Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.

The protocols described here efficiently direct human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of Gsk3 inhibitor followed by expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8–1.3 million cardiomyocytes/cm2) of virtually pure (80%–98%) functional cardiomyocytes from multiple hPSC lines without cell sorting or selection. Characterization of differentiated cells is performed in qualitative (immunostaining) and quantitative (flow cytometry) manners to assess expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjuction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery, toxicity testing, developmental studies, and regenerative medicine. Before these cells can be reliably utilized, characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4–99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy, and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent, which motivates their use in further applications such as drug evaluation and cardiac therapies.

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A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac Troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.