Abstract 3457: Derivation of Functional Cardiomyocytes from Parthenogenetic Stem Cells
Introduction: Parthenogenetic stem cells (PSCs) can be derived from unfertilized, chemically activated oocytes. They can principally give rise to derivatives of all three germ layers but evidence for differentiation of PSC into mature and functional cell types is scarce. In this study we assessed the cardiac differentiation capacity of PSCs in vitro and in vivo.
Methods: Oocytes from B6D2F1 transgenic mice, expressing enhanced green fluorescent protein (EGFP) under the control of the cardiac specific α-myosin heavy chain promoter (MHC-EGFP), were parthenogenetically activated with SrCl2 (10 mM) in the presence of cytochalasin B (5 μg/ml). Subsequently, PSCs were isolated from the inner cell mass of blastocyst stage parthenotes and cultured on murine embryonic feeder layers in the presence of leukemia inhibitory factor. Differentiation of PSCs was initiated in embryoid body (EB) cultures. Fluorescence-activated cell sorting (FACS) was used to enrich parthenogenetic cardiomyocytes (PCMs) from MHC-EGFP PSCs. In vitro PCMs were characterized by confocal laser scanning microscopy, intracellular calcium ([Ca2+]i) imaging, and action potential (AP) recordings. In vivo differentiation and function, respectively, of PCMs were assessed by histology and [Ca2+]i imaging with two-photon microscopy in chimeric mice generated by injection of PSCs into wild type blastocysts.
Results: PSC-derived EBs were beating spontaneously. FACS at the time of maximal α-MHC transcript abundance (EB culture days 19 –22) demonstrated cardiogenic potential in 1% of the PSC derivatives. The latter displayed spontaneous contractions as well as cardiotypic [Ca2+]i transients. PCMs displayed ventricle- (61%), atrial- (11%), and pacemaker-like APs (15%; n = 102 cells analyzed). Accordingly, EGFP-positive PCMs could also be identified in atrial, ventricular and AV-nodal tissue in chimeric hearts and showed simultaneous [Ca2+]i transients with non-transgenic, i.e. embryonic stem cells (ESC)-derived, myocytes.
Conclusion: PSCs show a similar cardiac differentiation potential as ESCs. Collectively, our findings may have important implications for the utility of PSCs in cell-based myocardial repair, tissue engineering, and drug screening.