Rationale Cardiomyocytes differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research including disease modeling and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix (ECM) is also fundamentally involved in cardiac development from the earliest morphogenetic events such as gastrulation. Objective We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using ECM in combination with growth factors known to promote cardiogenesis. Methods and Results PSCs were cultured as monolayers on Matrigel, an ECM preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin+ mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, BMP4, and bFGF) generated cardiomyocytes with high purity (up to 98%) and yield (up to 11 cardiomyocytes/input PSC) from multiple PSC lines. The resulting cardiomyocytes progressively mature over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial and ventricular cardiomyocytes were observed, and monolayers of electrically coupled cardiomyocytes modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions Dynamic ECM application promoted EMT of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Previous work in this laboratory has shown that endoderm cells in the heart forming region (HFR endoderm) of the chicken embryo induce terminal cardiac differentiation in explanted precardiac mesoderm cells. Immunostaining patterns indicating that HFR endoderm cells express Drosophila decapentaplegic (dpp)-like antigens prompted a degenerate polymerase chain reaction (PCR) screen to identify cDNAs in the dpp subgroup of the transforming growth factor-beta (TGF-beta) family. Among 50 clones of PCR products that have been sequenced, over half have identity with bone morphogenetic protein-2 (BMP-2). No other TGF-beta cDNAs have been detected, suggesting that BMP-2 is the major dpp subgroup protein synthesized by HFR endoderm cells. However, BMP-2 protein did not promote survival of either precardiac or non-precardiac mesoderm cells in culture. Whereas FGF-4 supports cardiogenesis in precardiac mesoderm, it did not induce cardiogenesis in nonprecardiac mesoderm, although explant viability was maintained. In contrast to the isolated effects of these growth factors, treatment of non-precardiac mesoderm with combined BMP-2 and FGF-4 induced cardiogenesis in the majority of explants, as revealed by the formation of a rhythmically contractile multicellular vesicle that expresses sarcomeric alpha-actin. These findings suggest that BMP-2 and FGF-4 possess respective differentiative and proliferative activities, the combination of which specifies cells to the cardiac lineage.

Serum response factor (SRF) homozygous-null embryos from our backcross of SRF LacZ/؉ "knock-in" mice failed to gastrulate and form mesoderm, similar to the findings of an earlier study (Arsenian, S., Weinhold, B., Oelgeschlager, M., Ruther, U., and Nordheim, A. (1998) EMBO J. 17, 6289 -6299). Our use of embryonic stem cells provided a model system that could be used to investigate the specification of multiple embryonic lineages, including cardiac myocytes. We observed the absence of myogenic ␣-actins, SM22␣, and myocardin expression and the failure to form beating cardiac myocytes in aggregated SRF null embryonic stem cells, whereas the appearance of transcription factors Nkx2-5 and GATA4 were unaffected. To study the role of SRF during heart organogenesis, we then performed cardiac-specific ablation of SRF by crossing the transgenic ␣-myosin heavy chain Cre recombinase line with SRF LoxP-engineered mice. Cardiac-specific ablation of SRF resulted in embryonic lethality due to cardiac insufficiency during chamber maturation. Conditional ablation of SRF also reduced cell survival concomitant with increased apoptosis and reduced cellularity. Significant reductions in SRF (>95%), atrial naturetic factor (>80%), and cardiac (>60%), skeletal (>90%), and smooth muscle (>75%) ␣-actin transcripts were also observed in the cardiac-conditional knock-out heart. This was consistent with the idea that SRF directs de novo cardiac and smooth muscle gene activities. Finally, quantitation of the knock-in LacZ reporter gene transcripts in the hearts of cardiac-conditional knock-out embryos revealed an ϳ30% reduction in gene activity, indicating SRF gene autoregulation during cardiogenesis.