**2. Stem cells and mesenchymal stem cells**

More than 200 different types of cells make up embryonic and adult tissues and are regulated by local and systemic environmental factors. Embryonic stem cells (ESC) derived from the internal cell mass of the blastocyst are constituted by ectoderm, endoderm, and mesoderm. Adult stem/progenitor cells, known as somatic SC, are undifferentiated cells located throughout the body. These cells have a high proliferative capacity and a differentiation potential limited to their lineage; they participate in regeneration, cell turnover, and homeostasis. The main function during life is to maintain the number of differentiated cells at a constant level and to replace dead cells or cells lost due to injury or disease [5].

antagonist, interferon-γ, low-density lipoprotein, monocyte chemotactic protein-1,

The mesenchymal stem cell (MSC) therapies offer new opportunities for confronting diseases that lack curative treatments through the properties of multipotentiality, self-renewal, and the secretion of paracrine factors derived from exosomes (cytokines, growth factors, microRNAs, and proteases), which act as mediators of intracellular communication and induce the repair

Cell therapy with MSCs is safe and effective in the treatment of degenerative and traumatic diseases; they are found *in vivo* in minimal quantities throughout the body and they have the ability to differentiate into bone, cartilage, and adipose tissue through stimuli and in culture. The MSCs are located in the perivascular environment, activating and creating a regenerative microenvironment, with the secretion of molecules to regulate the immune response; however, the therapeutic effects through paracrine interactions of the MSCs are of short duration. The response to changes in the environment is attributed to MSCs through the transcriptional regulation of mediators that control inflammation, remodeling, repair, and cellular recruitment. The repair process involves the regulation of extracellular matrix (ECM) deposition, collagen synthesis, fibroblast proliferation, platelet activation, fibrinolysis, and angiogenesis; the immune process suppresses T-cells, activates macrophages, and recruits neutrophils [2]. Cell differentiation and replacement is attributed to cellular secretions that function as therapeutic inducers. The secretions of extracellular vesicles (EV) are both local and systemic. To determine the functions of the factors secreted by the MSCs in regeneration, it is necessary to identify precisely the molecular profile of the secretome of the MSC constituted by growth factors (GF), cytokines and chemokines, proteases, ECM, hormones, and lipid mediators, and so on [3].

The secretome of MSCs contains multiple overlapping elements that make it difficult to identify them. The *in vivo* examination of the secretome of MSCs and the strategies to modulate it and the result of the analysis are essential for the design of the next generation of regenerative therapies without cells. In this way, questions arise about the regulatory function of the secretome of the MSC, such as (i) what are the most effective approaches to study the secretome both *in vitro* and *in vivo* and are new technologies necessary to achieve it? (ii) how do the properties of the secretome change or become manageable, and after the transplant how does it evolve in the local microenvironment? (iii) what are the best methods to achieve the sustain-

More than 200 different types of cells make up embryonic and adult tissues and are regulated by local and systemic environmental factors. Embryonic stem cells (ESC) derived from the

ability of the secretome and the control in the transplant? [4].

**2. Stem cells and mesenchymal stem cells**

nuclear factor κB, nitric oxide, NO synthase, endothelial NOS, NYHA

**1. Introduction**

and regeneration of organs and tissues [1].

186 Stromal Cells - Structure, Function, and Therapeutic Implications

SCs have a great capacity for self-renewal and the potential to produce a differentiated progeny. An SC can have the same phenotype but be less "mature" or less "differentiated" than its descendants and is classified into SCs/progenitors, "somatic", "adult", or "tissue" embryonic and nonembryonic cells. ESC are pluripotent, and most populations of progenitor cells arising during embryonic development cannot self-renovate and have common properties with adult SCs, such as the potential of differentiation and the capacity for asymmetric cell division [6].

SC can be differentiated into specific cell types. Their ability to self-renew is through indefinite replication, resulting in the creation of two identical SCs, and under appropriate conditions, differentiated into more specialized cells. The MSCs are spindle-shaped adherent plastic cells that can be isolated from the bone marrow (BM), adipose tissue, and other tissues; are multipotent; and have the ability to differentiate. *In vitro* they can differentiate into bone; a subset of the cells have a high proliferative potential colony-forming units (CFU-F) when they are grown in culture. Hematopoietic SCs regulate and maintain hematopoiesis in the microenvironment of BM [7].

The MSCs can produce blood cells, although they are derived from a different population called hematopoietic SCs. The MSCs are classified as nonhematopoietic multipotential SCs and have the ability to differentiate into mesenchymal as well as nonmesenchymal lineages. The MSCs have the capacity for self-renewal, colony formation, phenotypic expression pattern, and differentiation potential; they interact with cells of the innate and adaptive immune system in the modulation of immune response. They participate in physiological processes, such as tissue homeostasis and hematopoiesis, and in pathological processes such as diseases of aging, tissue damage, and degenerative, inflammatory, and autoimmune diseases. After administration *in vivo*, MSCs induce tolerance and migrate to injured tissues where they inhibit the release of proinflammatory cytokines and promote the survival of damaged cells [8].

The International Society of Cell Therapy has established the following minimum criteria to define multipotent MSCs: first, they must be adherent to the plastic, under standard culture conditions (minimal essential medium, plus 20% fetal bovine serum). Second, MSCs should express CD105, CD73, and CD90 and should not express surface molecules such as CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR. Third, they must be differentiated into osteoblasts, adipocytes, and chondroblasts *in vitro*. They can be isolated from many adult tissues, BM, and adipose tissue. They have the ability to differentiate and trans-differentiate into cells of different lineages and immunomodulation capacity. The term "mesenchymal stem cell" is used to refer to the subset of mesenchymal cells that demonstrate SC activity and meet these criteria [9, 10].

The main characteristics of MSCs are the potential for self-renewal, differentiation, and multipotency. Under appropriate microenvironmental conditions, they can proliferate and give rise to other types of cells, they can be trans-differentiated in cells of other lineages, and exert proregenerative, immunomodulatory, and antiinflammatory functions. Because of these characteristics, they can be an ideal therapeutic strategy for the treatment of inflammatory and systemic autoimmune diseases and are essential in the tissue regeneration of congenital, degenerative, and traumatic diseases [5].

(Th1, cytotoxic T lymphocyte and B lymphocyte), secreting factors such as TGF-β, IL-10, IDO, PGE-2, sHLA-G5. The MSCs are considered immune privileged cells due to the low expression of the major histocompatibility complex class II (MHC-II) and expressing costimulatory molecules on the cell surface and interfering with different pathways of the immune response. *In vitro*, MSCs inhibit cell proliferation of T-cells, B-cells, NK cells, and dendritic cells (DC), producing what is known as "division arrest anergy". On the other hand, MSCs can inhibit diverse key functions of the immune cells, such as the secretion of cytokines and the cytotoxicity of T-cells and NK cells; B-cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. In inflammation, MSCs must be activated to generate immunomodulation by suppression of molecules such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ. On the other hand, MSCs recruit regulatory T lymphocytes (Tregs) from both lymphoid and graft organs [15]. *In vivo* studies have shown differences with respect to the immunomodulatory properties of MSCs. Currently, the effectiveness of MSC treatment to suppress the abnormal immune response in scenarios such as prevention, treatment of allograft rejection periods, and autoimmune and inflammatory diseases is being investigated. Clinical trials in humans are being developed in the treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis, diabetes mellitus type 1, prevention of allograft rejection, survival of bone marrow and kidney grafts, and treatment of resistant graft versus host disease [16].

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*In vitro* the MSCs are able to differentiate to osteogenic, chondrogenic, adipogenic, and myogenic lineages, and they express markers of pericytes (CD146+, CD34-, CD45-, CD56). In vascular damage, released pericytes become MSCs, are activated by the lesion, and respond by secreting bioactive molecules that inhibit immune cells that produce tissue damage and prevent the development of autoimmune reactions. The secretion of these bioactive molecules

Activated MSCs also locally produce antimicrobial peptides such as LL37, which eliminate bacteria and attract macrophages and hematopoietic cells. Together, these function as therapeutic elements in the affected site and stimulate and increase TH2 and regulatory T-cells through inhibitory effects on the immune system. Thus, MSCs function as "medical signaling cells" with healing actions at sites of injury or inflammation. These trophic and immunomodulatory activities suggest that MSCs can serve as "pharmacies" regulated *in situ*. The MSCs act as "sentinels" in acute and chronic injuries; they function as multidrug dispensaries

In addition to the secretion of cytokines/chemokines, the MSCs show a great capacity for mitochondrial transfer and microvesicle secretion (exosomes) in response to injury. On the other hand, MSCs are recruited to the lesion to repair damaged tissues, an event intimately associated with tumorigenesis. Tumors are made up of different types of cancer cells that contribute to heterogeneity. Among these populations are the cancer stem cells (CSC) that participate in its onset and progression. A CSC population consists of MSCs that differ in cells with mesodermal characteristics. Resident or migratory MSCs favor angiogenesis and increase tumor aggressiveness. This interaction between MSCs and CSCs is fundamental in the development of carcinogenesis, progression, and metastasis. In cancer, tumor cells aberrantly secrete large amounts of exosomes to transport paracrine signals that contribute to

establishes a regenerative microenvironment in the injured tissue [17].

*in situ*, with "pharmacy" functions that promote natural regeneration [18].

tumor and distance interaction [19–21].

The origin of MSCs *in vivo* is controversial. They are located in the perivascular area of the adventitia from almost all vessels (arteries and veins). They are pericytes, which are in intimate contact with the basement membrane and the surrounding endothelial cells, forming the extensive network of the microvasculature. Phenotypic similarities are evident among microvessels, and pericytes can be isolated from any vascularized tissue, near smooth muscle cells of arterioles, venules, and larger vessels, and preserve the expression of pericyte markers such as NG2 and CD146 [11].

The immunomodulatory activity of MSCs is mediated by paracrine factors. Among these, the exosomes participate in the communication between the MSCs and the target tissue. To demonstrate this, one study investigated the effect of the exosomes derived from MSCs on peripheral blood mononuclear cells (PBMC), especially on T-cells. It was shown that the MSCderived exosomes extracted from the BM of healthy donors suppressed the secretion of the proinflammatory factor TNF-α and IL-1β and, conversely, increased the concentration of the antiinflammatory factor TGF-β *in vitro*. Exosomes can induce the conversion of T helper type 1 (Th1) into T helper type 2 (Th2) cells and reduce the potential of the T-cells to differentiate into effector T-cells producing interleukin 17 (Th17). In addition, the levels of regulatory T-cells (Treg) and protein 4 associated with cytotoxic T lymphocytes were increased. The results suggest that the exosomes derived from MSCs possess immunomodulatory properties [12].

Inflammation is a response of the organism to self-evolutionary harmful stimuli to maintain homeostasis. In the process, MSCs secrete paracrine factors that influence immune cells, dendritic cells, and macrophages, polarizing them toward a tolerogenic phenotype. Regulatory immune cells accumulate and converge in their regulatory pathways and create a tolerogenic environment conducive to immunomodulation [13].

During tissue regeneration, the regulation of the inflammatory process is essential, as is the control of local and systemic inflammatory response without causing damage in the injured tissues. The MSCs possess immunomodulatory properties that facilitate the repair of tissues by releasing exosomes, which generate an appropriate microenvironment to modulate inflammation. The exosomes contain bioactive molecules, which act as a cell-cell communication vehicle and influence the activities of receptor cells. During this process, the horizontal transfer of exosomal microRNA to recipient cells regulates the expression of the target gene and is essential to control inflammation and tissue homeostasis to develop new therapeutic approaches [14].

In MSC therapy, the following points should be kept in mind: (i) arrival at sites of ischemia or injury, when administered systemically and (ii) modulation of the immune responses mediated by T-cells, which express chemokine receptors and ligands in the migration of the cells and the homing process.

The MSCs induce immunomodulatory effects, interact with innate immune cells (dendritic cells, monocytes, natural killer [NK] cells, and neutrophils) and cells of the adaptive immune system (Th1, cytotoxic T lymphocyte and B lymphocyte), secreting factors such as TGF-β, IL-10, IDO, PGE-2, sHLA-G5. The MSCs are considered immune privileged cells due to the low expression of the major histocompatibility complex class II (MHC-II) and expressing costimulatory molecules on the cell surface and interfering with different pathways of the immune response. *In vitro*, MSCs inhibit cell proliferation of T-cells, B-cells, NK cells, and dendritic cells (DC), producing what is known as "division arrest anergy". On the other hand, MSCs can inhibit diverse key functions of the immune cells, such as the secretion of cytokines and the cytotoxicity of T-cells and NK cells; B-cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. In inflammation, MSCs must be activated to generate immunomodulation by suppression of molecules such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ. On the other hand, MSCs recruit regulatory T lymphocytes (Tregs) from both lymphoid and graft organs [15].

rise to other types of cells, they can be trans-differentiated in cells of other lineages, and exert proregenerative, immunomodulatory, and antiinflammatory functions. Because of these characteristics, they can be an ideal therapeutic strategy for the treatment of inflammatory and systemic autoimmune diseases and are essential in the tissue regeneration of congenital,

The origin of MSCs *in vivo* is controversial. They are located in the perivascular area of the adventitia from almost all vessels (arteries and veins). They are pericytes, which are in intimate contact with the basement membrane and the surrounding endothelial cells, forming the extensive network of the microvasculature. Phenotypic similarities are evident among microvessels, and pericytes can be isolated from any vascularized tissue, near smooth muscle cells of arterioles, venules, and larger vessels, and preserve the expression of pericyte markers

The immunomodulatory activity of MSCs is mediated by paracrine factors. Among these, the exosomes participate in the communication between the MSCs and the target tissue. To demonstrate this, one study investigated the effect of the exosomes derived from MSCs on peripheral blood mononuclear cells (PBMC), especially on T-cells. It was shown that the MSCderived exosomes extracted from the BM of healthy donors suppressed the secretion of the proinflammatory factor TNF-α and IL-1β and, conversely, increased the concentration of the antiinflammatory factor TGF-β *in vitro*. Exosomes can induce the conversion of T helper type 1 (Th1) into T helper type 2 (Th2) cells and reduce the potential of the T-cells to differentiate into effector T-cells producing interleukin 17 (Th17). In addition, the levels of regulatory T-cells (Treg) and protein 4 associated with cytotoxic T lymphocytes were increased. The results suggest that the exosomes derived from MSCs possess immunomodulatory properties [12]. Inflammation is a response of the organism to self-evolutionary harmful stimuli to maintain homeostasis. In the process, MSCs secrete paracrine factors that influence immune cells, dendritic cells, and macrophages, polarizing them toward a tolerogenic phenotype. Regulatory immune cells accumulate and converge in their regulatory pathways and create a tolerogenic

During tissue regeneration, the regulation of the inflammatory process is essential, as is the control of local and systemic inflammatory response without causing damage in the injured tissues. The MSCs possess immunomodulatory properties that facilitate the repair of tissues by releasing exosomes, which generate an appropriate microenvironment to modulate inflammation. The exosomes contain bioactive molecules, which act as a cell-cell communication vehicle and influence the activities of receptor cells. During this process, the horizontal transfer of exosomal microRNA to recipient cells regulates the expression of the target gene and is essential to control inflammation and tissue homeostasis to develop new therapeutic approaches [14]. In MSC therapy, the following points should be kept in mind: (i) arrival at sites of ischemia or injury, when administered systemically and (ii) modulation of the immune responses mediated by T-cells, which express chemokine receptors and ligands in the migration of the cells

The MSCs induce immunomodulatory effects, interact with innate immune cells (dendritic cells, monocytes, natural killer [NK] cells, and neutrophils) and cells of the adaptive immune system

degenerative, and traumatic diseases [5].

188 Stromal Cells - Structure, Function, and Therapeutic Implications

environment conducive to immunomodulation [13].

such as NG2 and CD146 [11].

and the homing process.

*In vivo* studies have shown differences with respect to the immunomodulatory properties of MSCs. Currently, the effectiveness of MSC treatment to suppress the abnormal immune response in scenarios such as prevention, treatment of allograft rejection periods, and autoimmune and inflammatory diseases is being investigated. Clinical trials in humans are being developed in the treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis, diabetes mellitus type 1, prevention of allograft rejection, survival of bone marrow and kidney grafts, and treatment of resistant graft versus host disease [16].

*In vitro* the MSCs are able to differentiate to osteogenic, chondrogenic, adipogenic, and myogenic lineages, and they express markers of pericytes (CD146+, CD34-, CD45-, CD56). In vascular damage, released pericytes become MSCs, are activated by the lesion, and respond by secreting bioactive molecules that inhibit immune cells that produce tissue damage and prevent the development of autoimmune reactions. The secretion of these bioactive molecules establishes a regenerative microenvironment in the injured tissue [17].

Activated MSCs also locally produce antimicrobial peptides such as LL37, which eliminate bacteria and attract macrophages and hematopoietic cells. Together, these function as therapeutic elements in the affected site and stimulate and increase TH2 and regulatory T-cells through inhibitory effects on the immune system. Thus, MSCs function as "medical signaling cells" with healing actions at sites of injury or inflammation. These trophic and immunomodulatory activities suggest that MSCs can serve as "pharmacies" regulated *in situ*. The MSCs act as "sentinels" in acute and chronic injuries; they function as multidrug dispensaries *in situ*, with "pharmacy" functions that promote natural regeneration [18].

In addition to the secretion of cytokines/chemokines, the MSCs show a great capacity for mitochondrial transfer and microvesicle secretion (exosomes) in response to injury. On the other hand, MSCs are recruited to the lesion to repair damaged tissues, an event intimately associated with tumorigenesis. Tumors are made up of different types of cancer cells that contribute to heterogeneity. Among these populations are the cancer stem cells (CSC) that participate in its onset and progression. A CSC population consists of MSCs that differ in cells with mesodermal characteristics. Resident or migratory MSCs favor angiogenesis and increase tumor aggressiveness. This interaction between MSCs and CSCs is fundamental in the development of carcinogenesis, progression, and metastasis. In cancer, tumor cells aberrantly secrete large amounts of exosomes to transport paracrine signals that contribute to tumor and distance interaction [19–21].

The MSCs represent an opportunity in cell therapy because: (i) they are easily accessible; (ii) the isolation is simple, they can be expanded to clinical scales in a short period; (iii) they can be preserved with a minimum loss of potency and stored for administration; and (iv) so far they have not shown adverse reactions to allogeneic transplantation compared with autotransplantation, and they can expand *in vitro*, without altering their main properties.

The paracrine signals of the extracellular environment influence the microenvironment of MSCs, both in proliferation and in differentiation. Many therapeutic strategies try to increase the effectiveness of regenerative therapies by direct application in the affected tissue or by differentiation in mature tissues. The MSCs have phenotypic plasticity and harbor an arsenal of bioactive molecules that are released by detecting signals in the local environment or pack-

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The rigidity and/or topography of the cellular environment controls the differentiation of the MSCs, the physical signals determining the target, and cellular differentiation, an environment with high rigidity that leads to osteogenic differentiation, while low rigidity induces lipogenic differentiation. These effects are independent of the chemical/biochemical inducers. Physical factors, such as tension, produce a reorganization of the cytoskeleton during the differentiation of the MSCs and affect the expression of the essential gene of the process. Physical signals control the lineage specification of the MSCs, reorganizing and adjusting the cytoskeleton, and the cells perceive physical signals and transform these into biochemical and biological signals. Specifically, biophysical signals can initiate and strengthen biochemical signaling for the determination and differentiation of the destination of MSCs. The physical properties of the cell environment direct the structural adaptation and functional coupling of

To facilitate the identification of terms that we use in the following section, we present here

"Extracellular vesicle" (EV), is synonymous with "membrane vesicle" (suggested for all

"Exosomes" are vesicles of 50–100 nm in diameter, generated by exocytosis of multivesicular bodies (MVB), and are a macromolecular complex involved in the degradation of RNA;

"Microparticle" (MV) is any small particle, regardless of its origin, and is more appropriate to

"Microvesicles" (ExMV) are larger extracellular membrane vesicles (100–1000 nm in diam-

**1.** Microvesicles/microparticles/ectosomes: these are produced by the formation of buds and

**2.** Exosomes: these form within the endosomal network and are released by fusing the mul-

The current nomenclature classifies the vesicles by their biogenesis. The criteria for classification

**3.** Apoptotic bodies: these are released as blisters of cells that undergo apoptosis.

"Ectosoma" is a microvesicle derived from neutrophils or monocytes;

aging in EVs [25, 26].

the cells to their environment [27].

abbreviations and meaning of the terms:

populations of vesicles derived from cells);

indicate membrane-bound structures;

The EVs are classified into three main classes:

tivesicular bodies with the plasma membrane; and

are according to their origin, function, or biogenesis.

the fusion of the plasma membrane;

eter) [28].
