Abstract 16884: Clonal Analysis of Hematopoietic Stem Cell Transdifferentiation in the Regenerated Myocardium
Early work documenting transdifferentiation of c-kit-positive hematopoietic stem cells (c-kit-HSCs) into myocytes has been difficult to reproduce. Conflicting results may be attributed to several factors, including the functional heterogeneity of the HSC pool, which may contain cells capable of forming myocytes and cells lacking this ability. We have addressed this issue by introducing clonal analysis of single HSCs. HSCs were infected with lentiviruses, and the unique site of viral insertion present in the infected HSC and its progeny was detected by immunolabeling or amplified by PCR. Freshly isolated FACS-sorted c-kit-HSCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins (RGB marking). Thus, single HSCs were marked by different combinations of inserted vectors, resulting in numerous clones of mixed colors. RGB-infected HSCs were injected in infarcted mice; at 4 days, engrafted HSCs showed a polyclonal pattern characterized by the presence of cells labeled by most of the color combinations seen in vitro. However, predominant expansion of homogeneously colored colonies derived from single HSCs was observed. At 2 weeks, only a few of the homogeneously colored colonies seen at 4 days persisted and formed large patches of cells composed of myocytes, endothelial cells (ECs), and smooth muscle cells. Thus, single c-kit-HSCs have different ability to survive, engraft, and transdifferentiate in the ischemic heart. Importantly, single HSCs gave rise to multiple cell types documenting their multipotency and the existence of a lineage relationship between HSCs and differentiated cardiac cells. These studies were complemented with a PCR-based method of detection of viral integrants. HSCs were transduced with an EGFP-lentivirus and injected in infarcted hearts. At 1 and 2 weeks, EGFP-positive myocytes, ECs, fibroblast, and c-kit-cells were sorted by FACS. Common insertion sites were found in the genomic DNA of all cell populations, documenting that single HSCs transdifferentiated into multiple cell lineages. Collectively, our findings demonstrate that HSCs are a functionally heterogeneous pool containing a cell subset with high transdifferentiation potential and regenerative ability.
- © 2012 by American Heart Association, Inc.