Proliferation of cardiomyocytes derived from embryonic stem cells

Introduction

Cardiomyocytes are the ideal cell type for cardiac grafting, but mature cardiomyocytes do not proliferate, thus inhibiting their potential as a cell source for myocardial repair. During embryological development, however, cardiomyocytes undergo mitosis, suggesting that early cardiomyocytes possess the mechanism to proliferate. To date, few studies have quantitatively examined the proliferation of cardiomyocytes derived from embryonic stem cells. Thus, we have been studying the proliferation of cardiomyocytes derived from human embryonic stem (ES) cells during the time course of in vitro cardiomyogenic differentiation.

Methods

Undifferentiated H1 and H7 human ES cells are cultured with mouse embryonic fibroblast conditioned-media and passaged weekly. ES clusters are dissociated with collagenase IV and cultured in suspension for 4 days to initiate differentiation as embryoid bodies (EBs). EBs are re-plated and cell outgrowths are cultured for several weeks thereafter. At different stages of differentiation, the EB outgrowths are dissociated with Blendzyme and the cells are fractionated over a Percoll gradient, according to previously described methods [1]. Enriched fractions of cardiomyocytes are cultured in 2-well chamber slides, pulsed with BrdU and fixed for immunohistochemical analysis.

Results

Beating cell foci, indicative of cardiomyogenesis, are readily apparent after 10-12 days of differentiation and continue to beat for several weeks. A readily identifiable proportion of proliferating (BrdU+) human cardiomyocytes within the enriched cultures have been observed at various stages of differentiation. Continued efforts are being made to quantify the percentage of BrdU+ cardiomyocytes and analyze key in vitro parameters that can be optimized to promote the proliferation of the human ES-derived cardiomyocytes.

PICTURES FOLLOWING:

Conclusions

In contrast to mouse ES cells, cardiomyocytes derived from human ES cells appear to proliferate extensively in vitro. This system represents the first in vitro model capable of elucidating the molecular mechanisms regulating the proliferation of human cardiomyocytes. Future molecular-based strategies to promote the proliferation of cardiomyocytes from human ES cells may facilitate the expansion of these cells for therapeutic purposes.

Literature Cited

[1] Chunhui Xu, Shailaja Police, Namitha Rao, Melissa K. Carpenter. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circulation Research 91: 501-508, 2002.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12242268&dopt=Abstract