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

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 of allogeneic lymphocytes *in vitro* [90, 92], and suppress proliferation of T cells activated by allogeneic cells or mitogens [91]. This immunomodulatory capacity supports the feasibility of using allogeneic MSCs for cardiovascular regeneration as well as other clinical applications [2, 95]. Furthermore, MSCs have been used to treat severe graft-vs-host disease (GVHD) [13, 96], decreasing the potential of graft rejection and/or GVHD, and supporting the concept that MSCs are a unique cell population for regenerative medicine with minimal immune reactivity. Allogeneic MSCs have proven both safe and effective [5, 7, 11, 29, 89], highlighting that MSCs engrafted in the cardiac tissue despite potential HLA mismatching. An advantage of allogeneic MSCs is their potential use as an "off-the-shelf" therapeutic agent, precluding the need to obtain and expand bone marrow or another tissue source from the patient, and providing consistency to the cell product [97]. In addition, autologous cells may have functional deficiencies due to the underlying diseases, co-morbidities, lifestyle, concomitant medications, or age [98–105]. Although allogeneic MSCs may be cleared more rapidly than autologous cells after differentiation [106], immunologic clearance might also offer the advantage of reducing any long-term risks of cell implantation [8, 94, 107].

A potential approach to improve therapeutic potential is the combination of MSCs with c-kit+ CSCs [28, 29, 79]. Using a porcine model of chronic ischemic cardiomyopathy, the combination of autologous or allogeneic swine MSCs and c-kit+ CSCs provides greater reverse remodeling, scar size reduction, and functional improvements than MSCs alone [29, 79]. The demonstrated safety of cell-based therapy using MSCs [7, 9, 115, 116] and c-kit+ CSCs [117, 118] in patients with ischemic cardiomyopathy combined with these preclinical findings revealed important biological interactions between these two stem cell types that enhance therapeutic responses and led to the initiation of the Cardiovascular Cell Therapy Research Network (CCTRN) sponsored, Combination of Mesenchymal and C-kit+ Cardiac Stem Cells as Regenerative

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There is evidence that senescence impairs the capacity of MSCs for multi-lineage differentiation, homing, immune modulation and wound healing [102, 103]. As stem cells age, they undergo a "quiescence-to-senescence switch" that impairs their function [102, 104, 119, 120] (**Figure 5**). The mechanisms underlying the age-related declines in stem cell function involve intrinsic aging as well as age-related changes in their tissue microenvironment, including extracellular matrix components and the stem cell niche [101, 104, 121], thereby adversely impacting self-renewal and therapeutic potential. This has implications when considering the age and comorbidities of patients and donors. For example, dysfunctional stem cell niches

**Figure 5.** Proposed mechanisms of aging-induced stem cell dysfunction. (A). Normal stem cell function involves activation of a quiescent stem cell to divide asymmetrically giving rise to a new stem cell (self-renewal) and another daughter cell that undergoes proliferation and differentiation. (B). Failure of self-renewal involves differentiation of both daughter cells, leading to a gradual depletion of the stem cell pool. (C). Aberrant differentiation may result from the abnormal skewing of the distribution of progeny toward one fate instead of various potential fates. Another potential mechanism involves the daughter cells acquiring abnormal fates that are not part of the normal repertoire. (D). Impaired stem cell response may be due to a decline or impairment in extrinsic or intrinsic signals. (E). Senescence and apoptosis of the quiescent stem cell or among the progeny following activation has also been described in aging. Adapted from

Jones DL et al., Nature Cell Biology, 2011.

Therapy for Heart Failure (CONCERT-HF; NCT02501811) clinical trial.

**2.6. MSC senescence and potential malignant transformation**

An important concern, and common exclusion criteria for participation in clinical trials is that the potential immunosuppressive effect of MSCs may lead to an increased risk of infection in patients who are already immunosuppressed due to medical therapy or concurrent chronic disease. In this regard, recent data has shown that MSCs exert significant anti-microbial effects through both direct and indirect mechanisms [108]. Indirect mechanisms include regulation of macrophages, neutrophils, phagocytes, and another pro- and anti-inflammatory cells of the immune system, whereas indirect mechanisms involve the secretion of anti-microbial peptides and proteins (AMPs) and the expression of indoleamine 2,3-dioxygenase, interleukin-17, and other molecules [94, 108]. Indeed, the anti-microbial effects of MSCs have been demonstrated in preclinical studies of sepsis, acute respiratory distress syndrome, and cystic fibrosis-related infections [108].

#### **2.5. Enhancement of MSC therapy**

Therapeutic interventions to optimize MSC function, such as growth factor administration [109–112], gene therapy [110], and modulation with small molecules or other pharmacologic approaches [110] are promising options under preclinical and clinical investigation to potentiate myocardial repair and regenerative capacity. For example, in the phase I cardiopoietic stem cell therapy in heart failure (C-CURE) trial and subsequent phase II/III congestive heart failure cardiopoietic regenerative therapy (CHART-1) study [72, 109, 113], autologous bone marrow-derived MSCs from patients with ischemic cardiomyopathy were treated exvivo with a cardiogenic cytokine cocktail to enhance their cardiac lineage commitment. In C-CURE, the authors reported significant improvement in cardiac function, physical performance, hospitalization, and event-free survival in the cell therapy group compared to controls [109]. However, the larger CHART-1 trial reported neutral results at 39 weeks of follow up with regards to composite and individual outcomes, including all-cause mortality, heart failure events, and surrogate cardiac structural and functional endpoints [113]. A sub-analysis of the CHART-1 study extended the follow-up period to 52 weeks at which point the anti-remodeling properties of the cardiopoietic MSCs became evident [72]. These findings are consistent with those of other clinical trials of MSC-based therapy for ischemic cardiomyopathy [7, 9, 114].

A potential approach to improve therapeutic potential is the combination of MSCs with c-kit+ CSCs [28, 29, 79]. Using a porcine model of chronic ischemic cardiomyopathy, the combination of autologous or allogeneic swine MSCs and c-kit+ CSCs provides greater reverse remodeling, scar size reduction, and functional improvements than MSCs alone [29, 79]. The demonstrated safety of cell-based therapy using MSCs [7, 9, 115, 116] and c-kit+ CSCs [117, 118] in patients with ischemic cardiomyopathy combined with these preclinical findings revealed important biological interactions between these two stem cell types that enhance therapeutic responses and led to the initiation of the Cardiovascular Cell Therapy Research Network (CCTRN) sponsored, Combination of Mesenchymal and C-kit+ Cardiac Stem Cells as Regenerative Therapy for Heart Failure (CONCERT-HF; NCT02501811) clinical trial.

#### **2.6. MSC senescence and potential malignant transformation**

of allogeneic lymphocytes *in vitro* [90, 92], and suppress proliferation of T cells activated by allogeneic cells or mitogens [91]. This immunomodulatory capacity supports the feasibility of using allogeneic MSCs for cardiovascular regeneration as well as other clinical applications [2, 95]. Furthermore, MSCs have been used to treat severe graft-vs-host disease (GVHD) [13, 96], decreasing the potential of graft rejection and/or GVHD, and supporting the concept that MSCs are a unique cell population for regenerative medicine with minimal immune reactivity. Allogeneic MSCs have proven both safe and effective [5, 7, 11, 29, 89], highlighting that MSCs engrafted in the cardiac tissue despite potential HLA mismatching. An advantage of allogeneic MSCs is their potential use as an "off-the-shelf" therapeutic agent, precluding the need to obtain and expand bone marrow or another tissue source from the patient, and providing consistency to the cell product [97]. In addition, autologous cells may have functional deficiencies due to the underlying diseases, co-morbidities, lifestyle, concomitant medications, or age [98–105]. Although allogeneic MSCs may be cleared more rapidly than autologous cells after differentiation [106], immunologic clearance might also

offer the advantage of reducing any long-term risks of cell implantation [8, 94, 107].

fibrosis-related infections [108].

cardiomyopathy [7, 9, 114].

**2.5. Enhancement of MSC therapy**

150 Stromal Cells - Structure, Function, and Therapeutic Implications

An important concern, and common exclusion criteria for participation in clinical trials is that the potential immunosuppressive effect of MSCs may lead to an increased risk of infection in patients who are already immunosuppressed due to medical therapy or concurrent chronic disease. In this regard, recent data has shown that MSCs exert significant anti-microbial effects through both direct and indirect mechanisms [108]. Indirect mechanisms include regulation of macrophages, neutrophils, phagocytes, and another pro- and anti-inflammatory cells of the immune system, whereas indirect mechanisms involve the secretion of anti-microbial peptides and proteins (AMPs) and the expression of indoleamine 2,3-dioxygenase, interleukin-17, and other molecules [94, 108]. Indeed, the anti-microbial effects of MSCs have been demonstrated in preclinical studies of sepsis, acute respiratory distress syndrome, and cystic

Therapeutic interventions to optimize MSC function, such as growth factor administration [109–112], gene therapy [110], and modulation with small molecules or other pharmacologic approaches [110] are promising options under preclinical and clinical investigation to potentiate myocardial repair and regenerative capacity. For example, in the phase I cardiopoietic stem cell therapy in heart failure (C-CURE) trial and subsequent phase II/III congestive heart failure cardiopoietic regenerative therapy (CHART-1) study [72, 109, 113], autologous bone marrow-derived MSCs from patients with ischemic cardiomyopathy were treated exvivo with a cardiogenic cytokine cocktail to enhance their cardiac lineage commitment. In C-CURE, the authors reported significant improvement in cardiac function, physical performance, hospitalization, and event-free survival in the cell therapy group compared to controls [109]. However, the larger CHART-1 trial reported neutral results at 39 weeks of follow up with regards to composite and individual outcomes, including all-cause mortality, heart failure events, and surrogate cardiac structural and functional endpoints [113]. A sub-analysis of the CHART-1 study extended the follow-up period to 52 weeks at which point the anti-remodeling properties of the cardiopoietic MSCs became evident [72]. These findings are consistent with those of other clinical trials of MSC-based therapy for ischemic There is evidence that senescence impairs the capacity of MSCs for multi-lineage differentiation, homing, immune modulation and wound healing [102, 103]. As stem cells age, they undergo a "quiescence-to-senescence switch" that impairs their function [102, 104, 119, 120] (**Figure 5**). The mechanisms underlying the age-related declines in stem cell function involve intrinsic aging as well as age-related changes in their tissue microenvironment, including extracellular matrix components and the stem cell niche [101, 104, 121], thereby adversely impacting self-renewal and therapeutic potential. This has implications when considering the age and comorbidities of patients and donors. For example, dysfunctional stem cell niches

**Figure 5.** Proposed mechanisms of aging-induced stem cell dysfunction. (A). Normal stem cell function involves activation of a quiescent stem cell to divide asymmetrically giving rise to a new stem cell (self-renewal) and another daughter cell that undergoes proliferation and differentiation. (B). Failure of self-renewal involves differentiation of both daughter cells, leading to a gradual depletion of the stem cell pool. (C). Aberrant differentiation may result from the abnormal skewing of the distribution of progeny toward one fate instead of various potential fates. Another potential mechanism involves the daughter cells acquiring abnormal fates that are not part of the normal repertoire. (D). Impaired stem cell response may be due to a decline or impairment in extrinsic or intrinsic signals. (E). Senescence and apoptosis of the quiescent stem cell or among the progeny following activation has also been described in aging. Adapted from Jones DL et al., Nature Cell Biology, 2011.

have been implicated in the aging frailty syndrome, which is characterized by decreased strength, endurance, physiologic function, and reserve capacity in multiple organ systems [122, 123]. Moreover, aging, renal failure, C-reactive protein (CRP) levels, and other adverse health parameters have been shown to correlate significantly with poor angiogenic potency of bone marrow stem cells [105, 124]. These studies suggest that the therapeutic potential of autologous MSCs obtained from patients may be limited, whereas more robust repair and regeneration would occur by using allogeneic MSCs from young, healthy donors. Indeed, two clinical trials in patients with ischemic and dilated cardiomyopathy, respectively, compared autologous to allogeneic MSCs and found that although both provided benefits in cardiac structural endpoints, the allogeneic MSCs provided greater cardiovascular functional benefits [5, 7, 81]. On the other hand, a study on the impact of recipient age on the efficacy of MSC therapy found that older (>60 years of age) patients responded just as effectively as younger (<60 years of age) patients when administered either autologous or allogeneic MSC therapy for chronic ischemic cardiomyopathy [125]. This finding is highly significant since the majority of the population with cardiovascular disease requiring cell-based therapy is aged.

Furthermore, in a mouse model of myocardial infarction, treatment with female MSCs produced greater improvement of cardiac functional endpoints than treatment with male MSCs [134]. Estradiol has been shown to contribute to these differences [135, 136]. A more complete understanding of how MSCs are influenced by donor sex and recipient hormonal environment is needed to address sex-related disparities in clinical outcomes as well as to optimize

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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 [41, 43]. However, subsequent studies have revealed that the MSC-mediated cardiac regenerative process is more complex than was

Multiple clinical trials suggest that MSCs can ameliorate left ventricular remodeling and improve cardiac function in patients with acute and chronic ischemic cardiomyopathy [7, 9, 11, 72, 84, 115, 116, 137–141]. The Transendocardial mesenchymal stem cells and mononuclear bone marrow cells for ischemic cardiomyopathy (TAC-HFT) trial demonstrated reverse remodeling and improved regional contractility of the scar as well as improved functional capacity and quality of life over 1 year in patients with chronic ischemic cardiomyopathy treated with transendocardial injection of autologous bone marrow-derived MSCs versus bone marrow mononuclear cells or placebo [9, 142]. The mesenchymal stromal cells in chronic ischemic Heart Failure (MSC-HF) trial showed that intramyocardial injection of autologous bone marrow-derived MSCs in patients with severe ischemic cardiomyopathy improved ventricular function and myocardial mass [140]. The same group showed that intramyocardial delivery of autologous MSCs into patients with coronary heart disease and refractory angina provided a sustained effect (3-year follow-up) in improving exercise capacity and ventricular function, and reducing hospitalization rates and revascularizations [143]. As mentioned previously, the CHART-1 study also demonstrated the anti-remodeling properties of cardiopoietic MSCs at the 1-year follow-up [72]. Encouraging results from preclinical studies with combination therapy [28, 79] have led to the initiation of the CONCERT-HF (NCT02501811) trial by the Cardiovascular Cell Therapy Research Network (CCTRN) in an effort to examine the effects of the transendocardial delivery of a combination of autologous bone marrowderived MSCs and cardiac progenitor cells into patients with ischemic cardiomyopathy.

Autologous adipose tissue-derived MSCs are also undergoing investigation in the cardiovascular field. The adipose-derived stromal cells for treatment of patients with chronic ischemic heart disease (MyStromalCell) trial was a phase II, first-in-man, single-center, double-blind, randomized, and placebo-controlled study of intramyocardial injections of autologous adipose-derived MSCs in patients with chronic ischemic heart disease and refractory angina but preserved ejection fraction [111, 112]. The MSCs were obtained from abdominal adipose tissue,

**3. MSCs as a regenerative therapeutic for cardiovascular diseases**

transplanted MSC function and survival.

initially envisioned (**Figure 6**).

**3.1. Clinical trials in cardiac disease**

Although the evidence is conflicting [126–130], clinical trials of MSC therapy usually exclude patients with a history of cancer due to concerns regarding the MSCs' potential for carcinogenesis. It remains unclear whether MSCs have the potential to undergo spontaneous malignant transformation and/or whether they interact with surrounding tumor stromal elements [129–131]. Spontaneous malignant transformation of human bone marrow-derived MSCs has been shown in vitro during long-term cultures [127]. These MSCs underwent faster proliferation, failed to undergo complete differentiation, and exhibited altered morphology and phenotype. Moreover, when these altered MSCs were administered to immunodeficient mice rapid-growing tumors throughout the lung tissue were found. On the other hand, in a separate study [128], human bone marrow-derived MSCs were grown in culture and assessed at different time points for expression of various tumor-related proteins until they reached senescence or passage 25. A progressive decrease in proliferative capacity with shortened telomeres was observed in most cultured MSCs until they reached senescence. In addition, the MSCs did not express telomerase activity or telomerase reverse transcriptase transcripts, and no chromosomal abnormalities or alternative lengthening of telomeres were observed, supporting the safety of in vitro MSC expansion, and therapeutic use. Despite these encouraging findings, the functional, phenotypic, and genetic characterization of culture-expanded MSCs merits further careful study [129, 131, 132]. In addition, recent findings indicate that various direct (e.g., cell fusion) and indirect (e.g., exosome or vesicle-mediated) interactions between MSCs and cancer cells can produce functional interference and/or mutual acquisition of new cellular properties [130]. These functional and phenotypic cellular alterations can lead to changes in metastatic behavior and induce new cancer stem cell development. On the other hand, exosomes and vesicle-mediated mechanisms may be a promising therapeutic tool against cancer.

### **2.7. Sex differences in MSCs**

Sex differences exist in many disease states as well as with respect to the role of MSCs in organ repair and regeneration after injury. There is evidence that female MSCs exhibit decreased apoptosis, interleukin-6, and tumor necrosis factor and increased endothelial growth factor and vascular endothelial growth factor expression compared to male donor MSCs [133]. Furthermore, in a mouse model of myocardial infarction, treatment with female MSCs produced greater improvement of cardiac functional endpoints than treatment with male MSCs [134]. Estradiol has been shown to contribute to these differences [135, 136]. A more complete understanding of how MSCs are influenced by donor sex and recipient hormonal environment is needed to address sex-related disparities in clinical outcomes as well as to optimize transplanted MSC function and survival.
