Stem Cell-Derived Exosomes

### **Chapter 1**

## Mesenchymal Stem Cell-Derived Exosomes for Myocardial Infarction Treatment

*Huifeng Zheng, Yimei Hong, Bei Hu, Xin Li and Yuelin Zhang*

### **Abstract**

Myocardial infarction (MI) is a major cause of morbidity and mortality in modern society. Over the past decades, mesenchymal stem cell (MSCs)-based therapy has shown promising results in the treatment of MI due to their unique properties of multi-differentiation ability, immune-privileged phenotype and paracrine activity. Recently, MSC-derived exosomes (MSC-EXO) have been proposed as a promising therapeutic strategy for MI with their ability to inhibit cardiomyocyte apoptosis and stimulate vascular angiogenesis. They also aid immunoregulation and rejuvenation of cardiomyocyte senescence by transporting their unique content such as proteins, lipids, and miRNAs. Compared with MSC transplantation, MSC-EXO administration has shown several advantages, including lower toxicity and immunogenicity and no risk of tumor formation. Nonetheless the potential mechanisms underlying MSC-EXO-based therapy for MI are not fully understood. In addition, lack of modification of MSC-EXOs can impact therapeutic efficacy. It is vital to optimize MSC-EXO and enhance their therapeutic efficacy for MI. We summarize the recent advances regarding biological characteristics, therapeutic potential and mechanisms, and optimal approaches to the use of MSC-EXOs in the treatment of MI.

**Keywords:** mesenchymal stem cells, exosome, myocardial infarction, treatment, therapeutic effect

### **1. Introduction**

Myocardial infarction (MI) results in irreversible loss of cardiomyocytes due to a restricted blood supply and is the major cause of morbidity and mortality worldwide. It has been estimated to account for 80% of deaths in patients with ischemic heart disease worldwide, and its prevalence continues to increase every year leading to a significant medical, social, and financial burden [1]. Despite the availability of advanced surgical interventions and medications including primary percutaneous coronary intervention, angiotensin-converting enzyme drugs and β-blockers, there remains no effective means to prevent cardiomyocyte loss due to myocardial ischemia [2]. The only cure for this devastating disease is heart transplantation but this is restricted by its high cost, a shortage of donor hearts, and the occurrence of immune

rejection following transplantation [3]. Exploration of novel therapies for left ventricle remodeling and dysfunction following infarction is urgently needed.

Over the past decades, stem cell-based therapy has become a promising strategy to treat MI with significant progress made in animal studies and clinical trials [4–6]. Among all types of stem cell under investigation, mesenchymal stem cells (MSCs) have garnered huge interest due to their easy isolation, high reproductive activity, differentiation capability and immunomodulatory properties [7, 8]. MSCs can be isolated from multiple tissues or cells including bone marrow, adipose tissue, umbilical cord blood and even pluripotent stem cells [9–12]. There is accumulating evidence that MSCs are promising candidates for MI treatment [13–15]. More importantly, it is now widely accepted that the cardioprotective effects of MSC-based therapy in MI are due to their strong paracrine effects, rather than trans-differentiation ability [7, 16–18]. Therefore, researchers are increasingly huge interested in the therapeutic efficacy of MSC-derived bioactive molecules, especially exosome (EXO), that are considered major components of the paracrine effect in MSC-based therapy [19, 20]. EXO, a subgroup of extracellular vesicles (EVs), are 40–160 nm diameter membranebound vesicles that can be found in almost all biological fluids. It has been well documented that MSC-EXO exert their cardioprotective effects in MI by delivering diverse biological molecules, including non-coding RNA, DNA, lipids and proteins [21–24]. More importantly, compared with MSC transplantation, MSC-EXO have several advantages such as easier storage and transplantation, less immune rejection, minimum risk of immunogenicity and no risk of tumor formation [25]. We discuss the current understanding of the biological characteristics, therapeutic effects and potential mechanisms of MSC-based therapy in MI. We also highlight the current challenges and potential approaches to improve the efficacy and production of MSC-EXO in regenerative medicine to guide their future clinical application.

### **2. Characterization and Isolation of MSC-EXO**

EVs are bilayer lipid membrane-bound subcellular vesicles released by all types of cells and present in all body fluids. According to MISEV2018, EVs are divided into "small EVs" (sEVs, <100 nm or <200 nm) and "medium/large EVs"(m/lEVs, >200 nm) respectively [26]. EXO are sEVs approximately 40–160 nm in diameter (100 nm on average) and the main subclass of EVs [27]. The biogenesis of EXO begins with inward budding to form an early endosome. Finally, EXO are built when multi vesicular bodies (MVBs, late endosomes) fuse with plasma membrane and are secreted into the extracellular space [28, 29]. MSC-EXO express EXO-specific markers CD9, CD63, CD81, Alix and Tsg101 as well as MSC surface markers including CD29, CD44, CD90 and CD73. Among these, CD29 and CD44 have been identified previously as the specific biomarkers for MSC-EXO [30, 31]. The size and concentration of EXO can be characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) [32]. Recently, plasmonic scattering microscopy has been applied to image exosomes and analyze biomarkers [33].

It is difficult to show whole landscape of EXO dispersed in solution. Therefore, purification of EXO is of importance for EXO definition. EXO are distributed throughout body fluids and this represents a challenge to their isolation. EXO are secreted into body fluids such as blood, urine, saliva, lymph, breast milk, cerebrospinal fluid and pericardial fluid etc. [34]. EXO components reflects the state of the original cell. Different methods of isolation of EXO varies from various body fluids. Meanwhile,

### *Mesenchymal Stem Cell-Derived Exosomes for Myocardial Infarction Treatment DOI: http://dx.doi.org/10.5772/intechopen.110736*

the extraction result differs from types of biological fluid. Which was optimal remains controversial [35]. Isolation of abundant EXO can help in the assessment of their biological functions [36]. Several recent alternative methods ranging from conventional to newly developed techniques to isolate and purify EXO are summarized in **Table 1**. Different methods for EXO isolation have different advantages and disadvantages. During isolation, ultracentrifugation and density gradient centrifugation are the most commonly used techniques [47]. Currently, several new methods have been established to facilitate high-throughput and high-purity manufacture of EXO. The characterization and isolation of MSC-EXO are summarized in **Figure 1**.



**Table 1.**

*Methods of MSC-EXO isolation.*

**Figure 1.**

*Characterization and isolation of MSC-EXO.*

### **3. The bioactive constituents of MSC-EXO for MI treatment**

MSC-EXO exert their benefits in various diseases by enclosing and transporting a vast array of molecules [48]. It has been demonstrated that exosomal components are almost dependent on the source cell and cellular conditions [25, 49, 50]. Generally, EXO contain multiple characteristic molecules with typical physiological functions [51–53]. MSC-EXO comprise a variety of substances, including many kinds of proteins and a lot of noncoding RNA, including microRNAs (miRNAs) and long noncoding RNAs (lncRNA) [54]. These components can act as paracrine factors, mediating cell-to-cell signaling and communication. More importantly, they can be used as prognostic and diagnostic markers [55, 56].

### **3.1 Exosomal miRNAs in MSC-EXO for MI treatment**

miRNAs are endogenous and 19–25 nucleotides in size. They can be isolated from cells, tissues and body fluids [57]. By pairing to the mRNAs of protein-coding genes, miRNAs play an important role in regulating post-transcriptional silencing of target genes [58, 59]. There is accumulating evidence that miRNAs are enriched in MSC-EXO and are the major bioactive constituents [60–62]. In the last few decades, the cardioprotective role of MSC-derived exosomal miRNAs has attracted huge attention [63]. It has been well documented that many MSC-derived exosomal miRNAs have beneficial functions in MI treatment [64]. Importantly, several potential mechanisms have been identified such as promotion of angiogenesis, reduction of cell death and an antifibrotic effect [65]. Enhanced angiogenesis is one of the important repair mechanisms underlying MSC-EXO-based therapy for MI [66–68]. Through direct miRNAs transfer, MSC-EXO convey their proangiogenic signals to injured cardiomyocytes [69]. Previous study has shown that silenced MSC-derived exosomal miR-210 largely lost its proangiogenic effect. Further experimental study revealed that exosomal miR-210 improves angiogenesis of MSC-EXO via targeting of Efna3 [70]. Zhu et al. demonstrated that macrophage migration inhibitory factor (MIF) could enhance the pro-angiogenic effect of MSC-EXO by enhancing the level of miR-133a-3p via regulation of the AKT signaling pathway [71]. miR-221 is one of the most studied miRNAs. A recent study reported that up-regulated exosomal miR-221-3p derived from senescent MSCs improved their ability of angiogenesis, migration and proliferation, and suppressed apoptosis by regulating the PTEN/AKT pathway [72]. Ma et al. revealed that miR-132-electroporated MSC-EXO could promote angiogenesis both *in vitro* and *in vivo* by downregulating RASA1 [23]. These studies show that MSC-EXO improve angiogenesis by transmitting miRNAs via various biological signaling pathway following MI.

There is increasing evidence that ameliorating cardiomyocyte death is another major mechanism by which EXO restore cardiac function following MI. MSC-EXO reduce myocardial cell death via multiple mechanisms including an anti-apoptosis action, inhibition of pyroptosis and an anti-inflammatory effect [73]. Apoptosis is programmed cell death that is strongly associated with myocardial ischemia [74]. Previous studies have proved that MSCs have an anti-apoptotic effect through secretion of exosomes enriched in miRNAs [75]. Hypoxia-elicited MSC-EXO (Hypo-EXO) facilitates cardiac repair by preventing cell death in MI via delivery of miR-125b.

Mechanistically, miR-125b-5p suppresses apoptosis of cardiomyocytes by downregulating the expression of apoptotic genes p53 and BAK1 [63]. Another study demonstrated that EXO derived from miR-146a-modified adipose-MSCs attenuated MI via inhibition of apoptosis, the inflammatory response, and fibrosis in a rat model of AMI by targeting early growth response factor 1(EGR1) [76]. Wang et al. reported that adipose-MSC-EXO carrying miR-671 reduced the apoptosis of cardiomyocytes and alleviated myocardial fibrosis and inflammation via inactivation of the TGFBR2/ Smad2 Axis [77]. miR-153-3p plays an important role in modulating cell proliferation, apoptosis and angiogenesis. It has been illustrated that EXO-miR-153-3p significantly reduces apoptosis of endothelial cells and cardiomyocytes and promotes their viability. By targeting ANGPT1, miR-153-3p can regulate the VEGF/VEGFR2/PI3K/AKT/ eNOS pathways to prevent hypoxic damage to endothelial cells and cardiomyocytes [78]. Furthermore, a growing number of studies have shown that stem cell-derived exosomal miRNAs, such as miR-150-5p, miR-126, and miR-486-5p, demonstrate antiapoptotic activity in MI treatment [79–81]. These findings indicate that the anti-apoptotic effect of MSC-EXO can be partly ascribed to the delivery of some antiapoptotic miRNAs.

Autophagy is a self-destructive process during which a cell degrades and recycles unnecessary or dysfunctional cellular components [82]. Autophagy is involved in promoting cell death and exacerbates myocardial dysfunction following severe ischemic stress. There is accumulating evidence that MSC-EXO reduce cell death by mediating autophagy. Xiao et al. determined that MSC-EXO reduced autophagic flux in infarcted hearts via exosomal transfer of miR-125b by interfering with p53/ Bnip3 signaling and protected cardiomyocytes against damage [83]. Liu et al. showed that miR-93-5p-enhanced ADSC-EXO had a greater cardioprotective effect by suppressing hypoxia-induced autophagy and inflammatory cytokine expression via targeting of Atg7 and Toll-like receptor 4 (TLR4), respectively [84]. Furthermore, Li et al. reported that exosomal miR-301 derived from MSCs protected against MI by inhibiting myocardial autophagy [85]. In addition, MSC-exosomal miRNAs exerted a cardioprotective effect in MI by attenuating cardiac fibrosis. Inflammation and subsequent fibrosis are important pathological reactions that result in scar formation post-MI. Human umbilical cord MSCs-EXO containing miR-29b have been shown to prevent cardiac fibrosis following MI, leading to a reduction in infarct size and improved cardiac function in a mouse model of MI [86]. Moreover, miR-671 carried by adipose-derived MSC-EXO has been proven to also reduce myocardial fibrosis and inflammation both *in vitro* and i*n vivo* [77]. The roles of MSC-exosomal miRNA and the potential mechanism for MI treatment are summarized in **Table 2**.

### **3.2 Exosomal lncRNAs in MSC-EXO for MI treatment**

LncRNAs are defined as RNA transcripts >200 nucleotides without proteincoding potential. lncRNAs play important roles in regulating a variety of biological processes. Recent studies have shown that they participate in the initiation and progression of MI through regulation of gene expression at the epigenetic, transcriptional and post-transcriptional levels [87]. Moreover, MSC-derived exosomal lncRNAs have been shown to have cardioprotective effects for MI. LncRNA KLF3-AS1 in human MSC-EXO ameliorated pyroptosis of cardiomyocytes in a rat model of MI via regulation of the miR-138-5p/Sirt1 axis [88]. A recent study has illustrated that hypoxia promoted MSCs to secret lncRNA-UCA1-enriched EXO


*Mesenchymal Stem Cell-Derived Exosomes for Myocardial Infarction Treatment DOI: http://dx.doi.org/10.5772/intechopen.110736*

### **Table 2.**

*MSC-exosomal miRNAs for MI treatment.*

that had a cardioprotective effect via the lncRNA-UCA1/miR-873-5p/XIAP axis. Furthermore, exosomal lncRNA-UCA1 in human plasma may be considered a potential noninvasive biomarker for the diagnosis of AMI [89]. Similarly, Huang et al. showed that Atorvastatin pretreatment enhanced the therapeutic efficacy of MSC-EXO in a rat MI model via up-regulation of LncRNA H19 by promoting endothelial cell function [90]. The roles of MSC-exosomal LncRNA and their potential mechanism in MI treatment are summarized in **Table 3**.


**Table 3.**

*MSC-exosomal LncRNAs for MI treatment.*

### **3.3 Exosomal proteins in MSC-EXO for MI treatment**

MSC-EXOs further elicit benefit by delivering their cargo of potentially therapeutic proteins to recipient cells [91]. To date, nearly two thousand proteins in MSC-EXO have been identified [92–96]. Like miRNAs and lncRNAs, proteins in MSC-EXO have the potential to protect cardiomyocytes against injury following MI. Proteins in MSC-EXO whose role is basic cellular function, include common proteins, enzymes and signaling molecules [97]. One study suggested that hucMSC-EXO protected myocardial cells against apoptosis and promoted cell proliferation and angiogenesis by improving the expression of Bcl-2 family [98]. EXO secreted from CXCR4 overexpressing MSCs have been shown to promote cardiomyocyte survival and angiogenesis in ischemic hearts following MI via the AKT signaling pathway [99]. Deng et al. reported that EXO from AD-MSCs could ameliorate cardiac damage following MI by activating S1P/SK1/S1PR1 signaling and promoting macrophage M2 polarization [100]. The roles of MSC-exosomal proteins and their potential mechanism for MI treatment are summarized in **Table 4**.

Taken together, although current knowledge is limited, it can be inferred that various proteins carried by MSC-EXO protect ischemic cardiomyocytes through different mechanisms.


### **Table 4.**

*MSC-Exosomal proteins for MI treatment.*

*Mesenchymal Stem Cell-Derived Exosomes for Myocardial Infarction Treatment DOI: http://dx.doi.org/10.5772/intechopen.110736*

### **4. Potential strategies to improve the therapeutic efficacy of MSC-EXO for MI**

Although MSC-EXO-based therapy has shown promising results in MI, their therapeutic efficacy is heavily restricted by the low production and concentration of biological molecules released by EXO derived from MSCs under routine culture conditions. The production and biological components of MSC-EXO vary depending on the different external stimuli surrounding MSCs and MSC status. Therefore, modifying and optimizing exosomal content in MSC-EXO *in vitro* prior to transplantation to enhance their therapeutic efficacy for MI is vital. Over the past decades, several novel strategies, including altering culture conditions and pretreatment with pharmacological compounds and molecules, have been explored to generate modified MSC-EXO with greater benefits for MI treatment [56, 101]. More importantly, genetic modification of MSCs has had a great impact on the release of MSC-EXO, directly modulating their therapeutic efficacy. The influence of these factors on production and function of MSC-EXO will be discussed in the following sections. Different strategies to improve the therapeutic effects of MSC-EXO in MI are summarized in **Figure 2**.

### **4.1 MSC-EXO generated from different culture conditions**

The status of MSCs is largely dependent on culture conditions. Changes to culture conditions may influence MSC-EXO content and its biological functions. As a key impact on MSC culture, oxygen concentration plays a critical role in the regulation of gene expression, exon splicing, and phenotype of MSCs [102]. Therefore, oxygen gradients control MSC functions and generate different biological functions of MSC-EXO. MSCs survive under hypoxic conditions after transplantation into the ischemic heart and then release EXO to exert their benefit. Nonetheless MSCs are usually cultured under normoxic conditions *in vitro.* Therefore, the EXO released from MSCs under normoxic conditions *in vitro* and under hypoxic conditions *in vivo* carry different biological molecules with correspondingly different therapeutic effects. It has been reported that transplantation of MSCs under hypoxic conditions results in an enhanced therapeutic effect for MI [103, 104], indicating that hypoxic preconditioning may be a potential approach to prime MSC-EXO for MI treatment. Accumulating

**Figure 2.** *Different strategies to improve the therapeutic effects of MSC-EXO in MI.*

evidence shows that EXO from hypoxia-primed MSCs used to treat MI are superior to EXO from MSCs cultured under normoxic conditions [63, 105, 106]. Hypoxic preconditioning can enrich some specific miRNAs in the MSC-EXO that protect against MI by promoting angiogenic potential, attenuating inflammation and ameliorating apoptosis of cardiomyocytes [101]. It has been documented that hypoxic preconditioning of MSC-EXO elicits better therapeutic efficacy for MI by reducing the apoptosis of cardiomyocytes via upregulation of miR-210 that targets AIFM3 protein [75]. Zhang et al. showed that EXO isolated from hypoxic MSCs improved myocardial function in a rat model of myocardial ischemia-reperfusion injury by suppressing oxidative stress and the inflammatory response via delivery of miR-98-5p [107]. More importantly, EXO derived from MSCs stably overexpressing hypoxia inducible factor (HIF)-1α displayed an increased angiogenic capacity, partially due to the high level of Jagged1. This may have potential applications for MI treatment [108]. Indeed, transplantation of EXO collected from HIF-1α overexpressing MSCs improved heart function by promoting angiogenic formation in a rat model of MI [109]. Apart from hypoxic conditions, culture medium with different types of serum influence the characteristics of MSCs, modulating the efficacy of MSC-EXO-based therapies. Compared with normal serum, MSCs cultured with serum collected from the blood of mice with middle cerebral artery occlusion robustly demonstrated an upregulated level of miR-20a in their EXO [110]. Whether culturing MSCs with special serum can improve the efficacy of MSC-EXO for MI remains to be determined. Recently, it has been reported that the production of MSC-EXO can be augmented using a 3D porous scaffold structure instead of the traditional 2D culture in plastic plates, providing a novel strategy to optimize MSC-EXO for MI treatment [111]. Therefore, exploring suitable culture conditions for MSCs will not only improve the yield of EXO but also modify the therapeutic components of the EXO, ultimately enhancing their efficacy for MI treatment.

### **4.2 MSC-EXO generated following preconditioning with pharmacological compounds and biomolecules of MSCs**

There is accumulating evidence that preconditioning with pharmacological agents and biomolecules robustly improves the therapeutic efficacy of MSCs in MI by enhancing MSC survival and paracrine effects [112–115]. These results prompted us to determine whether pharmacological preconditioning could be a novel approach to enhance the cardioprotective effects of MSC-EXO. Our group has shown that compared with MSC-EXO, EXO isolated from MSCs pretreated with hemin, a potent heme oxygenase-1 (HO-1) inducer, exhibited better cardioprotection for MI via inhibition of cardiomyocyte senescence by elevating the level of miR-183-5p [116]. Huang et al. demonstrated that EXO obtained from atorvastatin-pretreated MSCs had greatly enhanced therapeutic efficacy for MI treatment in terms of promoting angiogenesis and inhibiting inflammation [90]. In addition to pharmacological agents, preconditioning with specific biomolecules can contribute to the secretion of MSC-EXO. EXO derived from interferon-gamma (IFN-γ)-treated MSCs exhibited more potent cardioprotective function in a rat model of MI by increasing angiogenesis and inhibiting cardiomyocyte apoptosis through upregulation of miR-21 [117]. Interestingly, Xiao et al. found that compared with MSC-EXO, EXO derived from MSCs pretreated with ischemic rat heart extracts enriched with IL-22 promoted the angiogenic capacity of human umbilical vein endothelial cells, indicating a novel preconditioning approach to optimize MSC-EXO for MI treatment [99]. These reports confirm that preconditioning with pharmacological compounds or biomolecules can alter the surrounding microenvironment of the culture conditions of MSCs and influence their paracrine effects, ultimately affecting the action of their derived EXO.

### **4.3 MSC-EXO isolated from genetically modified MSCs**

Genetic modification of MSCs via knockdown or overexpression of some RNAs or proteins is another efficient approach to improve the therapeutic effect of MSC-EXO. Our previous study showed that compared with MSC-EXO, administration of EXO secreted by MSCs transduced with macrophage migration inhibitory factor, a proinflammatory cytokine, exhibited a better therapeutic efficacy for MI by downregulating cardiomyocyte mitochondrial fragmentation, reactive oxygen species generation, and apoptosis [118]. A recent report revealed that EXO collected from stromal-derived factor 1-overexpressing MSCs intravenously administered in a mouse model displayed enhanced heart protection by inhibiting apoptosis and autophagy of myocardial cells and increasing angiogenesis by the regulating PI3K signaling pathway [119]. In another study, EXO from MSCs transduced with lentiviral CXCR4 promoted restoration of cardiac function in a rat model of MI by ameliorating cardiomyocyte apoptosis and increasing angiogenesis via upregulation of IGF-1α and p-AKT levels and downregulation of active caspase 3 level [120]. As discussed above, miRNAs are important biological components that play a pivotal role in the cardioprotective effect of MSC-EXO in MI [121–123]. Therefore, overexpression of miRNAs in MSCs can enhance the efficacy of MSC-EXO for MI treatment. Direct injection of MSC-EXO with miR-183-5p overexpression has been shown to result in better cardiac function via suppression of apoptosis and oxidative stress of cardiomyocytes by targeting FOXO1 [124]. Administration of EXO derived from miR-129-5p-modified MSCs displayed enhanced cardiac function following MI in mice by downregulating apoptosis of cardiomyocytes and production of inflammatory cytokines via targeting of HMGB1 [125]. Moreover, EXO derived from miR-126-overexpressing adipose-MSCs demonstrated better beneficial effects by inhibiting cardiac fibrosis and inflammatory cytokine expression and increasing angiogenesis [80]. Thus, genetically modified MSC-EXO have been considered an effective means by which to enhance their cardioprotective effects in MI.

### **5. Limitations and challenges of MSC-EXO-based therapy for MI**

Despite several significant advantages over MSCs, there remain some limitations and challenges to the clinical application of MSC-EXO for MI treatment. First, the rapid clearance of MSC-EXO from ischemic heart tissue after transplantation limits the beneficial effects for MI. An optimum delivery route for administration of MSC-EXO is unavailable. Currently, intramyocardial transplantation is the most efficacious. Exploration of alternative approaches to optimize retention and engraftment of MSC-EXO in the ischemic heart is urgently needed. Second, although the biological components in MSC-EXO, including miRNAs, lncRNA, recombinant proteins, and cytokines, have been intensively investigated, the exact mechanisms underlying MSC-EXO-based therapy for MI require further investigation. Third, MSC-EXO are currently isolated mainly depending on their vesicle size. Different sizes of MSC-EXO may contain different components with corresponding different therapeutic outcomes for MI. A more accurate isolation and purification method for MSC-EXO

should be adopted. Fourth, multiple harmful and unwanted biological components in MSC-EXO may restrict their efficiency. Several strategies to modify and remove unwanted components are under investigation. Finally, although classic high-speed centrifugation is the most common method used for MSC-EXO isolation, it is limited by the disadvantages of low production of EXO, high heterogeneity and non-scalability. A scalable isolation protocol for mass production of homogenous MSC-EXO for clinical application is needed.

### **6. Conclusion**

Over the past decades, administration of MSC-EXO has been shown to attenuate cardiac remodeling and improve heart function recovery following MI by inhibiting cardiomyocyte apoptosis, stimulating vascular angiogenesis, immunoregulation and rejuvenating cardiomyocyte senescence. Although the great potential of MSC-EXO therapy for heart function recovery has been clearly demonstrated, the therapeutic role of MSC-EXO in MI is extremely complex. Many issues remain to be carefully addressed and evaluated including the need for a high quality isolation protocol, delivery routes, and optimum EXO dose. In addition, potential risks must be carefully evaluated prior to translation into clinical trials. MSC-EXO-based therapy is still in its infancy and most experimental studies have been in a small animal model. The therapeutic efficacy of MSC-EXO should be evaluated in a porcine model or pre-clinical large animal model. This may provide further evidence to support clinical translation of MSC-EXO-based therapy to humans. Despite the unresolved issues, with the advanced development and technical breakthroughs in EXO research, it is hoped that clinical translation of MSC-EXO to promote cardiac regeneration and repair will soon be a reality for patients with MI.

### **Acknowledgements**

This research was in part supported by the Natural Science Foundation for Distinguished Scholarship of Guangdong Province of China (2022B1515020104 to Y. Zhang), the Distinguished Scholarship of Guangdong Provincial People's Hospital (KY0120220132 to Y. Zhang), National Natural Science Grant of China (No. 82270253 to Y. Zhang, No. 82072225 to X. Li), Natural Science Foundation of Chongqing (No. cstc2020jycj-msxmX0301 to H. Zheng) and NSFC Incubation Program of GDPH (KY012021167 to Y. Hong).

### **Conflict of interest**

The authors confirm that they have no conflicts of interest.

### **Other declarations**

The authors thank Ms. S Aglionby for editing the manuscript.

*Mesenchymal Stem Cell-Derived Exosomes for Myocardial Infarction Treatment DOI: http://dx.doi.org/10.5772/intechopen.110736*

### **Author details**

Huifeng Zheng1,2†, Yimei Hong1†, Bei Hu1 , Xin Li1 \* and Yuelin Zhang1 \*

1 Department of Emergency Medicine, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China

2 Department of Intensive Care Unit, Chongqing General Hospital, Chongqing, China

\*Address all correspondence to: xlidoct@qq.com; zhangyuelin1999@163.com

† Huifeng Zheng and Yimei Hong contributed equally to this study.

© 2023 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.

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### **Chapter 2**

## MSC-Derived Exosomes for Tissue Engineering and Disease Intervention

*Leisheng Zhang, Xiaowei Gao, Shixun Ma, Miao Yu, Xianghong Xu, Yuanguang Zhao, Shuang Chen, Yonghong Li, Xiaonan Yang, Tiankang Guo and Hui Cai*

### **Abstract**

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells or medicinal signaling cells or multipotent stem cells, are heterogeneous cell populations with unique immunomodulatory feature and hematopoietic-supporting capacity. MSCs function through a variety of approaches including paracrine and autocrine, direct- or trans-differentiation, bidirectional immunomodulation, and serving as constitutive microenvironment. Of them, exosomes and microvesicles function as the pivotal vehicle for mediating the ameliorative and therapeutic effect of MSCs toward various recurrent and refractory diseases, such as xerophthalmia, radioactive nasal mucosa injury, acute-on-chronic liver failure (ACLF), dermal chronic ulcers, and intrauterine adhesions. State-of-the-art renewal has also highlighted the promising prospective of MSC-derived exosomes (MSC-exo) and diverse biomaterial composites in regenerative medicine. In this book chapter, we mainly focus on the concept, biological phenotypes, preclinical research, and clinical practice of MSC-derived exosomes (MSC-Exos) and/or biomaterials, which will collectively supply overwhelming new references for the further development of MSC-Exos-based biotherapy and disease diagnosis in future.

**Keywords:** exosomes, tissue engineering, mesenchymal stem cells, disease intervention, immunomodulation

### **1. Introduction**

Exosomes have been considered as cell-derived nanovesicles, which are indicated in disease diagnosis and treatment via the intercellular transportation of cellular constituents, such as proteins, mRNAs, microRNAs, lipids, and cytokines [1–3]. Longitudinal studies have indicated the secretion of exosome by mammalian cells and the wide distribution in cellular systems [1]. For instance, Heo et al. reported the effect of exosomes upon atherosclerosis by inducing or inhibiting progression of disease through cell-to-cell communication, which suggested the application in disease

diagnosis and treatment [4]. State-of-the-art literatures also highlighted the marked technological advances of exosome-based nanotechnology to bloom the further exploitation of exosome-associated biology, pathology, chemistry, and therapeutics [5]. Despite the small membrane vesicles can be released and generated by a variety of eukaryotic cells, yet one of the major obstacles for potential application of exosomes is the strategies for high-efficient and robust enrichment of large-scale preparation with high quantity [1, 6].

Of the numerous parental cells for exosome enrichment, mesenchymal stem/ stromal cells (MSCs) are considered with robust immunomodulatory property and therapeutic potential [7–9]. Differ from the relative cell types, MSCs are advantaged stem cells with high percentage of mesenchymal-associated biomarker expression (e.g., CD44, CD73, CD90, CD105), whereas with minimal expression of type II major histocompatibility complex (MHC-II) or hematopoietic-related surface marker expression (e.g., CD31, CD34, CD43, CD45) [10–12]. In this chapter, we summarize the basic and clinical research of MSC-derived exosomes (MSC-exo), including the definition, biological phenotypes and significance, preclinical and clinical investigations, and the concomitant molecular mechanism. Taken together, the contents in this chapter will benefit the further development of MSC-exo-based therapeutic regimens for refractory and recurrent disorder administration in future.

### **2. MSCs and exosomes**

Mesenchymal stem/stromal cells (MSCs), also known as medicinal signaling cells, are unique multipotent stem cells with multi-lineage differentiation capacity and bidirectional immunomodulatory property [13]. Meanwhile, enormous literatures have demonstrated the hematopoietic-supporting effect of MSCs upon the selfrenewal and lineage differentiation potential of hematopoietic stem cells (HSCs), which thus play a critical role in physiologic hematopoiesis and hematologic malignancies [14]. Since the first identification from clinical samples in 1968 [15], MSCs of different origins have been consecutively isolated from adult tissues (e.g., bone marrow, adipose tissue, dental pulp) and perinatal tissues (e.g., placenta tissue, amniotic membrane, umbilical cord, amniotic fluid) [16, 17]. Of them, bone marrow-derived MSCs (BM-MSCs) have been considered with the widest range of application, while umbilical cord-derived MSCs (UC-MSCs) have been recognized with the most robust long-term proliferation and immunoregulatory capacity, respectively [13, 18]. As shown by the ClinicalTrials.gov website, over 1400 trials have been registered for a variety of refractory and recurrent disease administration (**Figure 1**).

To date, MSCs have been considered with various origins, including mesoderm, endoderm, ectoderm, trophoblasts, and neural crest cells (NCCs) [19, 20]. Therewith, MSCs have been recognized as heterogeneous stem cells and show variations in multitudinous biofunctions. Since the year of 2005, pioneering investigators in the field have employed pluripotent stem cells (PSCs) including induced PSCs (iPSCs) and embryonic stem cells (ESCs) for the preparation of MSCs for large-scale application. For instance, Zhang et al. reported the high-efficiency MSC generation from human PSCs (hPSCs) within two weeks by a cell programming strategy, which was accomplished by the combination of a master transcription factor named MSX2 (Msh Homeobox 2) and small molecule cocktails (TGF-β, bFGF, decitabine) [19]. Subsequently, Wei et al. took advantage of two chemical compounds including OICR-9429 (a JAK signal and histone methyltransferase inhibitor) and decitabine

*MSC-Derived Exosomes for Tissue Engineering and Disease Intervention DOI: http://dx.doi.org/10.5772/intechopen.110530*

**Figure 1.** *Illustration of MSC-based clinical trials.*

(DAC) for the enhanced induction of MSCs from hESCs [21]. Very recently, we reported the high-efficiency MSC induction from hESCs within two weeks via the non-gene-editing cell programming with LLY-507 (a JAK/STAT or BRD4 inhibitor) and AZD5153 (an epigenetic reader domain inhibitor) [10]. Similar to UC-MSCs, hPSC-MSCs revealed superiority in ex vivo proliferation and immunomodulation over the counterparts from diverse adult tissues.

MSCs function via diverse modes of action, including differentiation, immunomodulation, and secretion. Of them, exosomes and small extracellular vesicles (sEVs) are considered as the two major forms of derivatives of MSCs, which thus play a pivotal role in mediating disease diagnosis and treatment [22, 23]. Exosomes are nanosized sEVs secreted by the parent cells and play a pivotal role in diverse physiological and pathological processes [24]. To date, a variety of components have been identified from exosomes, such as nucleic acids (e.g., mRNAs, microRNAs, tRNAs, circRNAs), proteins (e.g., cytokines, peptides, amino acids, anti-inflammatory factors), lipids, metabolites, and relative bioactive substances [25].

Distinguish from liposomes and nanoparticles, exosomes with endogeneity and heterogeneity reveal unique and extensive advantages in the field of pathologic diagnosis and disease treatment [26]. Exosomes are nanoparticles with a diameter ranging from 50 nm to 200 nm, which are adequate to interact with organelles and relative intracellular vesicles [27, 28]. As a unique nanoscale spherical lipid bilayer vesicles, exosomes exhibit a density of 1.13–1.19 g • mL−1 according to the sucrose density gradient solution [29]. The conception of "exosomes" is firstly proposed by Trams et al. and referred to the vesicles derived from plasma membrane, which is also regarded as the membrane vesicles with 5′-nucleotide enzyme activity. Currently, exosomes are recognized as nano-particles with multitudinous physiological functions, which can be isolated from the exudation and supernatant of various cells and breezily cross the extracellular matrix and blood vessel wall [7, 30]. For instance, a category of exosomal microRNAs have been involved in the pathogenesis and diagnosis of tumors and immune disorders, which are adequate to mediate exosome-inflammasome crosstalk and epithelial mesenchymal transition (EMT), together with chemoresistance and metastasis of tumor cells [27, 30]. In 2021, Patil et al. reported the novel mechanisms of MSC-exo-mediated phagocytosis and opsonization of dying cardiomyocytes during myocardial ischemic injury both in vitro and in vivo [31].

As reviewed by Zhang et al., the ubiquitous exosomes are advantaged cell-free therapeutic products, which are small in size and thus breezily cross the extracellular matrix and blood vessel wall [7]. For instance, MSC-exo have shown considerable safety and therapeutic effects upon various diseases such as atherosclerosis, and acute and chronic wound model [4, 26]. Govindappa and the colleagues found that diabetic milieu were adequate to stimulate RNA-binding proteins like human antigen R (HuR) expression via increasing pro-fibrogenic and inflammatory responses in fibroblasts and cardiac fibrosis mediated by macrophages-derived exosomes [32]. Meanwhile, the chitosan hydrogel has been proved effective in boosting the stability and retention of exosomes enriched from placenta-derived mesenchymal stem cells (P-MSC-exo) and the contents (e.g., proteins, lipid, microRNAs), together with the resultant enhanced efficacy for hindlimb ischemia treatment and remission [12]. As mentioned above, MSC-derived exosomes (MSC-exo) and the relative sEVs have been reported with diverse application prospects in a variety of refractory and recurrent disease administration, yet the large-scale application for disease management is far from satisfaction, which largely attributes to the inherent disadvantages of exosomes, including the low yield, storage stability, low purity, and weak targeting [27].

### **3. Biomaterials/MSC-exo composites**

Biomaterials with tissue compatibility and inflammatory response mainly function via in contact with biological tissue, which are mainly applied in the medical field for tissue engineering or developing artificial organs for regenerative purposes. Generally, biomaterials can be divided into synthetic polymer biomaterials (silicone rubber, polyurethane, polyester, polyacrylonitrile), natural polymer biomaterials (regenerated fibers, chitin, collagen, hyaluronic acid), medical metal materials (titanium and titanium alloys, stainless steel, titanium-nickel memory alloys), inorganic biomedical materials (bioactive ceramics, carbon materials, glass materials), hybrid biomaterials (e.g., cross-linked hybridization of collagen, polyvinyl alcohol), and composite biomaterials (e.g., bioceramics, glass reinforced with glass fibers).

Current progress in materials and cell biology has extensively indicated the superiority of multiple biomaterials for preclinical and clinical application upon diverse relapse and recurrent diseases, and in particular, the composites of biomaterials and MSC-exo for tissue engineering and regenerative purposes attribute to the unique biocompatibility. In details, biomaterials with biocompatible and biodegradable properties are capable of facilitating the efficacy of MSCs or MSC-exo and enhancing their manifestations during anti-tumor immunity by endowing the therapeutic ability of these encapsulated constituents [33–35]. To date, a variety of biomaterials have been introduced for MSC-exo-based regimens in biomedicine, such as hydrogel acid (HA) (e.g., ε-caprolactone (PCL)/nano-hydroxyapatite (nHA) scaffold, chitosan hydrogel, PCL/nHA + HPCH hybrid scaffolds), gelatin, and nanomaterials [12, 36]. These biomaterials with promising prospective have been proved with the ability to reinforce the biological properties or functions of the encapsulated objectives including

### *MSC-Derived Exosomes for Tissue Engineering and Disease Intervention DOI: http://dx.doi.org/10.5772/intechopen.110530*

MSC-exo [37]. Of them HA and gelatin are considered as the two major forms of extracellular matrix, which are widely distributed in tissues of the body and benefiting the preparation of the compatible hybrid hydrogels by orchestrating the specific composition, scaffold structure, immune microenvironment, and the concomitant physico-chemical property [38, 39]. For example, as reviewed by Celikkin et al. and Xiao et al., Gelatin methacrylate-based hydrogels exhibit preferable properties over other counterparts in tissue engineering and disease administration on the basis of their biofunctionality as well as unique mechanical tenability (e.g., chemical properties, porosity, physical strength, and conductivity) [40, 41]. Similarly, a number of investigators in the field have also observed the hydrogel encapsulation, exosomeloaded thermosensitive hydorgels, and MSC-exo/hydrogel hybrid patch in controlling the release of paracrine factors, enhancing the maintenance of the biological activity, enhancing corneal epithelium regeneration from MSCs and MSC-exos [42–44].

Longitudinal studies have also suggested the application of multifarious collagens with high biocompatibility is applied as natural scaffolds for tissue engineering, including extracellular matrix (ECM), elastin, proteoglycans, and glycoproteins [45, 46]. For example, the implantation of engineered collagen matrices or resorbable collagen scaffolds has been reported effectively for the remission of meniscus defects by Warth et al. and Patil et al. [47, 48]. Of note, the latest progress has also highlighted the bioprinting of pure collagen or in combination with MSCs and/or MSC-exo for regenerative purposes as well [49]. On the basis of the immunomodulatory properties, MSC-exo have been used as a dermatological nano-therapeutic agent for the administration of oxidative stress-induced skin injury by modulating the NRF2 defense system and H2O2-stimulated epidermal keratinocytes [50].

Nanomaterials are defined by their diameters ranging from 1 nm to 100 nm, together with the facilitating effect upon the permeability and retention of the encapsulated cells or cellular components including MSCs and the derivatives (e.g., exosomes, sEVs) [51]. Nanomaterials, together with the nanostructure-mediated physical signals, have been recognized as splendid sources for mimicking ECM and enhancing the therapeutic effect of MSC-exo, which are tightly orchestrated by activating specific signals [52–57]. For example, Luo and the colleagues verified the feasibility of MSC-exo for amelioration of the inflammation-induced astrocyte alterations via the Nrf2-NF-κB signaling pathway [58]. Instead, Zhang et al. verified BM-MSC-derived exosomes (BM-MSC-exo) for promoting remyelination and reducing neuroinflammation via inhibiting the TLR2/IRAK1/NF-κB signaling pathway and increasing polarization of M2 phenotype in the demyelinating central nervous system [59]. Additionally, DiStefano et al. took advantage of Lactic-co-Glycolic Acid and the resultant hydrogel-embedded poly microspheres for the efficient delivery of hMSC-derived exosomes and the promotion of bioactive annulus fibrosus repair [60]. Very recently, Geng et al. reported the generation of the multifunctional antibacterial MSC-Exos@CEC-DCMC HG hydrogel (carboxyethyl chitosan-dialdehyde carboxymethyl cellulose) and BM-MSC-exo composite for accelerating diabetic wound healing [61]. Additionally, with the aid of optimized BM-MSC-exo and unique hierarchical scaffolds, Liu et al. demonstrated the application for bone regeneration by modulating the Smad pathway activated by Bmpr2/Acvr2b competitive receptor [62]. As to the underlying molecular mechanism of various biomaterials in MSC-exobased regimens, various biomaterials function mainly via orchestrating a series of mode of action, including integrating or incorporating with the encapsulated MSCexo, benefiting the secretion and maintenance of MSC-exo, serving as substrates or scaffolds, and reinforcing the antioxidant property as well [63–65]. Collectively,

biomaterials of different kinds with high biocompatibility have been recognized as momentous components of the formulations for tissue engineering and regenerative medicine [66–70].

### **4. MSC-exo-based clinical trials**

Attributes to the advantaged property, MSC-exo have been extensively explored in clinical trials. According to the ClinicalTrials.gov website (https://www.clinicaltrials.gov/) of National Institute of Health (NIH), a total number of 164 interventional trials have been registered up to February 4th, 2023 (**Figure 2**). Of them, most are in the phase 1 and phase 2 stages, together with the recruiting status (**Table 1**). To date, MSC-exo have been involved in numerous disease administration, including respiratory diseases (e.g., COVID-19, acute respiratory distress syndrome), digestive diseases (e.g., perianal fistula with Crohn's disease, familial hypercholesterolemia, irritable bowel disease, non-alcoholic fatty liver disease), cutaneous diseases (e.g., alopecia, psoriasis, endothelial dysfunction, wounds and injuries, oral mucositis, diabetic foot), vascular diseases (e.g., cerebrovascular disorders, myocardial infarction, myocardial ischemia, myocardial stunning), reproductive diseases (e.g., extreme prematurity, preterm, polycystic ovary syndrome), neurodevelopmental disorders (e.g., neuralgia, refractory depression, anxiety disorders, post-stroke dementia, Alzheimer disease, major depressive disorder, bipolar disorder, mild cognitive impairment, stroke, acute ischemic stroke, Parkinson disease), movement disorders (e.g., knee osteoarthritis, meniscus tear, tibial and knee injuries, arthralgia), endocrine system diseases (e.g., type 1 diabetes mellitus, type 2 diabetes mellitus), immunological disorders (e.g., allergic asthma, severe eosinophilic asthma), and urinary diseases (e.g., chronic kidney failure, bladder cancer, polycystic kidney disease), and even tumors (e.g., metastatic melanoma, colon cancer, non-Hodgkin's

**Figure 2.** *Illustration of MSC-exo-based clinical trials.*




*MSC-Derived Exosomes for Tissue Engineering and Disease Intervention DOI: http://dx.doi.org/10.5772/intechopen.110530*



### *MSC-Derived Exosomes for Tissue Engineering and Disease Intervention DOI: http://dx.doi.org/10.5772/intechopen.110530*

**Table 1.** *MSC-exo-based clinical trials.* lymphoma, lung cancer, non-small cell lung cancer, metastatic pancreatic adenocarcinoma, squamous cell carcinoma of the head and neck, advanced breast cancer, triple negative breast cancer, gynecologic cancer, prostate cancer, intraductal papillary mucinous neoplasm, malignant glioma, advanced hepatocellular carcinoma, acute myeloid leukemia, T-cell lymphoma, squamous cell carcinoma, advanced gastric cancer, colorectal cancer).

### **5. Conclusions**

Biomaterials and MSC-exo composites are advantaged sources for tissue engineering and regenerative medicine, which hold superiority over other therapeutic regimens attributes to the unique characteristics. In this chapter, we detailed and introduced the basic conception and latest updates of biomaterials and MSC-exo composites for biomedicine, which will collectively facilitate the further development of MSC-exo-based cell-free regimens in future. Of the multitudinous biomaterials, those with preferable tissue compatibility and minimal inflammatory response would reveal more robust application prospect with MSC-exo in future.

### **Acknowledgements**

The authors would like to thank the members in Key Laboratory of Molecular Diagnostics and Precision Medicine for Surgical Oncology in Gansu Province & NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor, Gansu Provincial Hospital, and Chinese Academy of Sciences Hefei Institute of Physical Science for their kind suggestions. This study was supported by grants from, National Natural Science Foundation of China (82260031, 82160534), Gansu Provincial Hospital Intra-Hospital Research Fund Project (22GSSYB-6, 21GSSYB-8, 20GSSY5-2), The 2022 Master/Doctor/Postdoctoral program of NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor (NHCDP2022004, NHCDP2022008, NHCDP2022014), the project Youth Fund funded by Shandong Provincial Natural Science Foundation (ZR2020QC097), Science and technology projects of Guizhou Province (QKH-J-ZK[2021]-107), Natural Science Foundation of Jiangxi Province (20224BAB206077, 20212BAB216073), Key project funded by Department of Science and Technology of Shangrao City (2020AB002, 2020 K003, 2021F013, 2022AB003), Jiangxi Provincial Key New Product Incubation Program Funded by Technical Innovation Guidance Program of Shangrao (2020G002), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2019PT320005), The 2021 Central-Guided Local Science and Technology Development Fund (ZYYDDFFZZJ-1), Guiding plan for scientific and technological development of Lanzhou (2019-ZD-102), Gansu Key Laboratory of molecular diagnosis and precision treatment of surgical tumors (18JR2RA033), Key talent project of Gansu Province of the Organization Department of Gansu provincial Party committee (2020RCXM076), Young Science and Technology Talent Support Project of Gansu Association for Science and Technology (GXH202220530-17), Natural Science Foundation of Gansu Province (21JR11RA186, 20JR10RA415).

*MSC-Derived Exosomes for Tissue Engineering and Disease Intervention DOI: http://dx.doi.org/10.5772/intechopen.110530*

### **Conflict of interest**

The authors declare no conflict of interest.

### **Notes/thanks/other declarations.**

Not applicable.

### **Appendices and nomenclature**


### **Author details**

Leisheng Zhang1,2,3,4\* † , Xiaowei Gao5† , Shixun Ma1† , Miao Yu1 , Xianghong Xu1 , Yuanguang Zhao3 , Shuang Chen3 , Yonghong Li1 , Xiaonan Yang<sup>6</sup> , Tiankang Guo1 and Hui Cai1

1 Laboratory of Molecular Diagnostics and Precision Medicine for Surgical Oncology in Gansu Province and NHC Key Laboratory of Diagnosis and Therapy of Gastrointestinal Tumor, Key Gansu Provincial Hospital, Lanzhou, China

2 CAS Key Laboratory of Radiation Technology and Biophysics in Institute of Biology and Hefei Institute of Physical Science, Chinese Academy of Sciences, Hefei, China

3 Jiangxi Research Center of Stem Cell Engineering, Jiangxi Health-Biotech Stem Cell Technology Co., Ltd., Shangrao, China

4 Department of General Practice, Affiliated Hospital of Xiangnan University, Chenzhou, China

5 Department of Otorhinolaryngology, Second Hospital of Tianjin Medical University, Tianjin, China

6 Department of Plastic and Reconstructive Surgery and Department of Hemangioma and Vascular Malformation, Plastic Surgery Hospital Affiliated to Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

\*Address all correspondence to: leisheng\_zhang@163.com

† Co-first authors.

© 2023 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.

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### Section 2
