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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 protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor– and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8–1.3 million cardiomyocytes per cm 2 ) of virtually pure (80–98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction 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.

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of [beta]-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction 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 cells (hPSCs) have the capacity to infinitely self-renew or differentiate into any somatic cell type. Since heart failure is the world's leading cause of death, a great clinical need exists to differentiate hPSCs into cardiomyocytes, enabling exploration of new drugs or cell-based therapies. Various factors in the cellular microenvironment are known to influence self-renewal or differentiation trajectories of stem cells. In this work, we focused on substrate stiffness, as stiffening of the heart is a physiological phenomenon during embryonic development and progression of heart disease. To investigate effects of mechanics on differentiation to cardiomyocytes, we cultured hPSCs and their derivatives on polyacrylamide hydrogel substrates. In embryoid body and directed differentiation culture systems, hPSC differentiation to cardiomyocytes peaked on 49.4 kPa hydrogels. This was linked to an early peak in mesendoderm specification in hPSCs undergoing directed differentiation on hydrogels. Next we initiated differentiation on tissue culture polystyrene (TCPS) surfaces, split cells to hydrogels at the cardiac progenitor cell stage, and observed no ultimate difference in cardiomyocyte purity with stiffness. To manipulate cellular mechanics internally, we primed hPSCs on TCPS with Y27632, an inhibitor of cytoskeletal organization, and observed significant increases in cardiomyocyte purity. This work indicates that hPSCs are sensitive to mechanical modulation at early stages of differentiation, and proper mechanics can drastically alter their propensity to become cardiomyocytes. Although factors to obtain cardiomyocytes from hPSCs have been elucidated, the task remains to characterize the functionality of these beating cells. To this end, we developed an assay to quantify contraction stress of cardiomyocytes. We differentiated hPSCs to cardiomyocytes on TCPS and split them to polyacrylamide hydrogels embedded with fluorescent beads. Through traction force microscopy, we tracked bead displacements beneath contracting cardiomyocytes and converted them to contraction stresses. We found that, in hPSC-derived and neonatal rat cardiomyocytes, contraction stress increased with substrate stiffness. Next we demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline, a cardioactive drug, and remained stable in culture over a period of two months. This assay demonstrates appropriate functional responses of hPSC-derived cardiomyocytes and serves to motivate their use in drug studies and regenerative medicine.