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. 2015 Oct 6:87:21.3.1-21.3.15.
doi: 10.1002/0471142905.hg2103s87.

Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells

Paul W Burridge  1   2   3 , Alexandra Holmström  1   2   3 , Joseph C Wu  1   2   3
Affiliations

Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells

Paul W Burridge et al. Curr Protoc Hum Genet. .

Abstract

Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal-derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost-effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non-enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell-derived cardiomyocytes, cryopreservation, and thawing.

Keywords: cardiac; cardiomyocyte; chemically defined; differentiation; human induced pluripotent stem cells; monolayer.

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Figures

Figure 1
Figure 1. Human pluripotent growth and cardiac differentition
A, Time-course of pluripotent growth and subsequent cardiac differentiation showing the medium and small molcules used in each day (d) of differentiation. B, Representative images of hiPSCs seeded at a 1:15 split ratio in E8, followed by differentiation using the CDM3 protocol.
Figure 2
Figure 2. Characterization of cardiomycytes produced using CDM3
A, Flow cytometry of day 20 cardiomyocytes stained with troponin T (TNNT2) with or without metabolic purification. B, Immunofluorescent staining of day 15 cardiomyocytes for troponin T (TNNT2) and α-actinin (ACTN2). C, Measurement of contraction by video capture using Cellogy Pulse System.
See this image and copyright information in PMC

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