A study for cardiac stem cells from embryonic stem cells using the cell sorting system

Abstract

Recently, various kinds of tissue specific stem cells, such as hematopoietic stem cells or neural stem cells, have been identified. It has been well known the embryoid body formed by aggregated embryonic stem (ES) cells differentiate into cardiac muscle cells, but cardiac progenitor/stem cells, which are supposed to exist during cardiac differentiation, have not been identified. Here, we intended to characterize cardiac progenitor/stem cells from ES cells using FACS sorting system. We considered that flk 1 might be a good marker for isolating cardiac progenitor/stem cells. Because flk 1 is one of the early markers of the anterior lateral mesoderm where vasculogenesis and also cardiomyogenesis originate from paired regions of the cardiac crescent. Differentiation of ES cells was induced in suspension culture in medium without leukemia inhibitory factor (LIF) . At 3.75 day from induction, the cells were stained with anti-flk 1 antibody, and cells with flk 1+ or fikl- were sorted respectively. Each subpopulation was co-cultured on OP 9 stromal cell. The differentiation of cardiac muscle cells was examined at day 10. Simultaneously, the gene expression analysis for Nkx-2.5 was carried out using RT-PCR.No colonies of contracting cardiac muscle cells were identified on tissue culture plate or type 4-collagen corted plates. Contracting muscle cells were identified only after plating on OP 9 cells, although no colonies on NIH 3 T 3 cells. These results suggest that OP 9 cells supply a signal indispensable for cardiac cell differentiation. However, there were no significant difference in numbers of contracting colonies and expression of Nkx 2.5 between flk 1 + and flk 1- cells. In this sense, the significance of flk 1 for cardiac cell differentiation still requires further discussion. Further examination remains to be done about signaling molecules for cardiac muscle cell differentiation.