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Cardiac progenitor characterization and multipotential differentiation capacity.

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posted on 13.06.2013, 02:24 authored by Nicolas Christoforou, Brian Liau, Syandan Chakraborty, Malathi Chellapan, Nenad Bursac, Kam W. Leong

A. The percentage of total cells expressing cell-surface antigens cKit, Flk1, and Sca-1 or the combinations of cKit/Flk1, and cKit/Sca-1 were determined by fluorescence-activated cell sorting before (gray) and after (black) puromycin addition for enriching Nkx2-5(+) cardiac progenitor cells. No RFP(+) cells were detected before addition of puromycin. The percentage of the five cell subpopulations also expressing RFP following puromycin addition is noted above the black columns. Error bars represent standard deviation. *denotes significant change (p<0.05) as determined by t-test statistical analysis. Isotype control antibodies were used as negative control in order to set the gates for the three cell surface antibodies. B. When cultured in suspension the derived iPS cells readily aggregated to form three-dimensional embryoid bodies which temporally differentiated into various cell types including spontaneously contracting cardiomyocytes detected as early as differentiation day 7. C. Following three days of puromycin antibiotic selection aggregates of spontaneously contracting RFP(+) cardiac progenitors were allowed to attach on gelatin-coated polystyrene. The cells in these aggregates stained positive for cardiac-specific actinin. D. Enzymatically dissociated and puromycin selected iPS-derived cardiac progenitors formed large-scale monolayers of spontaneously contracting cells. E. RFP-expressing (Myh6 promoter) and spontaneously contracting cardiomyocytes stained positive for the cardiac-specific marker Tnnt2. F. Acta2(+)/Tagln(+) smooth muscle cells were detected interspersed within the cultures of Act2(+) cardiomyocytes. G–H. Rare colonies of endothelial cells with varying size were detected within the cell monolayers as determined by immunostaining for Vwf.