A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.
Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays.


CONCLUSIONS
Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia.
Hananeh
H
Fonoudi
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Hassan
H
Ansari
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Saeed
S
Abbasalizadeh
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Mehran Rezaei
MR
Larijani
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Sahar
S
Kiani
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Shiva
S
Hashemizadeh
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Ali Sharifi
AS
Zarchi
Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia.
Alexis
A
Bosman
Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia.
Gillian M
GM
Blue
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Sara
S
Pahlavan
St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia.
Matthew
M
Perry
Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.
Yishay
Y
Orr
The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.
Yaroslav
Y
Mayorchak
St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia.
Jamie
J
Vandenberg
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Mahmood
M
Talkhabi
Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia.
David S
DS
Winlaw
Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, New South Wales, Australia.
Richard P
RP
Harvey
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran.
Nasser
N
Aghdami
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research, Tehran, Iran Baharvand@RoyanInstitute.org.
Hossein
H
Baharvand
D000943:Antigens, Differentiation / Q000378:metabolism; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D060449:Wnt Signaling Pathway
D000943:Antigens, Differentiation
Bioreactor; Cardiomyocytes; Cell therapy; Directed differentiation; Embryonic stem; Human pluripotent stem cells; Induced pluripotent stem; Small molecules