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

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.

Encapsulation boosts islet-cell signature in differentiating human induced pluripotent stem cells via integrin signalling

“…This is in contrast with previous studies, which deliberately used clusters to assess the alginate encapsulation effect 29 . We showed here for the first time that The general impact of the 3D organization for the cell differentiation potential is also supported by the introduction of air liquid interface 1 or suspension culture 46 Our subsequent proteome analyses suggested that encapsulation strongly boosts β -cell fate in a stage-specific manner, by promoting early-stage differentiation and potentiating the islet signature during the later stages. To our knowledge, this is the first comparison by global proteomics of encapsulated and Matrigel differentiating cells.…”

Hydrogel platform for in vitro three-dimensional assembly of human stem cell-derived β cells and endothelial cells

“…These are currently being explored for cell replacement therapy, as these cells can spontaneously differentiate into SC-β cells after transplantation over the course of months [3,[27][28][29], but the mechanism of this maturation process is unknown and differs based on rodent species [27]. SC-β cells or earlier progenitors, particularly derived from diabetic patients through an induced pluripotent stem cell (iPSC) intermediate, are currently being studied for disease modeling and drug screening purposes [3,[30][31][32][33][34][35][36][37][38][39][40][41]. These studies would benefit from a cellular assembly that more closely reflects the native islet microenvironment [42].…”

Single Cell Transcriptomic Analysis of Pancreatic β Cell Development and Differentiation from Pluripotent Stem Cells

“…The various differentiation protocols for converting pluripotent stem cells into SC-Islets have been previously reviewed by Harb et al [55] and Millman et al [56]. Islets can be produced from hPSCs with six stage [57] or seven stage [58] differentiation protocols or through a combination of both protocols [59,60]. Protocols range between 21 and 35 days and generate between 30% and 60% SC-β cells.…”

“…Off-target cells include non-islet cells that are either endocrine or non-endocrine cell types [54]. Four studies have sequenced stem cell derived islets from both embryonic and induced pluripotent stem cells across various stages of differentiation (Stage 3 to Stage 7pancreatic progenitor to islet cells) [45,54,59,61]. Previous studies have sequenced cells prior to Stage 3 pancreatic progenitors, including pluripotent stem cells (Stage 0) [62,63] and definitive endoderm stage cells (Stage 1) [64,65].…”