Efficient generation of cardiomyocytes from pluripotent stem cells (PSCs) for multiple downstream applications such as regenerative medicine, disease modeling, and drug screening remains a challenge. Cardiomyogenesis may be regulated in vitro by a controlled differentiation process, which involves various signaling molecules and extracellular environment. Here, we describe a simple method to efficiently generate cardiomyocytes from human embryonic stem cells and human induced pluripotent stem cells.

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFβ receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC.

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.