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Stem Cell Reports. 2019 Aug 13;13(2):366-379. doi: 10.1016/j.stemcr.2019.06.004. Epub 2019 Jul 25.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

Author information

1
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
2
Institute of Molecular and Cell Physiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.
3
Institute for Neurophysiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
4
Division of Molecular and Translational Cardiology and Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
5
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Electronic address: henningkempf@outlook.com.
6
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic-, Transplantation and Vascular Surgery, REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Electronic address: zweigerdt.robert@mh-hannover.de.

Abstract

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.

KEYWORDS:

ROR1; WNT; cardiac differentiation; cardiomyocyte; chemically defined process; human pluripotent stem cells; process development; stirred tank bioreactor; suspension culture; upscaling

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