Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes

Abstract

Summary Directed differentiation of human pluripotent stem cells (hPSCs) into somatic counterparts is a valuable tool for studying disease. However, examination of developmental mechanisms in hPSCs remains challenging given complex multi-factorial actions at different stages. Here, we used TALEN and CRISPR-Cas-mediated gene editing and hPSC directed differentiation for a systematic analysis of the roles of 8 pancreatic transcription factors (PDX1, RFX6, PTF1A, GLIS3, MNX1, NGN3, HES1 and ARX). Our analysis not only verified conserved gene requirements between mice and humans, but also revealed a number of previously unsuspected developmental mechanisms with implications for type 2 diabetes. These include a role of RFX6 in regulating the number of pancreatic progenitors, a haploinsufficient requirement for PDX1 in pancreatic β cell differentiation, and a potentially divergent role of NGN3 in humans and mice. Our findings support use of systematic genome editing in hPSCs as a strategy for understanding mechanisms underlying congenital disorders.

Genetic regulatory signatures underlying islet gene expression and type 2 diabetes

“…S11) and involved in pancreatic progenitor specification, endocrine cell differentiation, maintenance of beta cell functional identity, and control of glucose homeostasis (28)(29)(30). Beta cell-specific deletion of RFX6 results in impaired insulin secretion (31,32).…”

Studying Kidney Disease Using Tissue and Genome Engineering in Human Pluripotent Stem Cells

“…Illustrating the power of CRISPR/Cas9 for functional human genetics, a recent study by Zhu et al [50] combined differentiation of hPSCs and gene editing to systematically study the role of 8 pancreatic transcription factors during pancreatic development and disease. The study uncovers the specific developmental step(s) affected by mutations in these genes and highlights the importance of developing human-specific models for studying human genetics [50].…”

Editing genetics, stem cells are prophetic, what’s the best way to model cells of diabetics?

“…In the June 2016 issue of Cell Stem Cell, Zhu et al elegantly combined gene-editing technologies in hPSCs to probe the relevance of previously discovered murine genetic mechanisms to human pancreatic development and disease (26). To study the role of genes during human development, they created gain of function and loss of function hPSC lines.…”

“…This system was used by Zhu et al [2016] to interrogate the function of eight pancreatic transcription factors, six of which (Pdx1, Rfx6, Ptf1a, Glis3, Mnx1, and Ngn3 but not Hes1 or Arx) are associated with permanent neonatal diabetes mellitus (31)(32)(33)(34)(35). A total of 62 hPSC mutant lines were created, with each gene targeted by two gRNAs, four mutant lines per gene, and two isogenic control lines per targeting experiment.…”

“…Zhu et al (26) devised a sophisticated platform enabling the analysis of a large number of genes during human development. Using these tools, a multitude of genes can be assessed, and the developmental steps that are perturbed can be teased out and further investigated for deep, mechanistic analysis.…”