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Cited by 211Pancreatic function and diabetesPluripotent Stem Cells ResearchDiabetes and associated disorders

Generation of insulin-producing pancreatic β cells from multiple human stem cell lines

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Hogrebe, Nathaniel J., Maxwell, Kristina G., Augsornworawat, Punn, Millman, Jeffrey R.·2021·Nature Protocols
DOI 10.1038/s41596-021-00560-yPMC PMC8529911
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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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References 52 · Cited by 211

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  1. [2]Shapiro J Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. New. Engl. J. Med 343, 230– 38 (2000). 10911004
  2. [3]D’Amour KA Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol 23, 1534– 41 (2005). 16258519
  3. [4]D’Amour KA Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol 24, 1392– 1401 (2006). 17053790
  4. [5]Nostro MC Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861– 71 (2011). 21270052 PMC3035090
  5. [6]Kunisada Y, Tsubooka-Yamazoe N, Shoji M & Hosoya M Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells. Stem Cell Res. 8, 274– 84 (2012). 22056147
  6. [7]Borowiak M Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell 4, 348– 58 (2009). 19341624 PMC4564293
  7. [8]Sneddon JB, Borowiak M & Melton DA Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme. Nature 491, 765– 68 (2012). 23041930 PMC6005657
  8. [9]Mahaddalkar PU Generation of pancreatic beta cells from CD177+ anterior definitive endoderm. Nat. Biotechnol (2020). 32341565
  9. [10]Xu X, Browning VL & Odorico JS Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells. Mech. Dev 128, 412– 27 (2011). 21855631 PMC3225072
  10. [11]Nostro MC & Keller G Generation of beta cells from human pluripotent stem cells: Potential for regenerative medicine. Semin. Cell Dev. Biol 23, 701– 10 (2012). 22750147 PMC4400853
  11. [13]Rezania A Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes 61, 2016– 29 (2012). 22740171 PMC3402300
  12. [14]Bruin JE Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice. Diabetologia 56, 1987– 98 (2013). 23771205
  13. [15]Bruin JE Accelerated maturation of human stem cell-derived pancreatic progenitor cells into insulin-secreting cells in immunodeficient rats relative to mice. Stem Cell Reports 5, 1081– 96 (2015). 26677767 PMC4682152
  14. [16]Rezania A Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo. Stem Cells 31, 2432– 42 (2013). 23897760
  15. [17]Kelly OG Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat. Biotechnol 29, 750– 56 (2011). 21804561
  16. [18]Schaffer AE, Freude KK, Nelson SB & Sander M Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors. Dev. Cell 18, 1022– 29 (2010). 20627083 PMC3133668
  17. [19]Nelson SB, Schaffer AE & Sander M The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells. Development 134, 2491– 500 (2007). 17537793
  18. [20]Nostro MC Efficient generation of NKX6–1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports 4, 591– 604 (2015). 25843049 PMC4400642
  19. [21]Veres A Charting cellular identity during human in vitro β -cell differentiation. Nature 569, 368– 73 (2019). 31068696 PMC6903417
  20. [22]Sharon N Wnt signaling separates the progenitor and endocrine compartments during pancreas development. Cell Rep. 27, 2281– 91 (2019). 31116975 PMC6933053
  21. [23]Johansson KA Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types. Dev. Cell 12, 457– 65 (2007). 17336910
  22. [25]Rezania A Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol 32, 1121– 33 (2014). 25211370
  23. [26]Russ HA Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J. 34, 1759– 72 (2015). 25908839 PMC4516429
  24. [28]Sui L β-Cell replacement in mice using human type 1 diabetes nuclear transfer embryonic stem cells. Diabetes 67, 26– 35 (2018). 28931519 PMC5741143
  25. [30]Zhou X LIN28B Impairs the transition of hESC-derived β cells from the juvenile to adult state. Stem Cell Reports 14, 9– 20 (2020). 31883920 PMC6962644
  26. [31]Cogger KF Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors. Nat. Commun 8, 331 (2017). 28835709 PMC5569081
  27. [32]Johannesson B, Sui L, Freytes DO, Creusot RJ & Egli D Toward beta cell replacement for diabetes. EMBO J. 34, 841– 55 (2015). 25733347 PMC4388595
  28. [33]Sneddon JB Stem cell therapies for treating diabetes: progress and remaining challenges. Cell Stem Cell 22, 810– 23 (2018). 29859172 PMC6007036
  29. [34]Millman JR & Pagliuca FW Autologous pluripotent stem cell-derived β-like cells for diabetes cellular therapy. Diabetes 66, 1111– 20 (2017). 28507211
  30. [35]Nair GG, Tzanakakis ES & Hebrok M Emerging routes to the generation of functional β-cells for diabetes mellitus cell therapy. Nat. Rev. Endocrinol 16, 506– 18 (2020). 32587391 PMC9188823
  31. [36]Latres E, Finan DA, Greenstein JL, Kowalski A & Kieffer TJ Navigating two roads to glucose normalization in diabetes: Automated insulin delivery devices and cell therapy. Cell Metab. 29, 545– 63 (2019). 30840911
  32. [37]Johnson JD The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds. Diabetologia 59, 2047– 57 (2016). 27473069
  33. [38]Tremmel DM, Mitchell SA, Sackett SD & Odorico JS Mimicking nature-made beta cells: recent advances towards stem cell-derived islets. Curr Opin Organ Transplant 24, 574– 81 (2019). 31433306 PMC6925596
  34. [39]Velazco-Cruz L, Goedegebuure MM & Millman JR Advances toward engineering functionally mature human pluripotent stem cell-derived β cells. Front. Bioeng. Biotechnol 8, 1– 7 (2020). 32733873 PMC7363766
  35. [41]Hogrebe NJ, Augsornworawat P, Maxwell KG, Velazco-Cruz L & Millman JR Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nat. Biotechnol 38, 460– 70 (2020). 32094658 PMC7274216
  36. [42]Maxwell KG Gene-edited human stem cell–derived β cells from a patient with monogenic diabetes reverse preexisting diabetes in mice. Sci. Transl. Med 12, eaax9106 (2020). 32321868 PMC7233417
  37. [44]Kilian KA, Bugarija B, Lahn BT & Mrksich M Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl. Acad. Sci 107, 4872– 7 (2010). 20194780 PMC2841932
  38. [45]McBeath R, Pirone DM, Nelson CM, Bhadriraju K & Chen CS Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 6, 483– 95 (2004). 15068789
  39. [46]Mamidi A Mechanosignalling via integrins directs fate decisions of pancreatic progenitors. Nature 1, 114– 18 (2018). 30487608
  40. [47]Rosado-Olivieri EA, Anderson K, Kenty JH & Melton DA YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells. Nat. Commun 10, 1464 (2019). 30931946 PMC6443737
  41. [48]Cebola I TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors. Nat. Cell Biol 17, 615– 26 (2015). 25915126 PMC4434585
  42. [50]Slaoui M & Fiette L Histopathology procedures: from tissue sampling to histopathological evaluation. Methods Mol. Biol 691, 69– 82 (2011). 20972747
  43. [51]Augsornworawat P, Maxwell KG, Velazco-Cruz L, & Millman JR Single-cell transcriptome profiling reveals β cell maturation in stem cell-derived islets after transplantation. Cell Reports 32, 108067 (2020). 32846125 PMC7491368
  44. Hogrebe N Nat Biotechnol 38, 460– 470 (2020): 10.1038/s41587-020-0430-6 32094658 PMC7274216 · DOI · Paper page
  45. Maxwell KG Sci. Transl. Med 12, eaax9106 (2020): 10.1126/scitranslmed.aax9106 32321868 PMC7233417 · DOI · Paper page

Authors
Hogrebe, N. J., Maxwell, K. G., Augsornworawat, P., & Millman, J. R.
Affiliations
Washington University in St. Louis (US)
Corresponding author
Millman, Jeffrey R.
ORCID
Venue
Nature Protocols
Publisher
Springer Science and Business Media LLC
Published
2021-08-04
Volume
16
Issue
9
Pages
4109-4143
ISSN
  • 1750-2799 (ISSN-L)
  • 1754-2189
DOI
10.1038/s41596-021-00560-y
PMC
PMC8529911
OpenAlex
W3189682742
Cited by
211
References
52
Citations by year
2026: 43 · 2025: 55 · 2024: 50 · 2023: 38 · 2022: 22 · 2021: 2 · 2020: 1
FWCI
27.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Protocols (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • JDRF (5-CDA-2017-391-A-N, 3-APF-2020-930-A-N)
  • U.S. Department of Health & Human Services | National Institutes of Health (R01DK114233, R01DK127497, T32DK108742)
Topics
  • Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Diabetes and associated disorders
Keywords
pancreatic beta cells, human pluripotent stem cells, pancreatic progenitors, islet-like clusters, insulin secretion, glucose-stimulated insulin secretion, retinoic acid, latrunculin A, islet transplantation, stem cell-derived islets
MeSH terms
Cell Differentiation, Cell Line, Humans, Cell Culture Techniques, Pluripotent Stem Cells, Insulin-Secreting Cells

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