**2.2. Cardiac differentiation**

Cardiomyogenic differentiation of MSCs is of key interest for cardiac regenerative medicine, particularly ischemic and non-ischemic cardiomyopathy [40, 67, 68]. Treating MSCs with 5-azacytidine produces spontaneous, synchronous beating cells in culture with ventricular myocyte-like potentials, suggesting that MSCs are able to transdifferentiate into cardiomyocytes [43]. Alternative and potentially safer factors that induce differentiation into a cardiomyocyte phenotype include conditioned media containing bone morphogenetic protein-2 (BMP-2) and FGF-4 [69] as well as insulin, dexamethasone, and ascorbic acid [70]. The combination of these factors induces overexpression of cardiomyocyte-specific proteins, leading to cardiomyogenic differentiation for possible use in disease processes of injured myocardium [69–72]. Indeed, expression of myotubules, α-actinin, SERCA2 and other cardiac-related proteins in transdifferentiated cells may serve to attenuate cardiac infarct size and enhance perfusion, and regional function as suggested by early *in vivo* studies [73, 74]. Co-culture of mouse or rat MSCs with rat neonatal ventricular myocytes also stimulates MSC transdifferentiation into cardiomyocytes [75, 76]. The necessity of cell-to-cell contact [1, 75] versus secreted factors within the cardiac microenvironment [76] as a requirement for cardiomyogenic differentiation remains unclear.

MSC therapy promotes cardiomyogenesis not only by direct cardiomyocyte differentiation, but also by stimulating endogenous c-kit+ cardiac progenitors (CPCs) to proliferate, undergo lineage commitment, and form transient amplifying cells [1, 28, 29, 77–79]. We demonstrated 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 cells for other lineages [80].

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