Structured Culture Scaffolds Improve the Calcium Handling Properties of Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells

Induced Pluripotent Stem Cells derived Cardiomyocytes (iPSC-CM) have been proposed as in vitro models of myocardial physiology and disease; however a significant obstacle is their immature phenotype which is poorly representative of adult myocardium. We hypothesise that culture scaffolds which force the alignment of iPSC-CM will improve important determinants of cellular function such as Ca2+ handling properties.iPSC-CM (Cellular Dynamics, Wisconsin) were seeded onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds (MS) fabricated using photolithography, or on unstructured PDMS membrane (UM). After two weeks in culture iPSC-CM were loaded with Fluo-4 AM. Confocal microscopy was used in line scanning mode whilst iPSC-CM were beating spontaneously, at 1Hz and 0.5Hz.iPSC-CM cultured on MS had a shorter time to peak Ca2+ transient amplitude (tP) when stimulated at 1Hz (Mean+/-SEM: MS: 145+/-6ms, n=37; UM: 200+/-10ms, n=58; p=0.0002, Mann Whitney Test) and time to 50% transient decay (t50) (Mean+/-SEM: MS: 222+/-8ms, n=37; UM: 258+/-8ms, n=58; p=0.0065) but no change in time to 90% transient decay (t90). At 0.5Hz there was a shorter tP (Mean+/-SEM: MS: 199+/-11ms, n=37; UM: 242+/-10ms, n=64; p=0.0073) but no changes in t50 or t90. Similarly while iPSC-CM were beating spontaneously, there was a reduced tP (Mean+/-SEM: MS: 223+/-19ms, n=18; UM: 280+/-11ms, n=37; p=0.00121). The spontaneous beating rate was unchanged between groups. SR Ca2+ release was elicited using 50mM caffeine and could be obtained consistently in 80% (n=38) of MS but only 10% (n=17) of UM suggesting that SR Ca2+ regulation is improved by culture on MS.We conclude that structured culture improves Ca2+ cycling in iPSC-CM to make them more representative of adult human myocardium, our provisional data suggests this is related to sarcoplasmic reticulum maturity however further characterization is required.