Diabetologia

, Volume 60, Issue 8, pp 1454–1466

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

  • 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
Article

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.

Keywords

Beta-like cells BMP4 Human iPSC Insulin–neurogenin 3 Small molecule Sodium cromoglicate 

Abbreviations

4Fs

Nicotinamide forskolin dexamethasone and ALK5 inhibitor

AMY

Amylase

BMP

Bone morphogenetic protein

CK

Cytokeratin

E

Embryonic day

ESC

Embryonic stem cell

GCG

Glucagon

hESC

Human embryonic stem cell

hiPSC

Human induced pluripotent stem cell

GHRE

Ghrelin

INS+

Insulin-positive

iPSC

Induced pluripotent stem cell

NGN3

Neurogenin 3

PDX1

Pancreatic and duodenal homeobox 1

PS

Penicillin/streptomycin

qRT-PCR

Quantitative real-time RT-PCR

SCG

Sodium cromoglicate

SOX17

SRY-box 17

SST

Somatostatin

STZ

Streptozotocin

Supplementary material

125_2017_4302_MOESM1_ESM.pdf (378 kb)
ESM 1(PDF 377 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Yasushi Kondo
    • 1
    • 2
  • Taro Toyoda
    • 1
  • Ryo Ito
    • 1
    • 2
  • Michinori Funato
    • 1
  • Yoshiya Hosokawa
    • 1
  • Satoshi Matsui
    • 1
  • Tomomi Sudo
    • 1
  • Masahiro Nakamura
    • 1
  • Chihiro Okada
    • 1
    • 3
  • Xiaotong Zhuang
    • 2
  • Akira Watanabe
    • 1
  • Akira Ohta
    • 1
  • Nobuya Inagaki
    • 2
  • Kenji Osafune
    • 1
  1. 1.Center for iPS Cell Research and Application (CiRA)Kyoto UniversityKyotoJapan
  2. 2.Department of Diabetes, Endocrinology and NutritionKyoto UniversityKyotoJapan
  3. 3.Mitsubishi Space Software Co., LtdAmagasakiJapan

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