Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes

Elife. 2018 Nov 9;7:e38519. doi: 10.7554/eLife.38519.

Abstract

Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.

Keywords: CRISPR-Cas9; Insulin gene mutations; beta-cell development; endoplasmic reticulum stress; human; induced pluripotent stem cells; mTORC1; regenerative medicine; stem cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / genetics
  • CRISPR-Cas Systems / genetics
  • Cell Differentiation / genetics
  • Cell Proliferation / genetics
  • Diabetes Mellitus / genetics*
  • Diabetes Mellitus / pathology
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum Stress / genetics*
  • Female
  • Humans
  • Induced Pluripotent Stem Cells / chemistry*
  • Induced Pluripotent Stem Cells / metabolism
  • Infant, Newborn
  • Insulin-Secreting Cells / chemistry
  • Insulin-Secreting Cells / metabolism
  • Male
  • Mice
  • Mutation
  • Proinsulin / chemistry
  • Proinsulin / genetics*
  • Protein Folding
  • Sequence Analysis, RNA
  • Signal Transduction
  • Single-Cell Analysis

Substances

  • Proinsulin

Supplementary concepts

  • Diabetes Mellitus, Permanent Neonatal

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.