Aberrant Splicing of <i>INS</i> Impairs Beta-Cell Differentiation and Proliferation by ER Stress in the Isogenic iPSC Model of Neonatal Diabetes

Abstract

One of the causes of diabetes in infants is the defect of the insulin gene (INS). Gene mutations can lead to proinsulin misfolding, an increased endoplasmic reticulum (ER) stress and possible beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. We generated induced pluripotent stem cells (iPSCs) from a patient diagnosed with neonatal diabetes mellitus carrying the INS mutation in the 2nd intron (c.188-31G>A) and engineered isogenic CRISPR/Cas9 mutation-corrected cell lines. Differentiation into beta-like cells demonstrated that mutation led to the emergence of an ectopic splice site within the INS and appearance of the abnormal RNA transcript. Isogenic iPSC lines differentiated into beta-like cells showed a clear difference in formation of organoids at pancreatic progenitor stage of differentiation. Moreover, MIN6 insulinoma cell line expressing mutated cDNA demonstrated significant decrease in proliferation capacity and activation of ER stress and unfolded protein response (UPR)-associated genes. These findings shed light on the mechanism underlying the pathogenesis of monogenic diabetes.

Keywords

• diabetes
• insulin
• induced pluripotent stem cells
• CRISPR/Cas9 genome editing
• isogenic cell lines
• proliferation