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Chemically defined generation of human cardiomyocytes.

ABSTRACT: Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

SUBMITTER: Burridge PW

PROVIDER: S-EPMC4169698 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Chemically defined generation of human cardiomyocytes.

Burridge Paul W PW Matsa Elena E Shukla Praveen P Lin Ziliang C ZC Churko Jared M JM Ebert Antje D AD Lan Feng F Diecke Sebastian S Huber Bruno B Mordwinkin Nicholas M NM Plews Jordan R JR Abilez Oscar J OJ Cui Bianxiao B Gold Joseph D JD Wu Joseph C JC

Nature methods 20140615 8

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic ...[more]

PMID: 24930130

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Project description:BackgroundExisting methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably.MethodsWe induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs.ResultsWNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro.ConclusionWe provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.

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Chemically defined generation of human cardiomyocytes.

Publications

Chemically defined generation of human cardiomyocytes.

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