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Isolation of mouse pancreatic islet Procr+ progenitors and long-term expansion of islet organoids in vitro

Nature Protocols volume 17, pages 1359–1384 (2022)Cite this article

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Abstract

Insulin production is required for glucose homeostasis. Pancreatic islet β cells are the only cells that produce insulin in humans; however, generation of functional β cells in vitro from embryonic or adult tissues has been challenging. Here, we describe isolation of pancreatic islet progenitors from adult mice, which enables the efficient generation and long-term expansion of functional islet organoids in vitro. This protocol starts with purification of protein C receptor (Procr)-expressing islet progenitors. Coculture with endothelial cells generates islet organoids in vitro that can be expanded by passage. Functional maturation is achieved as a consequence of a prolonged culture period and cyclic glucose stimulation. Primary islet organoids form in 7–10 days. Subsequently, each passage takes 1 week, with the final maturation step requiring 3 weeks of additional culture. The resulting organoids are predominantly composed of β cells but also contain small proportions of α, δ and pancreatic polypeptide cells. The organoids sense glucose and secrete insulin. This approach thus provides a strategy for β cell generation in vitro and an organoid system to study islet regeneration and diseases.

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Fig. 1: Schematic diagram of the time line for generating islet organoids.
Fig. 2: Expression of Procr in mouse pancreatic cells.
Fig. 3: Pancreas perfusion and islet purification by density gradient centrifugation and handpicking.
Fig. 4: FACS sorting of Procr+ islet progenitors and Cd31+ ECs for in vitro culture.
Fig. 5: Bright-field images of the islet organoids at different time points.
Fig. 6: Characterization of the islet organoids in vitro and the organoid cells in vivo.

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Data availability

The data supporting the findings of this study are available within the article. The single-cell RNA-sequencing data shown in Fig. 2a–c can be found at https://www.biosino.org/node/index, with access no. OEP000249 (https://www.biosino.org/node/project/detail/OEP000249). Source data are provided with this paper.

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Acknowledgements

This research was supported by grants from the Chinese Academy of Sciences (XDA16020200 to Y.A.Z.), the National Key Research and Development Program of China (2020YFA0509002 to Y.A.Z.) and the National Natural Science Foundation of China (31830056, and 31861163006 to Y.A.Z.).

Author information

Author notes

  1. Daisong Wang

    Present address: Hubrecht Institute and Oncode Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Centre Utrecht, Netherlands, Utrecht University and Princess Maxima Center, Utrecht, Netherlands

  2. These authors contributed equally: Jingqiang Wang, Daisong Wang.

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Jingqiang Wang, Daisong Wang, Xinyi Chen, Shubo Yuan, Lanyue Bai, Chunye Liu & Yi Arial Zeng

  2. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, China

    Yi Arial Zeng

  3. Bio-Research Innovation Center, Institute of Biochemistry and Cell Biology, Suzhou, China

    Yi Arial Zeng

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Contributions

Y.A.Z. and D.W. envisioned the original idea and developed the protocol. J.W., L.B., C.L. and S.Y. helped protocol optimization. X.C. drew illustrations. J.W., D.W. and Y.A.Z. wrote the manuscript.

Corresponding author

Correspondence to Yi Arial Zeng.

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Competing interests

Y.A.Z. and D.W. have a patent on islet organoid technology.

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Nature Protocols thanks Ekaterine Berishvili, Jeffrey R. Millman, Fong Cheng Pan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Wang, D. et al. Cell 180, 1198–1211.e19 (2020); https://doi.org/10.1016/j.cell.2020.02.048

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Wang, J., Wang, D., Chen, X. et al. Isolation of mouse pancreatic islet Procr+ progenitors and long-term expansion of islet organoids in vitro. Nat Protoc 17, 1359–1384 (2022). https://doi.org/10.1038/s41596-022-00683-w

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