Mesenchymal Stromal Cells (MSCs) Acquire Cardiac-Specific Markers but Do Not Become Functional Cardiomyocytes and Retain the Stromal Phenotype.

Abstract

Although marrow-derived MSCs may be beneficial in treating models of ischemic heart disease, their ability to transdifferentiate into functional cardiomyocytes remains unclear. MSCs from female transgenic mice expressing green fluorescent protein (GFP) under the control of the cardiac-specific α-myosin heavy chain promoter were co-cultured with male rat embryonic cardiomyocytes (rCMs). This novel in vitro model permits identification and functional analysis of putatively transdifferentiated MSCs based on GFP expression. In co-culture, 6.3% of MSCs became GFP+. Quantitative PCR using murine-specific primers revealed that co-cultured MSCs express the cardiac-specific genes atrial natriuretic factor, Nkx2.5 and α-cardiac actin. Furthermore, immunohistochemistry on GFP+ MSCs established co-expression of the sarcomeric proteins troponin I and α-actinin, but without a clear sarcomeric pattern. Despite expression of cardiac genes, patch-clamp experiments illustrate that GFP+ MSCs did not fire action potentials and did not express voltage gated sodium and calcium currents (INa and ICa). In contrast, action potentials, INa and ICa were readily observed in all GFP− rCMs studied in the co-cultures. When investigated using slow voltage ramps, GFP+ MSCs displayed electrical properties typical of non-excitable cells, suggesting retention of a stromal cell phenotype. Indeed, detailed immunophenotyping of GFP+ MSCs demonstrated expression of all antigens used to characterize MSCs as well as the acquisition of additional markers of cardiomyocytes with the phenotype: CD45− CD34+ CD73+ CD105+ CD90+ CD44+ SDF1+ CD134L+ collagen type IV+ vimentin+ troponin T+ troponin I+ a-actinin+ connexin 43+. Although cell fusion between rCMs and MSCs was detectable by fluorescence in situ hybridization, the very low frequency (0.68%) cannot account for the phenotype of the GFP+ MSCs. In conclusion, we have identified a MSC population that shows plasticity towards the cardiomyocyte lineage but that retains mesenchymal stromal cell properties, including a non-excitable electrophysiological phenotype. The demonstration of a MSC-derived population co-expressing cardiac and stromal cell markers may explain the conflicting results in the literature regarding transdifferentiation and indicates the importance of extensive immunophenotypic and functional analysis of the manipulated cells. The data also infer the need to identify mechanisms other than cardiomyocyte differentiation that underlie the effects of MSCs on myocardial injury.