Abstract 15793: Transdifferentiation of Bone Marrow Cells and Cardiomyogenesis in the Infarcted Heart

A major debate of the last decade involves the plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) and their ability to acquire cell lineages different from the organ of origin. Conflicting results obtained in different laboratories may depend on the functional heterogeneity and distinct transdifferentiation potential of single c-kit-BMCs. To address this issue, two methodologies were employed to label individual c-kit-BMCs and define their clonal fate in vivo: viral gene-tagging and fluorescent protein-based multicolor cell marking. c-kit-BMCs were infected with EGFP lentiviruses and injected in infarcted hearts. At 2 weeks, the regenerated myocardium was enzymatically digested and EGFP-labeled myocytes, endothelial cells, fibroblasts, and c-kit-cells were sorted. By employing a PCR-based method of detection of viral integrants, we searched for unique sites of viral insertion in the infected c-kit-BMCs and their progeny. Common insertion sites were found in the DNA of c-kit-BMCs and specialized cells, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, only specific subsets of c-kit-BMCs generated cardiomyocytes in vivo. This finding strongly indicated that c-kit-BMCs are functionally heterogeneous, being composed of myogenic and non-myogenic cells. To strengthen these observations, freshly isolated FACS-sorted c-kit-BMCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins. Different combinations of inserted vectors in individual c-kit-BMCs resulted in a variety of mixed colors, each indicative of the development of clonal populations. RGB-infected c-kit-BMCs were delivered to infarcted rats; at 4 days, engrafted undifferentiated c-kit-BMCs displayed most of the color combinations seen in vitro. However, only few of the color combinations present at 4 days persisted at the later time-points. These homogeneously colored colonies consisted of large patches of myocytes, which derived from the expansion and transdifferentiation of single c-kit-BMCs. Our findings document that c-kit-BMCs are functionally heterogeneous and have different ability to survive, engraft, and transdifferentiate in the ischemic heart.