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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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.
References
Zhou, Q. & Melton, D. A. Pancreas regeneration. Nature 557, 351–358 (2018).
Greggio, C. et al. Artificial three-dimensional niches deconstruct pancreas development in vitro. Development 140, 4452–4462 (2013).
Sugiyama, T. et al. Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation. Proc. Natl Acad. Sci. USA 110, 12691–12696 (2013).
Ramiya, V. K. et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6, 278–282 (2000).
Jin, L. et al. Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel. Proc. Natl Acad. Sci. USA 110, 3907–3912 (2013).
Bonner-Weir, S. et al. In vitro cultivation of human islets from expanded ductal tissue. Proc. Natl Acad. Sci. USA 97, 7999–8004 (2000).
Gao, R., Ustinov, J., Korsgren, O. & Otonkoski, T. In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells. Diabetologia 48, 2296–2304 (2005).
Yatoh, S. et al. Differentiation of affinity-purified human pancreatic duct cells to beta-cells. Diabetes 56, 1802–1809 (2007).
Rovira, M. et al. Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc. Natl Acad. Sci. USA 107, 75–80 (2010).
Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721 (2013).
Minami, K. et al. Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells. Proc. Natl Acad. Sci. USA 102, 15116–15121 (2005).
Seaberg, R. M. et al. Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat. Biotechnol. 22, 1115–1124 (2004).
Smukler, SimonR. et al. The adult mouse and human pancreas contain rare multipotent stem cells that express insulin. Cell Stem Cell 8, 281–293 (2011).
Hogrebe, N. J., Augsornworawat, P., Maxwell, K. G., Velazco-Cruz, L. & Millman, J. R. Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nat. Biotechnol. 38, 460–470 (2020).
Pagliuca, FeliciaW. et al. Generation of functional human pancreatic β cells in vitro. Cell 159, 428–439 (2014).
Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121–1133 (2014).
Yoshihara, E. et al. Immune-evasive human islet-like organoids ameliorate diabetes. Nature 586, 606–611 (2020).
González, B. J., Creusot, R. J., Sykes, M. & Egli, D. How safe are universal pluripotent stem cells? Cell Stem Cell 26, 307–308 (2020).
Valdez, I. A., Teo, A. K. K. & Kulkarn, R. N. Cellular stress drives pancreatic plasticity. Sci. Transl. Med. 7, 273ps272 (2015).
Wang, D. et al. Long-term expansion of pancreatic islet organoids from resident Procr+ progenitors. Cell 180, 1198–1211.e19 (2020).
Gittes, G. K. Developmental biology of the pancreas: a comprehensive review. Dev. Biol. 326, 4–35 (2009).
Lammert, E., Cleaver, O. & Melton, D. Induction of pancreatic differentiation by signals from blood vessels. Science 294, 564–567 (2001).
Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 455, 627–632 (2008).
Lemper, M. et al. Reprogramming of human pancreatic exocrine cells to β-like cells. Cell Death Differ. 22, 1117–1130 (2014).
Lee, J. et al. Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells. eLife 2, e00940 (2013).
Pan, F. C. & Wright, C. Pancreas organogenesis: from bud to plexus to gland. Dev. Dyn. 240, 530–565 (2011).
Furuyama, K. et al. Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells. Nature 567, 43–48 (2019).
Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).
McCall, M. D., Maciver, A. H., Pawlick, R. L., Edgar, R. & Shapiro, A. M. J. Histopaque provides optimal mouse islet purification kinetics: comparison study with Ficoll, iodixanol and dextran. Islets 3, 144–149 (2014).
Zmuda, E. J., Powell, C. A. & Hai, T. A method for murine islet isolation and subcapsular kidney transplantation. J. Vis. Exp. 2011, 2096 (2011).
Akira Shiroi, M. et al. Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone. Stem Cells Int. 20, 284–292 (2002).
Crowley, L. C., Marfell, B. J., Christensen, M. E., and Waterhouse, N. J. Measuring cell death by trypan blue uptake and light microscopy. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot087155 (2016).
Corbin, K. L. et al. A practical guide to rodent islet isolation and assessment revisited. Biol. Proced. Online 23, 7 (2021).
Huang, C. & Gu, G. Effective isolation of functional islets from neonatal mouse pancreas. J. Vis. Exp. 2017, 55160 (2017).
Junod, A., Lambert, A. E., Stauffacher, W. & Renold, A. E. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J. Clin. Invest. 48, 2129–2139 (1969).
Lenzen, S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 51, 216–226 (2007).
Deeds, M. C. et al. Single dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models. Lab. Anim. 45, 131–140 (2011).
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.).
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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.
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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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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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DOI: https://doi.org/10.1038/s41596-022-00683-w
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