Chemically defined and growth-factor-free culture system for the expansion and derivation of human pluripotent stem cells

Abstract

The large-scale and cost-effective production of quality-controlled human pluripotent stem cells (hPSCs) for use in cell therapy and drug discovery would ideally require a chemically defined xenobiotic-free culture system. Towards the development of such a system, costs associated with the use of recombinant proteins as supplements in basal culture media need to be reduced. Here, we describe a growth-factor-free culture medium that uses just three chemical compounds and a lower number of recombinant proteins than used in commercially available media. We show that the culture medium supports the long-term propagation of hPSCs, as confirmed by karyotype, the expression of pluripotency markers and the capacity to differentiate into cell types derived from the three embryonic germ layers. hPSCs growing in the medium were less dependent on glycolytic pathways than cells grown in medium containing growth factors. Moreover, the medium supported the generation of induced pluripotent stem cells derived from either human dermal fibroblasts or peripheral blood mononuclear cells. Our findings should facilitate the ongoing development of a completely xeno-free, chemically defined, synthetic culture system for hPSCs.

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Fig. 1: Development of a bFGF/TGF-β-free culture system.
Fig. 2: Characterization of hESCs and hiPSCs after long-term culture in AKIT medium.
Fig. 3: Transcriptomic and functional analysis of hPSCs during AKIT culture.
Fig. 4: Feeder-free hiPSC derivation during AKIT culture.

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Acknowledgements

We thank N. Nakatsuji (Kyoto University), M. F. Pera (The University of Melbourne), M. Kahn (University of Southern California) and J. Wu (The Salk Institute) for their critical discussion and helpful advice. We also thank C. Wells (The University of Melbourne), O. Korn and R. Mosbergen (Stemformtics) for their valuable input on transcriptome analysis, M. Kanno (CiRA, Kyoto University) for technical advice on iPSC derivation and A.-L. Skaltsounis (University of Athens) for providing materials. We also thank all lab members, especially N. Asari and J. Ueda, for experimental support and helpful discussions. This project was supported in part by a Start-Up Grant for Young Scientists (Kyoto University), an iCeMS Accelerated Project Grant (iCeMS, Kyoto University), and JSPS Grants-in-Aid for Young Scientist A and Scientific Research B (Japan Society for Promotion Science; grant numbers 24680052 and 15H03022) to K.H.; New Energy and Industrial Technology Development Organization (NEDO) Japan (P10027 and P14006) and Japan Agency for Medical Research and Development (AMED) to H.Su. and K.H.; and an iCeMS Overseas Visit Program for Young Researchers to S.-Y.Y.

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Contributions

K.H. designed the project. S.-Y.Y., T.I., H.Sh., N.Y., M.H., B.N., H.Su. and K.H. performed the experiments. S.-Y.Y., T.I., H.Sh., N.V.-S., B.N. and K.H. analysed the data. S.-Y.Y., T.I., H.Sh., N.V.-S., B.N. and K.H. wrote the manuscript.

Corresponding author

Correspondence to Kouichi Hasegawa.

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K.H., S.-Y.Y., H.Sh. and N.Y. have applied for a patent on the culture system.

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Supplementary Information

Supplementary figures, tables, and movie and data captions.

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Supplementary Dataset

Copy number variation and loss of heterozygosity of KhES‐1 and H9 cells after prolonged culture in AKIT, E8 and KSR/bFGF on feeder layer.

Supplementary Video 1

Beating cardiomyocytes differentiated from KhES‐1 cells at passage 33 in the AKIT system.

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Yasuda, S., Ikeda, T., Shahsavarani, H. et al. Chemically defined and growth-factor-free culture system for the expansion and derivation of human pluripotent stem cells. Nat Biomed Eng 2, 173–182 (2018). https://doi.org/10.1038/s41551-018-0200-7

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