Efficient Generation of Cardiac Purkinje-like Cells from Embryonic Stem Cells by Activating cAMP Signaling.
Strategies to derive cardiac conduction system (CCS) cells including Purkinje cells (PC) would facilitate models for mechanistic studies and drug discovery, and also provide new cellular materials for regenerative therapies. However, using current cardiac differentiation protocols, the differentiation efficiency of CCS cells is extremely low, typically below 1% of the culture. High-throughput chemical screening is a powerful strategy for identifying small molecules that can activate signaling pathways to enhance embryonic stem cell (ESC) differentiation. We describe how to carry out a high- throughput screen to identify small molecules that can efficiently promote CCS generation from mouse ESCs. We also describe several assays, including immunofluorescence staining, electrophysiology, FACS analysis and quantitative real- time PCR, to characterize the phenotype of ESC-derived PC. In summary, we describe an efficient way to identify small molecules that enhance cardiac PC generation. These protocols can be adapted to identify other rare cell lineages by directed differentiation from ESCs. © 2017 by John Wiley & Sons, Inc.
Department of Life Science, National Taiwan University, Taipei, Taiwan.
Su-Yi
SY
Tsai
Department of Surgery, Weill Cornell Medical College, New York, New York.
Shuibing
S
Chen
Department of Surgery, Weill Cornell Medical College, New York, New York.
Todd
T
Evans
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D057166:High-Throughput Screening Assays; D051379:Mice; D009206:Myocardium / Q000166:cytology*; D011689:Purkinje Cells / Q000166:cytology*; D015398:Signal Transduction
D000242:Cyclic AMP
directed differentiation; heart; high-throughput chemical screen; protocol