**2. Biology of mesenchymal stem cells**

MSCs are non-hematopoietic stem cells with multilineage potential that originate from the mesodermal germ layer. The pioneering studies conducted by Friedenstein et al. provided the first evidence that these fibroblast-like cells, described as spindle-shaped and clonogenic in culture conditions could be isolated from bone marrow via their inherent adherence to plastic in culture [31, 32]. MSCs are an integral part of the stromal microenvironment and support hematopoietic stem cells and regulate hematopoiesis, although they comprise only ~0.01–0.001% of the total nucleated cells in the bone marrow [33, 34]. Moreover, MSCs have been isolated from virtually every tissue type, including adipose tissue, liver, lung, skeletal and heart muscle, synovial membrane, amniotic fluid, placenta, umbilical cord blood, and dental pulp, suggesting that they reside in all organs [35–37].

MSCs are readily expanded *in vitro* and have the capacity, as classically defined, to differentiate into osteoblasts, chondrocytes, and adipocytes [38, 39]. Studies also strongly support a role for MSCs in neovascularization, with the capacity for differentiation into both endothelial [40, 41] and vascular smooth muscle cells [40]. Finally, MSCs can differentiate into myocytes: skeletal myocyte differentiation is widely accepted, whereas there is ongoing controversy as to whether MSCs have a robust ability to form cardiomyocytes [40, 42–45].

A key question regarding postnatal MSC function is the degree to which they participate in tissue homeostasis. For example, in the case of an osteogenic lineage, multiple investigators [61–63] have shown that exposure of MSCs to dexamethasone, β-glycerol phosphate, and ascorbic acid can lead to expression of alkaline phosphatase by the differentiated osteogenic cells with subsequent formation of a mineralized extracellular matrix [61]. Importantly, MSCs do retain the capacity for bone differentiation in vivo [38, 64]. For example, we have shown that subcutaneously implanting MSCs leads to osteoblast differentiation [38]. On the other hand, chondrogenic differentiation of MSCs can be achieved by treating MSCs with dexamethasone and TGF-β3 [58]. Similarly, dexamethasone together with insulin, indomethacin, and 1-methyl-3-isobutylxanthine can stimulate MSC differentiation into adipocytes, which express adipocyte-specific markers including peroxisome proliferator-activated receptor (PPAR)-γ [65].

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Cao et al. [38] studied the regulation of MSC differentiation into adipocytes and osteoblasts with relation to PPAR-γ, an essential checkpoint regulator of the "adipogenesis-osteogenesis balance." The study showed that S-nitrosoglutathione reductase (GSNOR)-deficient mice have reduced adipogenesis and increased osteoblastogenesis compared to normal mice (**Figure 3**). Notably, GSNOR MSCs had improved differentiation capacity for bone and reduced propensity for adipocytes. This is due to higher levels of S-nitrosylated PPAR-γ protein with subsequent inhibition of its transcriptional activity, suggesting a negative feedback regulation by NO-mediated S-nitrosylation. In addition, S-nitrosylation of PPAR-y inhibits binding affinity to its downstream target fatty acid-binding protein 4 (FABP4) promoters (**Figure 4**). Importantly, the MSC differentiation affected the phenotype on the whole animal level. GSNOR deficient mice have lower body weight and fat mass, accompanied by elevated bone formation. In another study regarding osteogenic regulation, investigators found that modulation of specific microRNAs (-148b, -27a, and -489) plays a crucial role in MSC early osteogenic differentiation [66]. This has a tremendous corollary in bone diseases such as osteoporosis by providing both pathophysiological and therapeutic insights. Indeed, MSC differentiation into other cell lines of mesenchymal origin can offer further understanding

into many other human disease processes, in support of future treatment strategies.

**Figure 3.** GSNOR deficient mice have reduced weight and body mass with increased bone formation.

No single cell surface marker specifically identifies MSCs. The International Society for Cellular Therapy has provided minimum criteria for defining multipotent human MSCs including (1) plastic-adherence under standard culture conditions; (2) expression of CD105, CD73, and CD90 and absence of hematopoietic cell surface markers, CD34, CD45, CD11a, CD19, and HLA-DR; and (3) *in vitro* differentiation into osteocytes, adipocytes, and chondrocytes under specific culture conditions [46]. However, MSCs can lose/acquire surface markers as they are isolated and expanded [47]. Furthermore, MSCs isolated from different tissues may exhibit a molecular fingerprint specific for their tissue of origin and thus vary in their differentiation capacity [48–50].

Bone marrow-derived MSC precursors (MPCs) have also been identified based upon specific cell surface marker expression, the most important being stromal precursor antigens (STRO-1, STRO-3) and CD271 [51–56]. *In vitro* studies suggest that the STRO-1 and STRO-3-enriched MPC populations have superior proliferative ability, multilineage regenerative capacity, and paracrine activity compared to MSCs [51, 54, 55], whereas CD271+ selection significantly increases clonogenic outgrowth of MPCs [52]. Preclinical studies using large animals have shown the efficacy of MPCs in acute MI and chronic ischemic and non-ischemic models of cardiomyopathy. Intracoronary injection of allogeneic MPCs in sheep after acute MI produced a 40% decrease in scar size and a 50% increase in vascular density [57]. Similarly, using echocardiography to guide the catheter-based endomyocardial injection of allogeneic MPCs into sheep 4 weeks post-MI resulted in an increase in left ventricular ejection fraction (LVEF), wall thickness, and vascular density. In a model of non-ischemic cardiomyopathy, transendocardial administration of ovine allogeneic cells produced decreased left ventricular end-systolic volume, stabilization of LVEF, decreased myocardial fibrosis and increased myocardial regeneration [53].

#### **2.1. Osteogenic, chondrogenic, and adipogenic differentiation**

As mentioned above, MSCs can be readily expanded *in vitro* and can differentiate into osteoblasts, chondrocytes, and adipocytes [38, 39]. Various growth factors and molecules promote MSC differentiation. For instance, global gene expression profiling arrays were utilized to identify RNA transcripts, which led to the identification that TGF-β, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) signaling pathways regulate MSC differentiation into adipogenic, osteogenic, and chondrogenic lineages [58, 59]. Adipogenic and osteogenic differentiation of MSCs were enhanced *in vitro* upon inhibition of TGF-β signaling but prevented chondrogenic differentiation. In contrast, inhibition of PDGF signaling decreased osteogenic differentiation, whereas inhibition of FGF receptor signaling completely blocked osteogenic differentiation and reduced chondrogenic differentiation. Moreover, inhibition of any one of these pathways decreased MSC proliferation. Differentiation thus depends substantially on the microenvironment [60].

A key question regarding postnatal MSC function is the degree to which they participate in tissue homeostasis. For example, in the case of an osteogenic lineage, multiple investigators [61–63] have shown that exposure of MSCs to dexamethasone, β-glycerol phosphate, and ascorbic acid can lead to expression of alkaline phosphatase by the differentiated osteogenic cells with subsequent formation of a mineralized extracellular matrix [61]. Importantly, MSCs do retain the capacity for bone differentiation in vivo [38, 64]. For example, we have shown that subcutaneously implanting MSCs leads to osteoblast differentiation [38]. On the other hand, chondrogenic differentiation of MSCs can be achieved by treating MSCs with dexamethasone and TGF-β3 [58]. Similarly, dexamethasone together with insulin, indomethacin, and 1-methyl-3-isobutylxanthine can stimulate MSC differentiation into adipocytes, which express adipocyte-specific markers including peroxisome proliferator-activated receptor (PPAR)-γ [65].

MSCs are readily expanded *in vitro* and have the capacity, as classically defined, to differentiate into osteoblasts, chondrocytes, and adipocytes [38, 39]. Studies also strongly support a role for MSCs in neovascularization, with the capacity for differentiation into both endothelial [40, 41] and vascular smooth muscle cells [40]. Finally, MSCs can differentiate into myocytes: skeletal myocyte differentiation is widely accepted, whereas there is ongoing controversy as

No single cell surface marker specifically identifies MSCs. The International Society for Cellular Therapy has provided minimum criteria for defining multipotent human MSCs including (1) plastic-adherence under standard culture conditions; (2) expression of CD105, CD73, and CD90 and absence of hematopoietic cell surface markers, CD34, CD45, CD11a, CD19, and HLA-DR; and (3) *in vitro* differentiation into osteocytes, adipocytes, and chondrocytes under specific culture conditions [46]. However, MSCs can lose/acquire surface markers as they are isolated and expanded [47]. Furthermore, MSCs isolated from different tissues may exhibit a molecular fingerprint specific for their tissue of origin and thus vary in their

Bone marrow-derived MSC precursors (MPCs) have also been identified based upon specific cell surface marker expression, the most important being stromal precursor antigens (STRO-1, STRO-3) and CD271 [51–56]. *In vitro* studies suggest that the STRO-1 and STRO-3-enriched MPC populations have superior proliferative ability, multilineage regenerative capacity, and para-

clonogenic outgrowth of MPCs [52]. Preclinical studies using large animals have shown the efficacy of MPCs in acute MI and chronic ischemic and non-ischemic models of cardiomyopathy. Intracoronary injection of allogeneic MPCs in sheep after acute MI produced a 40% decrease in scar size and a 50% increase in vascular density [57]. Similarly, using echocardiography to guide the catheter-based endomyocardial injection of allogeneic MPCs into sheep 4 weeks post-MI resulted in an increase in left ventricular ejection fraction (LVEF), wall thickness, and vascular density. In a model of non-ischemic cardiomyopathy, transendocardial administration of ovine allogeneic cells produced decreased left ventricular end-systolic volume, stabilization

As mentioned above, MSCs can be readily expanded *in vitro* and can differentiate into osteoblasts, chondrocytes, and adipocytes [38, 39]. Various growth factors and molecules promote MSC differentiation. For instance, global gene expression profiling arrays were utilized to identify RNA transcripts, which led to the identification that TGF-β, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) signaling pathways regulate MSC differentiation into adipogenic, osteogenic, and chondrogenic lineages [58, 59]. Adipogenic and osteogenic differentiation of MSCs were enhanced *in vitro* upon inhibition of TGF-β signaling but prevented chondrogenic differentiation. In contrast, inhibition of PDGF signaling decreased osteogenic differentiation, whereas inhibition of FGF receptor signaling completely blocked osteogenic differentiation and reduced chondrogenic differentiation. Moreover, inhibition of any one of these pathways decreased MSC proliferation. Differentiation thus depends sub-

of LVEF, decreased myocardial fibrosis and increased myocardial regeneration [53].

selection significantly increases

to whether MSCs have a robust ability to form cardiomyocytes [40, 42–45].

crine activity compared to MSCs [51, 54, 55], whereas CD271+

**2.1. Osteogenic, chondrogenic, and adipogenic differentiation**

stantially on the microenvironment [60].

differentiation capacity [48–50].

146 Stromal Cells - Structure, Function, and Therapeutic Implications

Cao et al. [38] studied the regulation of MSC differentiation into adipocytes and osteoblasts with relation to PPAR-γ, an essential checkpoint regulator of the "adipogenesis-osteogenesis balance." The study showed that S-nitrosoglutathione reductase (GSNOR)-deficient mice have reduced adipogenesis and increased osteoblastogenesis compared to normal mice (**Figure 3**). Notably, GSNOR MSCs had improved differentiation capacity for bone and reduced propensity for adipocytes. This is due to higher levels of S-nitrosylated PPAR-γ protein with subsequent inhibition of its transcriptional activity, suggesting a negative feedback regulation by NO-mediated S-nitrosylation. In addition, S-nitrosylation of PPAR-y inhibits binding affinity to its downstream target fatty acid-binding protein 4 (FABP4) promoters (**Figure 4**). Importantly, the MSC differentiation affected the phenotype on the whole animal level. GSNOR deficient mice have lower body weight and fat mass, accompanied by elevated bone formation. In another study regarding osteogenic regulation, investigators found that modulation of specific microRNAs (-148b, -27a, and -489) plays a crucial role in MSC early osteogenic differentiation [66]. This has a tremendous corollary in bone diseases such as osteoporosis by providing both pathophysiological and therapeutic insights. Indeed, MSC differentiation into other cell lines of mesenchymal origin can offer further understanding into many other human disease processes, in support of future treatment strategies.

**Figure 3.** GSNOR deficient mice have reduced weight and body mass with increased bone formation.

that transendocardial injections of allogeneic MSCs in swine following myocardial infarction (MI) results in cardiogenic differentiation of MSCs accompanied by increased proliferation and enhanced lineage commitment of endogenous CPCs, and reconstitution of niche-like structures [1]. This stimulation of endogenous CPCs by MSCs requires a complex molecular interaction and is a crucial component of the beneficial cell therapeutic effects [1, 28, 29, 77–79]. Histologic examination revealed chimeric clusters (niches) comprised of adult cardiomyocytes, transplanted MSCs and CPCs expressing connexin-43 gap junctions, and N-cadherin mechanical connections between cells. These findings support the notion that MSCs act both as progenitors for certain cell lineages and through their participation in niches, as supporting

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Stimulation of endogenous precursors may be a general mechanism underlying MSC bioactivity. We recently showed that in humans with endothelial dysfunction MSCs can trigger endogenous EPC activation increasing their number and functional quality [81]. Thus MSCs can serve as a powerful therapeutic tool by reconstituting endogenous stem cell niches as well

The hypothesis that exogenously delivered stem cells would promote organ regeneration through transdifferentiation into tissue-specific cells sparked interest in stem cell research and cell-based therapy and was originally supported by studies in the heart [82] where MSCs become cardiomyocyte-like cells and endothelial cells [40, 41, 43]. However, subsequent studies have revealed that the MSC-mediated regenerative process is more complex than was initially envisioned, and that several mechanisms underlie the ability of MSCs to reduce scar size and improve left ventricular structure and function after myocardial injury [33, 83, 84]. MSCs engraft and persist for several months in myocardium when delivered by transendocardial injection [1, 33, 40] and they reduce cardiac fibrosis and promote neovascularization and cardiomyogenesis [40, 77, 85, 86]. Importantly, cardiac magnetic resonance imaging (MRI) documented a reduction of infarct size, improvement in left ventricular shape (measured as sphericity index of the left ventricle), and improvement in tissue perfusion and regional contractility [87]. Together, these preclinical studies support the anti-fibrotic and proangiogenic

Preclinical studies have demonstrated that MSCs can differentiate into cardiomyocytes and/or vascular structures in both allogeneic [1, 40, 87] and xenotransplantation [88] models, contributing to cardiac functional improvement and reduction of infarct size. Remarkably, there has been no evidence of rejection in animals subjected to allogeneic transplantation of MSCs [1, 29, 40, 87]. These studies reveal that allogeneic MSCs represent a unique cell population for cellular therapy due to their anti-proliferative, immunomodulatory, and anti-inflammatory effects [2, 33, 89]. The absence of major histocompatibility class (MHC) II antigens [90–92] and the secretion of T helper type 2 cytokines characterize MSCs as both immunoprivileged and immunosuppressive [2, 92–94]. MSCs fail to induce proliferation

as enabling and augmenting the reparative abilities of endogenous stem cells.

cells for other lineages [80].

**2.3. Anti-fibrotic and proangiogenic effects**

role of MSCs in the repair of the injured myocardium.

**2.4. Immunomodulatory, anti-inflammatory, and anti-microbial effects**

**Figure 4.** Regulation of adipogenesis-osteogenesis by MSCs. GSNOR deficiency with ensuing elevated levels of S-nitrosylated PPAR-γ leads to a decrease in PPAR- γ transcriptional activity and binding affinity to FABP4 promoter. This results in increased osteogenesis and decreased adipogenesis, which has strong implications in bone disease. *Reproduced from Cao Y et al., JCI, 2015.*
