Bone Marrow Cells Transdifferentiate into Cardiomyocytes and Repair the Infarcted Heart

A major controversy concerns whether c-kit-positive bone marrow cells (c-kit-BMCs) can acquire cell lineages different from their organ of origin. Technical challenges and variability in the protocols employed for cell isolation and detection of donor-derived structures may account only in part for the conflicting results obtained in different laboratories. We raised the hypothesis that c-kit-BMCs are functionally heterogeneous and possess distinct transdifferentiation potential. 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 sites of viral insertion in the isolated cells. 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, strongly indicating that the c-kit-BMC pool is composed of myogenic and non-myogenic cells. To strengthen these observations, 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 and gave rise to homogeneously colored colonies containing fluorescently labeled myocytes and vascular cells. The homogenous pattern of expression of the fluorescent proteins documented that the regenerated structures derived from the expansion and transdifferentiation of single c-kit-BMCs. Our findings demonstrate that c-kit-BMCs are functionally heterogeneous and have a different ability to survive, engraft, and transdifferentiate in the ischemic heart.