Theranostics 2019; 9(24):7222-7238. doi:10.7150/thno.32058

Research Paper

Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations

Sarkawt Hamad1,2, Daniel Derichsweiler1, Symeon Papadopoulos1,3, Filomain Nguemo1, Tomo Šarić1, Agapios Sachinidis1, Konrad Brockmeier5, Jürgen Hescheler1, Bastiaan J Boukens4, Kurt Pfannkuche1,5✉

1. Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Medical Faculty, Cologne, Germany
2. Biology Department, Faculty of Science, Soran University, Soran, Kurdistan region-Iraq
3. Center for Physiology and Pathophysiology, Institute for Vegetative Physiology, University of Cologne, Medical Faculty, Cologne, Germany
4. Department of Medical Biology, Amsterdam University Medical Center, Amsterdam, The Netherlands
5. Department of Pediatric Cardiology, University Clinics of Cologne, Cologne, Germany

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Citation:
Hamad S, Derichsweiler D, Papadopoulos S, Nguemo F, Šarić T, Sachinidis A, Brockmeier K, Hescheler J, Boukens BJ, Pfannkuche K. Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations. Theranostics 2019; 9(24):7222-7238. doi:10.7150/thno.32058. Available from http://www.thno.org/v09p7222.htm

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Abstract

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are promising candidates to treat myocardial infarction and other cardiac diseases. Such treatments require pure cardiomyocytes (CMs) in large quantities.

Methods: In the present study we describe an improved protocol for production of hiPSC-CMs in which hiPSCs are first converted into mesodermal cells by stimulation of wingless (Wnt) signaling using CHIR99021, which are then further differentiated into CM progenitors by simultaneous inhibition of porcupine and tankyrase pathways using IWP2 and XAV939 under continuous supplementation of ascorbate during the entire differentiation procedure.

Results: The protocol resulted in reproducible generation of >90% cardiac troponin T (TNNT2)-positive cells containing highly organized sarcomeres. In 2D monolayer cultures CM yields amounted to 0.5 million cells per cm2 growth area, and on average 72 million cells per 100 mL bioreactor suspension culture without continuous perfusion. The differentiation efficiency was hardly affected by the initial seeding density of undifferentiated hiPSCs. Furthermore, batch-to-batch variations were reduced by combinatorial use of ascorbate, IWP2, and XAV939.

Conclusion: Combined inhibition of porcupine and tankyrase sub-pathways of Wnt signaling and continuous ascorbate supplementation, enable robust and efficient production of hiPSC-CMs.

Keywords: Human induced pluripotent stem cells, iPS cells, hiPSCs, differentiation, cardiomyocytes, regenerative medicine, bioreactor suspension culture, Wnt signaling, ascorbate, robust method