed the basis for understanding the mechanisms of the action of NK cells (**Figure 1**). According to this hypothesis, the function of NK cells is to recognize and kill autologous cells which are deficient in MHC Class I expression, a

Impact of the Donor KIR Genotype on the Clinical Outcome of Hematopoietic Stem Cell Unrelated Transplants...

http://dx.doi.org/10.5772/67047

145

KIRs belong to the immunoglobulin superfamily and are structurally characterized by two or three extracellular immunoglobulin-like domains. KIRs recognize the MHC Class I molecules such as HLA-A, HLA-B and HLA-C. There are two distinct groups of KIRs: inhibitors and activators. The family of KIR genes, located on chromosome 19q13.4, includes 15 different loci [9]. Receptors are monomeric (single chain) with two (KIR2D) or three (KIR3D) immunoglobulin domains, which can be further divided into those with a long (Long) cytoplasmic tail (KIR2DL and KIR3DL) and those with a short (Short) one (KIR2DS and KIR3DS). The short tail generates an activation signal, while the long one generates an inhibition signal. Each group has the same extracellular domain, and consequently each group binds the same ligands. However, due to differences in their transmembrane and intracellular domains, a group of KIRs determines an inhibitory response, while the other group determines an activating response [10].

A very important aspect of the HSC transplant is the graft versus leukemia effect (GvL), which is a GvHD directed toward the leukemic cells: donors' T lymphocytes attack the cells of the tissues causing GvHD but they can also attack and eliminate residual leukemic cells [4]. If the donor-recipient HLA compatibility is high, the GvL effect is low, consequently, the possibility of relapse is high; on the contrary, when the HLA compatibility is low, the GvHD reaction will be great. A major challenge in improving the success of allogeneic hematopoietic stem cells in the treatment of leukemia is to minimize GvH reactions and simultaneously preserve and optimize GvL reactions. It has also been demonstrated that the GvL effect may be mediated by NK cells [5].

It has been shown that only 25% of patients who need a HSC transplant find an HLA-identical sibling donor; the remaining 75% do not have an HLA-identical donor in their own family; in this case, the alternative is to look for a HLA-matched, unrelated volunteer donor (matched unrelated donor (MUD)) in the worldwide register of bone marrow donors. If there is no compatible donor, the choice of the haploidentical donor is a viable alternative, because there might often be half-identical donors such as parents or siblings [6]. Recently, the number of so-called alternative donor transplants, MUD and haploidentical transplants with one or more HLA mismatches has significantly increased. This is due to the rise in the average age of the patients and therefore the impossibility of finding a compatible donor in their own family. For this reason research has steered toward the study of factors other than HLA matching, which could have a positive influence on the outcome of the transplants, including the role of natural killer (NK) cells in hematopoietic stem cell transplants and, consequently, the study of killer-cell immunoglobulin-like receptors (KIRs) genes.

#### **1.2. Natural killer cells and KIRs**

**1. Introduction**

144 Stem Cells in Clinical Practice and Tissue Engineering

recipient [1–3].

by NK cells [5].

**1.1. Hematopoietic stem cell transplantation (HSCT)**

Hematopoietic stem cell transplantation (HSCT) has emerged as one of the most useful therapeutic strategies in the treatment of hematologic malignancies (acute or chronic myeloid or lymphoid leukemia) or hereditary (Thalassemia major) for which conventional therapies offer little or no chance of cure. HSCT consists of the replacement of the diseased or nonfunctional bone marrow with healthy stem cells capable of regenerating all blood cells and restoring the normal hematologic and immunologic functions. The source of hematopoietic stem cells (HSC) can be bone marrow (BMT), peripheral blood after appropriate stimulation (PBHSCT) or cord blood (CBHSCT). The transplant can be autologous (the patient's own HSC transplant after suitable treatment) or allogeneic (HSC transplant from a healthy donor). In the latter case, it is essential to find a donor with genetic characteristics similar to those of the receiver because one of the most important variables that influence the success of HSCT is the compatibility of the genes of the HLA system. The Class I and II HLA matching between donor and recipient is crucial to prevent alloreactivity and, consequently, rejection, graft failure and, above all, the graft versus host disease (GVHD), because the transplanted cells from the donor are immunologically competent and can attack cells and tissues of the

A very important aspect of the HSC transplant is the graft versus leukemia effect (GvL), which is a GvHD directed toward the leukemic cells: donors' T lymphocytes attack the cells of the tissues causing GvHD but they can also attack and eliminate residual leukemic cells [4]. If the donor-recipient HLA compatibility is high, the GvL effect is low, consequently, the possibility of relapse is high; on the contrary, when the HLA compatibility is low, the GvHD reaction will be great. A major challenge in improving the success of allogeneic hematopoietic stem cells in the treatment of leukemia is to minimize GvH reactions and simultaneously preserve and optimize GvL reactions. It has also been demonstrated that the GvL effect may be mediated

It has been shown that only 25% of patients who need a HSC transplant find an HLA-identical sibling donor; the remaining 75% do not have an HLA-identical donor in their own family; in this case, the alternative is to look for a HLA-matched, unrelated volunteer donor (matched unrelated donor (MUD)) in the worldwide register of bone marrow donors. If there is no compatible donor, the choice of the haploidentical donor is a viable alternative, because there might often be half-identical donors such as parents or siblings [6]. Recently, the number of so-called alternative donor transplants, MUD and haploidentical transplants with one or more HLA mismatches has significantly increased. This is due to the rise in the average age of the patients and therefore the impossibility of finding a compatible donor in their own family. For this reason research has steered toward the study of factors other than HLA matching, which could have a positive influence on the outcome of the transplants, including the role of natural killer (NK) cells in hematopoietic stem cell transplants and, consequently, the study

of killer-cell immunoglobulin-like receptors (KIRs) genes.

The human NK cells are a part of the immune system with an important role in the host's defense against infections from pathogens and in "immune surveillance" against cancer cells. Their function depends on several families of activating and inhibitory receptors including KIR (killer-cell immunoglobulin-like receptors). The "missing-self" concept [7, 8], put forward by Karré and colleagues in the 1980s, formed the basis for understanding the mechanisms of the action of NK cells (**Figure 1**). According to this hypothesis, the function of NK cells is to recognize and kill autologous cells which are deficient in MHC Class I expression, a frequent event in tumor or virus-infected cells.

**Figure 1.** "Missing-self hypothesis" explains the mechanism of action of NK cells.

KIRs belong to the immunoglobulin superfamily and are structurally characterized by two or three extracellular immunoglobulin-like domains. KIRs recognize the MHC Class I molecules such as HLA-A, HLA-B and HLA-C. There are two distinct groups of KIRs: inhibitors and activators. The family of KIR genes, located on chromosome 19q13.4, includes 15 different loci [9]. Receptors are monomeric (single chain) with two (KIR2D) or three (KIR3D) immunoglobulin domains, which can be further divided into those with a long (Long) cytoplasmic tail (KIR2DL and KIR3DL) and those with a short (Short) one (KIR2DS and KIR3DS). The short tail generates an activation signal, while the long one generates an inhibition signal. Each group has the same extracellular domain, and consequently each group binds the same ligands. However, due to differences in their transmembrane and intracellular domains, a group of KIRs determines an inhibitory response, while the other group determines an activating response [10]. KIRs and their ligands are summarized in **Table 1**.


**1.3. Role of NK Cells in HSCT**

at http://www.ebi.ac.uk/ipd/kir/.

HSC transplants to be studied is the one from MUD.

selection for HSCT.

**2. Research aims**

In recent years, the antileukemic potential of natural killer (NK) cells and their role in hematologic malignancies and in HSCT has been attracting ever greater interest; however, it has not yet been clearly established whether the typing of KIR genes may be a useful tool in the

Impact of the Donor KIR Genotype on the Clinical Outcome of Hematopoietic Stem Cell Unrelated Transplants...

http://dx.doi.org/10.5772/67047

147

The beneficial GvL effect, promoted by alloreactive NK cells derived from the donor, was demonstrated for the first time in the HSC haploidentical transplants in patients with acute myeloid leukemia (AML); the best donor is NK mismatched with the recipient and therefore the donor's KIRs do not recognize HLA class I molecules expressed by the host. In this way, the donor's NK cells are responsive to the direction GvH and this results in a better antileukemic

More recently, Cooley et al. [13] have demonstrated that AML patients who received HSC transplants from unrelated Group B KIR haplotype donors had a better outcome after transplant, whereas recipient KIR genotype had no effect. The clinical outcome was better when the donors had one or two KIR B haplotypes (KIR B/x donors) rather than donors who had two KIR A haplotypes (KIR A/A donors); with a KIR B/x donor, relapse was reduced and the leukemia-free survival rate (LFS) was increased. This is because the haplotypes of Group B have a larger number of activating receptors, and this translates into better antileukemic activity. A subsequent study [14] sought to determine whether the protective effect of KIR B could be mapped to either the centromeric or the telomeric region of the KIR locus. The centromeric region contains genes encoding the inhibitory receptors for the C1 and C2 epitopes of HLA-C, whereas the telomeric region contains genes encoding the inhibitory receptors for the Bw4 and A3/11 epitopes and the activating C2 receptor. It was found that both the centromeric and telomeric regions of KIR B correlated with protective effect, but the much stronger association was with the centromeric region. As a consequence of these results, an algorithm was formulated, called "KIR B-content score", based on the number of centromeric and telomeric Group B KIR haplotypes gene-content motifs. The KIR B-content score defines three categories of donors: neutral donors have none or one KIR B motifs, better donors have two or more B motifs without KIR Cen B/B and best donors have two or more B motifs with KIR Cen B/B. A calculator for classification of the donor KIR B status (best, better and neutral) may be found

Recently, these results have been confirmed in HSCT from HLA-identical sibling donor [15]. Together, these results highlight the need for further studies of KIR polymorphisms, possibly at allelic level, to determine whether the typing of the donor KIR genes may be useful in donor

The aim of our research is a retrospective analysis of HSC transplants performed at the Bone Marrow Transplant Unit (UTMO) of the "V. Cervello" Hospital in Palermo. The category of

selection of an HSC donor to promote a better outcome of the transplant [11].

effect with a better overall survival rate and a lower rate of relapse of leukemia [12].

**Table 1.** KIRs and their ligands.

In normal cells the expression of Major Histocompatibility Complex (MHC) Class I molecules and their binding to NK inhibitory receptors inhibits the lysis, while in the virus-infected or cancer cells the lack of expression of self MHC molecules determines their susceptibility to lysis mediated by NK cells. In tumor or virus-infected cells, stress-induced molecules are expressed on the cell surface and are recognized by the activating receptors that enhance the lysis. NK cells can also lyse allogeneic cells expressing HLA Class I antigens that are not recognized by their inhibitory NK receptors.

The KIR genes are polymorphic in humans and there are different allelic variants; each individual possesses his own KIRs repertoire depending on the allelic variants possessed. Different haplotypes contain a different number of KIR genes; some have only one or a few activating receptors, others have more activating receptors. In humans, two groups of KIR haplotypes have been identified, A and B, based on the content of different KIR genes. Haplotypes of group A are present in the entire population and consist of six KIR inhibitory genes KIR3DL3, KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1 and KIR3DL2 and one activator KIR2DS4 that is often present in a null allele variant that is not expressed on the cell surface. Group B comprises haplotypes with a different genetic content including genes KIR2DS2, KIR2DL2, KIR2DL5, KIR2DS3, KIR2DS1, KIR2DS5 and KIR3DS1 that are not part of the haplotypes of Group A. As a consequence, many haplotypes in Group B encode for a greater number of activating receptors compared to haplotypes in Group A.

All individuals can be classified on the basis of the two KIR genotypes possessed: A/A, which is homozygous for the haplotypes of Group A, B/x, which contains one (heterozygous A/B) or two haplotypes of Group B (homozygous B/B). Individuals B/x possessing a larger number of activating receptors theoretically should have a better response against cancer or virus-infected cells.

### **1.3. Role of NK Cells in HSCT**

In recent years, the antileukemic potential of natural killer (NK) cells and their role in hematologic malignancies and in HSCT has been attracting ever greater interest; however, it has not yet been clearly established whether the typing of KIR genes may be a useful tool in the selection of an HSC donor to promote a better outcome of the transplant [11].

The beneficial GvL effect, promoted by alloreactive NK cells derived from the donor, was demonstrated for the first time in the HSC haploidentical transplants in patients with acute myeloid leukemia (AML); the best donor is NK mismatched with the recipient and therefore the donor's KIRs do not recognize HLA class I molecules expressed by the host. In this way, the donor's NK cells are responsive to the direction GvH and this results in a better antileukemic effect with a better overall survival rate and a lower rate of relapse of leukemia [12].

More recently, Cooley et al. [13] have demonstrated that AML patients who received HSC transplants from unrelated Group B KIR haplotype donors had a better outcome after transplant, whereas recipient KIR genotype had no effect. The clinical outcome was better when the donors had one or two KIR B haplotypes (KIR B/x donors) rather than donors who had two KIR A haplotypes (KIR A/A donors); with a KIR B/x donor, relapse was reduced and the leukemia-free survival rate (LFS) was increased. This is because the haplotypes of Group B have a larger number of activating receptors, and this translates into better antileukemic activity. A subsequent study [14] sought to determine whether the protective effect of KIR B could be mapped to either the centromeric or the telomeric region of the KIR locus. The centromeric region contains genes encoding the inhibitory receptors for the C1 and C2 epitopes of HLA-C, whereas the telomeric region contains genes encoding the inhibitory receptors for the Bw4 and A3/11 epitopes and the activating C2 receptor. It was found that both the centromeric and telomeric regions of KIR B correlated with protective effect, but the much stronger association was with the centromeric region. As a consequence of these results, an algorithm was formulated, called "KIR B-content score", based on the number of centromeric and telomeric Group B KIR haplotypes gene-content motifs. The KIR B-content score defines three categories of donors: neutral donors have none or one KIR B motifs, better donors have two or more B motifs without KIR Cen B/B and best donors have two or more B motifs with KIR Cen B/B. A calculator for classification of the donor KIR B status (best, better and neutral) may be found at http://www.ebi.ac.uk/ipd/kir/.

Recently, these results have been confirmed in HSCT from HLA-identical sibling donor [15]. Together, these results highlight the need for further studies of KIR polymorphisms, possibly at allelic level, to determine whether the typing of the donor KIR genes may be useful in donor selection for HSCT.

### **2. Research aims**

In normal cells the expression of Major Histocompatibility Complex (MHC) Class I molecules and their binding to NK inhibitory receptors inhibits the lysis, while in the virus-infected or cancer cells the lack of expression of self MHC molecules determines their susceptibility to lysis mediated by NK cells. In tumor or virus-infected cells, stress-induced molecules are expressed on the cell surface and are recognized by the activating receptors that enhance the lysis. NK cells can also lyse allogeneic cells expressing HLA Class I antigens that are not rec-

The KIR genes are polymorphic in humans and there are different allelic variants; each individual possesses his own KIRs repertoire depending on the allelic variants possessed. Different haplotypes contain a different number of KIR genes; some have only one or a few activating receptors, others have more activating receptors. In humans, two groups of KIR haplotypes have been identified, A and B, based on the content of different KIR genes. Haplotypes of group A are present in the entire population and consist of six KIR inhibitory genes KIR3DL3, KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1 and KIR3DL2 and one activator KIR2DS4 that is often present in a null allele variant that is not expressed on the cell surface. Group B comprises haplotypes with a different genetic content including genes KIR2DS2, KIR2DL2, KIR2DL5, KIR2DS3, KIR2DS1, KIR2DS5 and KIR3DS1 that are not part of the haplotypes of Group A. As a consequence, many haplotypes in Group B encode for a greater

All individuals can be classified on the basis of the two KIR genotypes possessed: A/A, which is homozygous for the haplotypes of Group A, B/x, which contains one (heterozygous A/B) or two haplotypes of Group B (homozygous B/B). Individuals B/x possessing a larger number of activating receptors theoretically should have a better response against cancer or virus-infected cells.

number of activating receptors compared to haplotypes in Group A.

ognized by their inhibitory NK receptors.

**KIRs KIR ligands**

146 Stem Cells in Clinical Practice and Tissue Engineering

KIR3DL1 HLA-Bw4 KIR3DL2 HLA-A3,-A11 KIR2DL4 HLA-G KIR2DL5 Unknown

KIR2DS1

KIR2DL3 KIR2DS2

KIR3DL7 KIR2DS4 KIR2DS5 KIR3DS1

**Table 1.** KIRs and their ligands.

KIR2DL1 Group 2 HLA-C Asn77 Lys80 (w2, w4, w5, w6)

KIR2DL2 Group 1 HLA-C Ser77 Asn80 (w1, w3, w7, w8)

The aim of our research is a retrospective analysis of HSC transplants performed at the Bone Marrow Transplant Unit (UTMO) of the "V. Cervello" Hospital in Palermo. The category of HSC transplants to be studied is the one from MUD.

The objectives of this study are the following:

