**5. Regenerative medicine and EVS**

Regenerative medicine is a relatively new concept and a complex domain that involves the restoration of damaged tissues using multiple techniques (e.g., SCs, biomaterials, differentiated autologous cells, or combinations of the aforementioned techniques). Regenerative medicine is focusing on repairing, regrowing, or replacing injured, malfunctioning, or missing tissue and addresses many tissular types: skin, heart tissue, cartilage tissue, bone tissue, adipose tissue etc. [101]. Thus, SC research focuses on their properties of repairing damaged tissues, either by producing new tissues by division and differentiation or by their partial repair.

Stem cells represent a highly interesting resource and were considered the ideal choice for regenerative therapies. SCs are defined as non-specialized cells, characterized by an enormous capacity of differentiation, which varies depending on their origin (embryonic, fetal, or adult). SCs are capable of differentiation into adipocytes, osteocytes, chondrocytes, endothelial cells, cardiomyocytes, pericytes, and smooth muscle cells [102–107]. They can also differentiate into neurogenic, cardiovascular, and neovascular pathways [108–113]. Allogeneic transplantation can be used in other applications due to the immunosuppressive properties of SCs [114].

Over the last decades, in an attempt to better understand SCs to use them in the processes of tissue repair and regeneration, multiple classifications have been made, depending on many aspects: the organism of origin (embryo, fetus, infant, adult), the tissue of origin within the adult-origin cells (mesenchymal tissue, hematopoietic, nervous, gastrointestinal, cutaneous, etc.), and the ability to divide (totipotent, pluripotent, multipotent, etc.) [115–117]. The understanding, even partial, of SCs' ability to divide, especially the asymmetric division of adult SCs has opened new horizons in terms of reparative and regenerative medicine [115]. Overpassing the initial idea that considers EVs as cellular debris, nowadays they are seen as tools for intercellular communication and as possible therapeutic vehicles. However, the same cannot be said about SCs. In the last decades, the interest for their properties has gained more and more interest. However, they have been regarded as cells at the origin of many pathologies since 1933, when Sabin *et al.* emphasize the possibility of radioactive damage to lymphoid tissue by affecting SCs [115, 118].

Although at the beginning researchers, scientists, and clinical doctors considered that the success of stem cell transplantation depends on the purity of the transplanted cells, not always the purer means also the better. Over time, it has become increasingly clear that the success of SC therapy depends on EVs and the soluble secreted factors because they play important paracrine roles. Our recent work also demonstrated that some other cellular types, such as the newly discovered telocytes, can act as cellular adjutants participating in the regenerative processes possibly through the released EVs influencing the microenvironment of the stem cell niche [118, 119]. Other additional evidence suggests that EVs can have not only regenerative properties, but also immunomodulatory roles, consequently summing up the therapeutic effects of stem cells. EVs, by contrast to stem cells, are nonimmunogenic and are not able to self-replicate [120]. In addition, EVs display powerful therapeutic potential, with positive outcomes regarding regeneration in many tissues (**Table 2**).

The central point of the pathophysiological mechanisms by which SCs contribute to the tissular repair are EVs that function as carriers of many biomolecules, such as miRNA, mRNA, cytokines, growth factors, differentiating factors with a key role in the main processes involved in tissue regeneration: immunomodulation, angiogenesis, differentiation [2]. Thus, multiple preclinical studies performed *in vitro* or *in vivo* on animal subjects have tried to identify the molecules involved and their role, important advances being made in diseases with high mortality and morbidity such as myocardial infarction, neuronal degeneration, osteoarticular diseases, skin ulcers, corneal damage etc. [121, 133, 134].

The use of SCs, despite promising results, has many disadvantages that require careful control of this procedure and the formation of very specific microenvironments to induce the differentiation of these cells [135]. Among the disadvantages mentioned before, those given by the ethical considerations of embryo use, the risk of uncontrolled differentiation, and the appearance of teratomas and genetic instability (especially those of embryonic origin) are the most important. To these disadvantages, a lower capacity for division and differentiation is added, as well as a laborious procedure for adult SCs acquirement [136, 137].

Among SCs, MSCs secrete growth factors and cytokines, with autocrine and paracrine properties. These substances inhibit the local immune system, fibrosis, and apoptosis, amplifying mitosis and differentiation of tissue-intrinsic reparative cells. These phenomena are known as trophic effects and differ from the direct differentiation of MSCs for tissue repair [138].

The numerous functions of MSCs in tissue regeneration and implicitly in the possible treatment of many diseases are mainly achieved by the secretion of exosomes loaded with key molecules (cytokines, growth factors, miRNA) and by molecules secreted directly into the extracellular environment with paracrine action [138–141].

However, they cannot produce infinite numbers of exosomes, repetitive isolation of cells being needed. The advantages of MSCs exosomes are their non-immunogenic


*Extracellular Vesicles as Intercellular Communication Vehicles in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.101530*

*MSCs—mesenchymal stem cells; CSCs—cardiac stem cells; BM-MSCs—bone marrow mesenchymal stem cells; ADSc—Adipose tissue stem cells; miRNA—microRNA; TCA-3—T-cell activation gene-3; SDF-1—stromal derived factor 1; VEGF—vascular endothelial growth factor; bFGF—basic fibroblast growth factor; SCF—F box containing complex; DKK 1—Dickkopf-related protein 1; TGF beta—transforming growth factor 1; MMP-13—matrix metalloproteinase; IL—interleukin; TNF—tumor necrosis factor; CCL—CC chemokine ligand; BMP—bone morphogenic protein.*

#### **Table 2.**

*The role of stem cell-derived EVs depending on their content and tissue type.*

property, the intrinsic therapeutic capacity of reducing tissue damage, large *ex vivo* expansion, conveniently reachable source, and clinically tested cell source [18].

MSCs are a source of small EVs which can favor angiogenesis and cell proliferation in infarcted myocardium, can inhibit cardiac remodeling, and improve ventricular functions [121, 142, 143].

Moreover, MSCs can repair infarcted myocardium through paracrine interactions. EVs derived from MSCs have a better therapeutic effect than simple MSCs therapy. In animal subjects, suffering from myocardial infarction, exosomes derived from MSCs

diminished inflammation, improved cardiac function, stimulated cardiomyocyte H9C2 cell proliferation, inhibited apoptosis induced by H2O2 and cardiac fibrosis, and slowed down the transformation of fibroblasts into myofibroblasts mediated by TGF-β [144].

Although macrovascular reperfusion is the gold standard therapy for acute myocardial infarction, heart failure developed due to deficient cardiac remodeling is still a major issue for long-term therapeutic management. Angiogenesis is crucial for tissular regeneration, therefore, interest for therapeutic enhancement of angiogenesis has increased. Preclinical and human clinical trials showed conflicting results, the use of one growth factor not being enough to promote adequate angiogenesis [144–147]. Cell transplantation could be an alternative/another solution [148, 149]. Stem cells were utilized as sources for new cardiac cells production (endothelial progenitor cells, MSCs, cardiac progenitor cells). Paracrine factors secreted by transplanted cells seem to influence endogenous repair of damaged tissues [121].

*In vivo* studies indicate that small EVs from MSCs that overexpress Akt can amplify neovascularization, ameliorating the left ventricle ejection fraction [150]. The angiogenic involvement is supported by the treatment of renal ischemic reperfusion injury with small EVs derived from umbilical cord MSCs ameliorated capillary density through promoting VEGF up-regulation, independently from HIF-1α [151]. Small EVs can also deliver miRNAs (miR-125a) to endothelial cells, favoring angiogenesis [152]. Comparable outcomes were also obtained from the use of MSCs which overexpress hypoxia-inducible factors (HIF-1α). Injection of exosomes from MSCs, containing Jagged1, and hypoxia-inducible factor—MSCs cultures led to angiogenesis *in vivo* and *in vitro*. Exosomes derived from HIF-1α-overexpressing MSCs have a strong angiogenic function, through an expansion in the packaging of Jagged1 [153]. In addition, the immune system has a big role in the repair of the ischemic myocardium, in the inflammatory and angiogenesis phases. Chemokines, cytokines, and the release of EVs with paracrine actions sustain this restoration. EVs favor tissular regeneration and angiogenesis, therefore, research in this area is of high interest for patients suffering from acute myocardial infarction.

A study evaluating the effect of intracoronary administration of cardiac-derived SCs-secreted small EVs showed a lower number and altered polarization state of CD68+ macrophages in the infarcted myocardium, with elevated expression of antiinflammatory genes (Arg1, IL4ra, Tgfb1, Vegfa). Macrophages primed with EVs from cardiac-derived SCs displayed high levels of miR-181b, which targets protein kinase C δ. Therefore, exosomal transfer of miR-181b into macrophages lowered the levels of protein kinase C δ transcript, underlining the cardioprotective properties of stem cell infusion after reperfusion [154].

According to L. Cambier and colleagues, cardiosphere-derived cells proved to reduce myocardial infarction size through secreted EVs-Y RNA fragment, found in generous concentrations in EVs from cardiosphere-derived cells, correlated with the potency of these cells *in vivo*. This fragment can be transferred from cardiac cells to target macrophages through EVs, inducing transcription and secretion of IL-10, offering cardioprotection. *In vivo* injection of EV-Y RNA fragment after reperfusion reduced the infarct size [121, 155].

One of the major issues in diabetic patients is inadequate myocardial angiogenesis, which is responsible for an elevated risk for ischemic heart disease in these patients. Exosomes loaded with miRNAs (miR-320-3p or 320a) derived from diabetic cardiomyocytes proved, they can influence angiogenesis in endothelial cell cultures. Moreover, miR-320-3p, together with the miR-29 family and miR-7a can regulate insulin secretion and its signaling pathways [156, 157].

*Extracellular Vesicles as Intercellular Communication Vehicles in Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.101530*
