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Cardiac differentiation of human pluripotent stem cells in scalable suspension culture

Abstract

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are a potential cell source for regenerative therapies, drug discovery and disease modeling. All these applications require a routine supply of relatively large quantities of in vitro–generated CMs. This protocol describes a suspension culture–based strategy for the generation of hPSC-CMs as cell-only aggregates, which facilitates process development and scale-up. Aggregates are formed for 4 d in hPSC culture medium followed by 10 d of directed differentiation by applying chemical Wnt pathway modulators. The protocol is applicable to static multiwell formats supporting fast adaptation to specific hPSC line requirements. We also demonstrate how to apply the protocol using stirred tank bioreactors at a 100-ml scale, providing a well-controlled upscaling platform for CM production. In bioreactors, the generation of 40–50 million CMs per differentiation batch at >80% purity without further lineage enrichment can been achieved within 24 d.

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Figure 1: Schematic illustration of the expansion in 2D followed by aggregate formation and cardiomyogenic differentiation in either the 12-well screening or the production platform in a stirred tank bioreactor.
Figure 2: Morphology of the 2D maintenance and expansion culture.
Figure 3: Flow cytometric analysis of the pluripotency markers TRA-1-60 and SSEA-3 for the hHSC_1285T_iPS2 cell line.
Figure 4: Morphology of cells and aggregates in the 12-well screening platform.
Figure 5: Bioreactor setup for aggregate generation under cyclic perfusion.
Figure 6: Assessment of the cardiac differentiation from bioreactor-derived aggregates on day 10.
Figure 7

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Acknowledgements

We thank A. Franke and D. Robles-Diaz for their excellent technical assistance; A. Haase for providing the induced pluripotent cell lines; and D.A. Elliott for providing the HES3 NKX2-5eGFP/w cell line. We thank Eppendorf for providing the cyclic perfusion script, impeller prototypes and comprehensive bioreactor schematic. This work was funded by the Cluster of Excellence REBIRTH (DFG EXC62/3; ZW 64/4-1, MA2331/16-1), the German Ministry for Education and Science (BMBF; grant no. 13N12606) and StemBANCC (support from the Innovative Medicines Initiative joint undertaking under grant agreement no. 115439-2, whose resources are composed of financial contribution from the European Union (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies' in-kind contribution).

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H.K., R.O. and R.Z. designed the experiments; H.K., C.K. and R.O. performed the experiments and analyzed the data; H.K., C.K. and R.Z. wrote the manuscript; U.M and R.Z. supervised the project; and all authors approved the final paper.

Corresponding author

Correspondence to Robert Zweigerdt.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data

The cyclic perfusion script for the DASbox Mini Bioreactor System. The script controls automatic interruption of the stirring before the medium exchange. (TXT 2 kb)

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Kempf, H., Kropp, C., Olmer, R. et al. Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nat Protoc 10, 1345–1361 (2015). https://doi.org/10.1038/nprot.2015.089

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