Abstract
We detail a six-stage planar differentiation methodology for generating human pluripotent stem cell–derived pancreatic β cells (SC-β cells) that secrete high amounts of insulin in response to glucose stimulation. This protocol first induces definitive endoderm by treatment with Activin A and CHIR99021, then generates PDX1+/NKX6-1+ pancreatic progenitors through the timed application of keratinocyte growth factor, SANT1, TPPB, LDN193189 and retinoic acid. Endocrine induction and subsequent SC-β-cell specification is achieved with a cocktail consisting of the cytoskeletal depolymerizing compound latrunculin A combined with XXI, T3, ALK5 inhibitor II, SANT1 and retinoic acid. The resulting SC-β cells and other endocrine cell types can then be aggregated into islet-like clusters for analysis and transplantation. This differentiation methodology takes ~34 d to generate functional SC-β cells, plus an additional 1–2 weeks for initial stem cell expansion and final cell assessment. This protocol builds upon a large body of previous work for generating β-like cells. In this iteration, we have eliminated the need for 3D culture during endocrine induction, allowing for the generation of highly functional SC-β cells to be done entirely on tissue culture polystyrene. This change simplifies the differentiation methodology, requiring only basic stem cell culture experience as well as familiarity with assessment techniques common in biology laboratories. In addition to expanding protocol accessibility and simplifying SC-β-cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-β cells from a wide variety of cell lines from various sources, broadening its applicability.
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Acknowledgements
This work was supported by the JDRF Career Development Award (5-CDA-2017-391-A-N), the National Institutes of Health (NIH) (R01DK114233, R01DK127497) and startup funds from the Washington University School of Medicine Department of Medicine. N.J.H. was supported by the JDRF (3-APF-2020-930-A-N). K.G.M. was supported by the NIH (T32DK108742). Microscopy was performed through the Washington University Center for Cellular Imaging, which is supported by the Washington University School of Medicine, the Children’s Discovery Institute (CDI-CORE-2015-505) and the Foundation for Barnes-Jewish Hospital (3770). Microscopy analysis was supported by the Washington University Diabetes Research Center (P30DK020579).
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N.J.H., K.G.M. and P.A. collected data for all experiments. N.J.H., K.G.M., P.A. and J.R.M. designed the experiments, wrote the manuscript and revised the manuscript.
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N.J.H., K.G.M., P.A., and J.R.M. are inventors on patents and patent applications related to SC-β cells. J.R.M. is a consultant for Sana Biotechnology. K.G.M., P.A. and J.R.M. are cofounders of Salentra Biosciences.
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Key references using this protocol
Hogrebe, N. et al. Nat. Biotechnol. 38, 460–470 (2020): https://doi.org/10.1038/s41587-020-0430-6
Maxwell, K. G. et al. Sci. Transl. Med. 12, eaax9106 (2020): https://doi.org/10.1126/scitranslmed.aax9106
Augsornworawat, P. et al. Cell Reports 32, 108067 (2020): https://doi.org/10.1016/j.celrep.2020.108067
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Hogrebe, N.J., Maxwell, K.G., Augsornworawat, P. et al. Generation of insulin-producing pancreatic β cells from multiple human stem cell lines. Nat Protoc 16, 4109–4143 (2021). https://doi.org/10.1038/s41596-021-00560-y
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DOI: https://doi.org/10.1038/s41596-021-00560-y
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