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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Published: July 25th, 2016

DOI:

10.3791/54276

1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, 2Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, 3St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales, 4School of Biotechnology and Biomolecular Sciences, University of New South Wales, 5Department of Developmental Biology, University of Science and Culture, 6Heart Centre for Children, The Children´s Hospital at Westmead, 7Sydney Medical School, University of Sydney, 8Department of Developmental Biology, University of Science and Culture, Tehran, Iran
* These authors contributed equally

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-β, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability of self-renewal and the capacity to differentiate into cells of the three embryonic germ layers 1,2. Due to these characteristics, hPSCs provide a valuable and unlimited source for the generation and scalable production of disease-relevant cell types for modeling human disease 3-5, for high-throughput drug screening and toxicity assays 6,7 and potentially for clinical applications 8. Generation of cardiomyocytes from hPSCs provides the opportunity to specifically investigate the mec....

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1. Preparation of Culture Media, Coating of Cell Culture Plates and Maintenance of Undifferentiated hPSCs

  1. Media Preparation
    Note: Sterilize media using a 0.22 µm filtration device and store at 4 °C protected from light for up to 4 weeks. Reagent names, suppliers and catalog numbers are listed in Materials Table.
    1. For Mouse Embryonic Fibroblasts (MEF) Medium, combine 445 ml DMEM, 50 ml Fetal Bovine Serum (FBS) and 5 ml cell culture .......

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In order to establish a simple method for the large-scale differentiation of cardiomyocytes from hPSCs, we created a protocol in which cells were treated initially with a WNT/β-catenin activator (CHIR99021)16 and subsequently with inhibitors of the WNT/β-catenin and transforming growth factor-β (TGF-β) pathways (IWP216 and SB43154217, respectively) and finally an activator of the sonic hedgehog (SHH) pathway (purmorphamine)17

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Cardiomyocytes derived from hPSCs are an extremely attractive source for use in human disease modeling, drug screening/toxicity testing and, perhaps in the future, regenerative therapies. One of the major hurdles to using these cells however, is the ability to provide enough high quality material for their effective use. Using our described protocol, we offer a method that overcomes this limitation.

Recently, synthetic small molecules targeting specific signaling pathways involved in cardiogen.......

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This study was funded by grants provided from Royan Institute, Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), the National Health and Medical Research Council of Australia (NHMRC; 354400), the National Heart Foundation of Australia/Heart Kids Australia (G11S5629), and the New South Wales Cardiovascular Research Network. HF was supported by a University International Postgraduate Scholarship from the University of New South Wales, Australia. RPH was supported by a NHMRC Australia Fellowship. The authors express their gratitude to the human subjects who participated in this research.

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Name Company Catalog Number Comments
Knockout DMEM Life Technologies 10829018
Knockout Serum Replacement (KO-SR) Life Technologies 10828028
Glutamax Life Technologies 35050061
MEM Non-essential Amino Acids Life Technologies 11140-050
β-Mercaptoethanol Life Technologies 21985-023
Basic Fibroblast Growth Factor (bFGF) Miltenyi Biotec 130-093-843
RPMI1640 Life Technologies 11875093
DPBS, no calcium, no magnesium Life Technologies 14190144
DPBS Life Technologies 14287072
Attachment Factor (AF) Life Technologies S006100
ECM Gel Sigma-Aldrich E1270
Laminin Invitrogen 23017-015
DMEM Life Technologies 11965-092                                                                                                       
Fatal Bovine Serum (FBS) Life Technologies 16140-071
B27 minus insulin Gibco A18956-01
Penicillin/Streptomycin Life Technologies 15070063
0.05% Trypsin/EDTA Life Technologies 25300-054
Collagenase Type IV Life Technologies 17140-019
Calcium Chloride (CaCl2) Sigma-Aldrich C7902
Mitomycin C Bioaustralis BIA-M1183
CHIR99021 Miltenyi Biotec 130-104-172
IWP2 Miltenyi Biotec 130-105-335
SB431542 Miltenyi Biotec 130-095-561
Purmorphamine Miltenyi Biotec 130-104-465
ROCK inhibitor Y-27632 Miltenyi Biotec 130-104-169
Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich E6758
Poly Vinyl Alcohol (PVA) Sigma-Aldrich 363073
Gelatin Sigma-Aldrich G1890
Trypan Blue Bio-Rad 145-0013
Accumax  Innovative Cell Technologies Inc. AM105
Sigmacote  Sigma-Aldrich SL2 
CELLSPIN Integra Biosciences 183 001
Spinner flask with 1 pendulum, 100 ml  Integra Biosciences 182 023
Mouse Embryonic Fibroblasts (MEF) Prepared in-house (or commercially available)
Human pluripotent stem cell (hPSC) lines Prepared in-house (or commercially available)

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