Abstract 521: Generation of Functional Cardiomyocytes from Adult Mouse Spermatogonial Stem Cells
Cell therapy is a promising approach for the treatment of cardiac diseases. Recently, we report the successful establishment of undifferentiated spermatogonial stem cells (SSCs) from adult mouse testis with the pluripotency of embryonic stem cells (ESCs). Here we show the complex structural and functional properties of cardiomyocytes derived from SSCs. We found that cardiomyocytes derived from SSCs express myocytic marker genes and proteins. The expression of connexin 43 at cell-to-cell contacts in beating cardiac clusters indicates the presence of gap junctions. Gap junction functional assay using fluorescence recovery after photobleaching (FRAP) shows fluorescence recovery to 25% after 30 seconds corresponding to a recovery rate of k = 0.47±0.06 min-1 (n=15), a measure of gap junction permeability (within the first 6 minutes). The addition of 100 μmol/L of carbenoxolone, a gap junction uncoupler, results in significantly decreased recovery (4%; k = 0.04±0.04 min-1; n=9; P<0.05 each) further proving the existence of functional gap junctions among cardiomyocytes. Action potential (AP) analyses (n=64 cells) demonstrate the presence of pacemaker- (33%), atrial- (14%) ventricular- (41%) and Purkinje-like cardiomyocytes (12%) with distinct morphologies at differentiation stages (days 10 to 15). Beta-adrenergic stimulation with 1.0 μmol/L isoproterenol results in a significant increase in spontaneous beating rate from 0.7975±0.1476 Hz to 1.62±0.2131 Hz (n=5, P<0.05). The addition of 0.5 mmol/L cadmium chloride, a non-specific blocker of L-type Ca2+ and Na+ channels, abolishes the cardiac AP. Confocal microscopy analysis of intracellular Ca2+ (fluo4) in SSC-derived cardiomyocytes exhibits that calcium increases homogenously throughout the cell pointing to a fine regulated Ca2+ release from intracellular Ca2+ stores, most likely the sarcoplasmic reticulum (SR). Using line scan mode, rhythmic Ca2+ transients are found and even small elementary Ca2+ release events (Ca2+ sparks), which are mainly due to SR Ca2+ release through a cluster of RyRs, appear. Thus, adult SSCs provide a new source of distinct types of cardiomyocytes for basis research and potentially therapeutic application.