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01-08-2017 | Article

Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells

Authors: Yasushi Kondo, Taro Toyoda, Ryo Ito, Michinori Funato, Yoshiya Hosokawa, Satoshi Matsui, Tomomi Sudo, Masahiro Nakamura, Chihiro Okada, Xiaotong Zhuang, Akira Watanabe, Akira Ohta, Nobuya Inagaki, Kenji Osafune

Published in: Diabetologia | Issue 8/2017

Abstract

Aims/hypothesis

Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS)⁺ cells using novel small-molecule inducers.

Methods

We screened small molecules that increased the induction rate of INS⁺ cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1)⁺ pancreatic progenitor cells. The differentiation protocol to generate INS⁺ cells from hiPSCs/hESCs was optimised using hit compounds, and INS⁺ cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS⁺ cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation.

Results

One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS⁺ cells increased from a mean ± SD of 5.9 ± 1.5% (n = 3) to 16.5 ± 2.1% (n = 3). SCG induced neurogenin 3-positive cells at a mean ± SD of 32.6 ± 4.6% (n = 3) compared with 14.2 ± 3.6% (n = 3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling.

Conclusions/interpretation

SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS⁺ cells from hiPSCs/hESCs.

Data availability

The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) with series accession number GSE89973.

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Ryan EA, Paty BW, Senior PA et al (2005) Five-year follow-up after clinical islet transplantation. Diabetes 54:2060–2069CrossRefPubMed

Speight J, Reaney MD, Woodcock AJ, Smith RM, Shaw JW (2010) Patient-reported outcomes following islet cell or pancreas transplantation (alone or after kidney) in type 1 diabetes: a systematic review. Diabet Med 27:812–822CrossRefPubMed

Shapiro AM, Ricordi C, Hering BJ et al (2006) International trial of the Edmonton protocol for islet transplantation. N Engl J Med 355:1318–1330CrossRefPubMed

Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872CrossRefPubMed

Okita K, Matsumura Y, Sato Y et al (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412CrossRefPubMed

Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147CrossRefPubMed

D’Amour KA, Bang AG, Eliazer S et al (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24:1392–1401CrossRefPubMed

Jiang J, Au M, Lu K et al (2007) Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 25:1940–1953CrossRefPubMed

Kroon E, Martinson LA, Kadoya K et al (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26:443–452CrossRefPubMed

10.

Tateishi K, He J, Taranova O, Liang G, D’Alessio AC, Zhang Y (2008) Generation of insulin-secreting islet-like clusters from human skin fibroblasts. J Biol Chem 283:31601–31607CrossRefPubMed

11.

Maehr R, Chen S, Snitow M et al (2009) Generation of pluripotent stem cells from patients with type 1 diabetes. Proc Natl Acad Sci U S A 106:15768–15773CrossRefPubMedPubMedCentral

12.

Chen S, Borowiak M, Fox JL et al (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5:258–265CrossRefPubMed

13.

Thatava T, Nelson TJ, Edukulla R et al (2011) Indolactam V/GLP-1-mediated differentiation of human iPS cells into glucose-responsive insulin-secreting progeny. Gene Ther 18:283–293CrossRefPubMed

14.

Kelly OG, Chan MY, Martinson LA et al (2011) Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat Biotechnol 29:750–756CrossRefPubMed

15.

Nostro MC, Sarangi F, Ogawa S et al (2011) Stage-specific signalling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138:861–871CrossRefPubMedPubMedCentral

16.

Schulz TC, Young HY, Agulnick AD et al (2012) A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS One 7:e37004CrossRefPubMedPubMedCentral

17.

Kunisada Y, Tsubooka-Yamazoe N, Shoji M, Hosoya M (2012) Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells. Stem Cell Res 8:274–284CrossRefPubMed

18.

Rezania A, Bruin JE, Riedel MJ et al (2012) Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes 61:2016–2029CrossRefPubMedPubMedCentral

19.

Pagliuca FW, Millman JR, Gürtler M et al (2014) Generation of functional human pancreatic β cells in vitro. Cell 159:428–439CrossRefPubMedPubMedCentral

20.

Rezania A, Bruin JE, Arora P et al (2014) Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32:1121–1133CrossRefPubMed

21.

Bruin JE, Erener S, Vela J, Hu X (2014) Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells. Stem Cell Res 12:194–208CrossRefPubMed

22.

Toyoda T, Mae S, Tanaka H et al (2015) Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells. Stem Cell Res 14:185–197CrossRefPubMed

23.

Araoka T, Mae S, Kurose Y et al (2014) Efficient and rapid induction of human iPSCs/ESCs into nephrogenic intermediate mesoderm using small molecule-based differentiation methods. PLoS One 9:e84881CrossRefPubMedPubMedCentral

24.

Kajiwara M, Aoi T, Okita K et al (2012) Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proc Natl Acad Sci U S A 109:12538–12543CrossRefPubMedPubMedCentral

25.

Suemori H, Yasuchika K, Hasegawa K, Fujioka T, Tsuneyoshi N, Nakatsuji N (2006) Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. Biochem Biophys Res Commun 345:926–932CrossRefPubMed

26.

Hara M, Wang X, Kawamura T et al (2003) Transgenic mice with green fluorescent protein-labeled pancreatic β-cells. Am J Physiol Endocrinol Metab 284:e177–e183CrossRefPubMed

27.

Mae S, Shono A, Shiota F et al (2013) Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells. Nat Commun 4:1367CrossRefPubMedPubMedCentral

28.

Gotoh M, Maki T, Satomi S et al (1987) Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection. Transplantation 43:725–730CrossRefPubMed

29.

Yuengsrigul A, Chin TW, Nussbaum E (1999) Immunosuppressive and cytotoxic effects of furosemide on human peripheral blood mononuclear cells. Ann Allergy Asthma Immunol 83:559–566CrossRefPubMed

30.

Osafune K, Caron L, Borowiak M et al (2008) Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 26:313–315CrossRefPubMed

31.

Cox JS (1967) Disodium cromoglycate (FPL 670) (‘Intal’): a specific inhibitor of reaginic antibody-antigen mechanisms. Nature 216:1328–1329CrossRefPubMed

32.

Theoharides TC, Sieghart W, Greengard P, Douglas WW (1980) Antiallergic drug cromolyn may inhibit histamine secretion by regulating phosphorylation of a mast cell protein. Science 207:80–82CrossRefPubMed

33.

Mazurek N, Berger G, Pecht I (1980) A binding site on mast cells and basophils for the anti-allergic drug cromolyn. Nature 286:722–723CrossRefPubMed

34.

Okada M, Itoh H, Hatakeyama T, Tokumitsu H, Kobayashi R (2003) Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox. Biochem J 374:433–441CrossRefPubMedPubMedCentral

35.

Romanin C, Reinsprecht M, Pecht I, Schindler H (1991) Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells. EMBO J 10:3603–3608PubMedPubMedCentral

36.

Reinsprecht M, Pecht I, Schindler H, Romanin C (1992) Potent block of Cl- channels by antiallergic drugs. Biochem Biophys Res Commun 188:957–963CrossRefPubMed

37.

Heinke S, Szücs G, Norris A, Droogmans G, Nilius B (1995) Inhibition of volume-activated chloride currents in endothelial cells by chromones. Br J Pharmacol 115:1393–1398CrossRefPubMedPubMedCentral

38.

Jenkins L, Brea J, Smith NJ et al (2010) Identification of novel species-selective agonists of the G-protein-coupled receptor GPR35 that promote recruitment of β-arrestin-2 and activate Gα13. Biochem J 432:451–459CrossRefPubMed

39.

Yang Y, Lu JY, Wu X et al (2010) G-protein-coupled receptor 35 is a target of the asthma drugs cromolyn disodium and nedocromil sodium. Pharmacology 86:1–5CrossRefPubMed

40.

Hrvatin S, O’Donnell CW, Deng F et al (2014) Differentiated human stem cells resemble fetal, not adult, β cells. Proc Natl Acad Sci U S A 111:3038–3043CrossRefPubMedPubMedCentral

41.

Goulley J, Dahl U, Baeza N, Mishina Y, Edlund H (2007) BMP4-BMPR1A signaling in β cells is required for and augments glucose-stimulated insulin secretion. Cell Metab 5:207–219CrossRefPubMed

42.

Szabat M, Johnson JD, Piret JM (2010) Reciprocal modulation of adult beta cell maturity by activin A and follistatin. Diabetologia 53:1680–1689CrossRefPubMed

43.

Saito H, Takeuchi M, Chida K, Miyajima A (2011) Generation of glucose-responsive functional islets with a three-dimensional structure from mouse fetal pancreatic cells and iPS cells in vitro. PLoS One 6:e28209CrossRefPubMedPubMedCentral

44.

Logothetopoulos J (1972) Islet cell regeneration and neogenesis. In: Geiger SR (ed) Handbook of physiology. American Physiological Society, Washington, p 67

45.

Bonner-Weir S, Weir GC (2005) New sources of pancreatic β-cells. Nat Biotechnol 23:857–861CrossRefPubMed

46.

Dor Y, Brown J, Martinez OI, Melton DA (2004) Adult pancreatic β-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429:41–46CrossRefPubMed

47.

Wang S, Jensen JN, Seymour PA et al (2009) Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function. Proc Natl Acad Sci U S A 16:9715–9720CrossRef

48.

Rezania A, Bruin JE, Xu J et al (2013) Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo. Stem Cells 31:2432–2442CrossRefPubMed

49.

Bruin JE, Rezania A, Xu J et al (2013) Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice. Diabetologia 56:1987–1998CrossRefPubMed

50.

Furuta Y, Hogan BL (1998) BMP4 is essential for lens induction in the mouse embryo. Genes Dev 12:3764–3775CrossRefPubMedPubMedCentral

Title: Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells
Authors: Yasushi Kondo
Taro Toyoda
Ryo Ito
Michinori Funato
Yoshiya Hosokawa
Satoshi Matsui
Tomomi Sudo
Masahiro Nakamura
Chihiro Okada
Xiaotong Zhuang
Akira Watanabe
Akira Ohta
Nobuya Inagaki
Kenji Osafune
Publication date: 01-08-2017
Publisher: Springer Berlin Heidelberg
Published in: Diabetologia / Issue 8/2017
Print ISSN: 0012-186X
Electronic ISSN: 1432-0428
DOI: https://doi.org/10.1007/s00125-017-4302-7

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Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells

Abstract

Aims/hypothesis

Methods

Results

Conclusions/interpretation

Data availability

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