Differentiation of iPSCs into Cardiomyocytes

Human induced pluripotent stem cells (iPSC) were acquired from the Coriell Institute (GM23338), as characterized previously^{102 (link)}. iPSC colonies were maintained on growth-factor reduced Matrigel (Corning)-coated tissue culture plates in mTeSR1 medium (Stem Cell Technologies). iPSCs were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) following a sequential protocol of manipulating the WNT pathway^{103 (link)}, using 12.5 μM CHIR99021 (Stem Cell Technologies) for 24 hours and 5 μM IWP4 (Tocris Bioscience) for 48 hours on day 3 after the induction of differentiation. Subsequently, the differentiating cultures were maintained in RPMI + B2-insulin until day 11 when spontaneous beating was observed. Media was then changed to RPMI + B27 + insulin for 2 days, followed by 4 days of metabolic selection using DMEM without glucose and 4 mM lactate^{104 (link)}. iPSC-CMs were kept in RPMI + B27 + insulin until day 21 after the start of differentiation when they were dissociated and re-plated on a 12-well plate coated with 50 μg/ml fibronectin (bovine, Sigma-Aldrich)^{105 (link)}. iPSC-CMs were cultured for a subsequent 2 days in RPMI + B27 + insulin before experiments were performed.

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Brill A.L., Fischer T.T., Walters J.M., Marlier A., Sewanan L.R., Wilson P.C., Johnson E.K., Moeckel G., Cantley L.G., Campbell S.G., Nerbonne J.M., Chung H.J., Robert M.E, & Ehrlich B.E. (2020). Polycystin 2 is increased in disease to protect against stress-induced cell death. Scientific Reports, 10, 386.

Publication 2020

Matrigel mTeSR1 medium CHIR99021

Bovine Cardiomyocytes Chir99021 Culture medium Fibronectin Glucose Growth factor Human induced pluripotent stem cells Induced pluripotent stem cells Insulin Matrigel Stem cell Tissue

Corresponding Organization :

Other organizations : Yale University, University of Illinois Urbana-Champaign, Washington University in St. Louis

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Variable analysis

independent variables

CHIR99021 (12.5 μM) for 24 hours
IWP4 (5 μM) for 48 hours

dependent variables

Differentiation of iPSCs into iPSC-derived cardiomyocytes (iPSC-CMs)
Spontaneous beating of differentiating cultures observed on day 11

control variables

Growth-factor reduced Matrigel-coated tissue culture plates
MTeSR1 medium for iPSC culture
RPMI + B2-insulin medium for iPSC-CM differentiation
RPMI + B27 + insulin medium for iPSC-CM culture
DMEM without glucose and 4 mM lactate for metabolic selection

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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