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Production and cryopreservation of definitive endoderm from human pluripotent stem cells under defined and scalable culture conditions

Nature Protocols volume 16, pages 1581–1599 (2021)Cite this article

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Abstract

The endodermal germ layer gives rise to respiratory epithelium, hepatocytes, pancreatic cells and intestinal lineages, among other cell types. These lineages can be differentiated from human pluripotent stem cells (hPSCs) via a common definitive endoderm (DE) intermediate that is characterized by the co-expression of the cell surface markers CXCR4, c-KIT and EPCAM and the transcription factors SOX17 and FOXA2. Here we provide a detailed protocol for mass production of DE from hPSCs in scalable and easy-to-handle suspension culture using a rotating Erlenmeyer flask or a sophisticated, fully controllable, 150-ml stirred tank bioreactor. This protocol uses two different media formulations that are chemically defined and xeno free and therefore good manufacturing practice ready. Our protocol allows for efficient hPSC-derived DE specification in multicellular aggregates within 3 days and generates up to 1 × 108 DE cells with >92% purity in one differentiation batch when using the bioreactor. The hPSC-derived DE cells that are generated can be cryopreserved for later downstream differentiation into various endodermal lineages. This protocol should facilitate the flexible production of mature DE derivatives for physiologically relevant disease models, high-throughput drug screening, toxicology testing and cellular therapies.

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Fig. 1: Schematic illustration of hPSC-scalable DE differentiation.
Fig. 2: Morphology of hPSCs in 2D culture.
Fig. 3: Morphology of cell aggregates during DE differentiation.
Fig. 4: Gating strategy and analysis of DE marker expression by flow cytometry.

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Data availability

Data regarding cell viability are included as Supplementary Data. The other data presented here were previously published and are available in the original publication13.

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Acknowledgements

We thank members of the Zweigerdt group, especially C. Halloin and W. Löbel, for help and suggestions regarding the experimental setup of the bioreactor. We also thank T. Scheper for providing bFGF and A. Kirschning and G. Dräger for providing CHIR99021. Parts of Fig. 1 were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License. This research was funded by the German Center for Lung Research (DZL, BREATH 82DZL002A1) and Federal State of Lower Saxony R2N (74ZN1574).

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Author notes

  1. These authors contributed equally: Anais Sahabian, Julia Dahlmann, Ulrich Martin, Ruth Olmer.

Authors and Affiliations

  1. Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), REBIRTH–Research Center for Translational and Regenerative Medicine, Hannover Medical School, Hannover, Germany

    Anais Sahabian, Julia Dahlmann, Ulrich Martin & Ruth Olmer

  2. Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover Medical School, Hannover, Germany

    Anais Sahabian, Julia Dahlmann, Ulrich Martin & Ruth Olmer

  3. Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany

    Anais Sahabian, Julia Dahlmann, Ulrich Martin & Ruth Olmer

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  1. Anais Sahabian
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Contributions

A.S. conceptualized and performed all experiments and analysis. A.S., J.D., R.O. and U.M. wrote the manuscript. R.O. and U.M. conceptualized and supervised the project and provided project administration and funding acquisition. All authors approved the final manuscript.

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Correspondence to Ulrich Martin or Ruth Olmer.

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Key reference using this protocol

Sahabian, A. et al. Cells 8, 1571 (2019): https://doi.org/10.3390/cells8121571

Key data used in this protocol

Sahabian, A. et al. Cells 8, 1571 (2019): https://doi.org/10.3390/cells8121571

Supplementary information

Supplementary Data

Source data for cell viability results are included in the ‘Anticipated Results’ section. Cell viability was assessed by Beckman Coulter Vi-CELL X.

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Sahabian, A., Dahlmann, J., Martin, U. et al. Production and cryopreservation of definitive endoderm from human pluripotent stem cells under defined and scalable culture conditions. Nat Protoc 16, 1581–1599 (2021). https://doi.org/10.1038/s41596-020-00470-5

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