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

Zengrong Zhu; Qing V Li; Kihyun Lee; Bess P Rosen; Federico González; Chew-Li Soh; Danwei Huangfu

doi:10.1016/j.stem.2016.03.015

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

Cell Stem Cell. 2016 Jun 2;18(6):755-768. doi: 10.1016/j.stem.2016.03.015. Epub 2016 Apr 28.

Authors

Zengrong Zhu¹, Qing V Li², Kihyun Lee³, Bess P Rosen⁴, Federico González¹, Chew-Li Soh¹, Danwei Huangfu⁵

Affiliations

¹ Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.
² Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA; Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
³ Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA; Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, 1300 York Avenue, New York, NY 10065, USA.
⁴ Biochemistry, Cell and Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, 1300 York Avenue, New York, NY 10065, USA.
⁵ Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA. Electronic address: huangfud@mskcc.org.

Abstract

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 eight 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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence
Basic Helix-Loop-Helix Transcription Factors / metabolism
CRISPR-Cas Systems / genetics
Cell Differentiation / drug effects
Cell Line
Cell Lineage / drug effects
Cell Lineage / genetics
Diabetes Mellitus / pathology*
Gene Editing*
Gene Knockout Techniques
Genome, Human*
Glucose / pharmacology
Haploinsufficiency / drug effects
Homeodomain Proteins / metabolism
Human Embryonic Stem Cells / cytology
Human Embryonic Stem Cells / drug effects
Human Embryonic Stem Cells / metabolism
Humans
Infant, Newborn
Insulin-Secreting Cells / drug effects
Insulin-Secreting Cells / metabolism
Insulin-Secreting Cells / pathology
Mutation / genetics
Nerve Tissue Proteins / metabolism
Pancreas / embryology*
Pancreas / pathology*
Phenotype
Pluripotent Stem Cells / cytology*
Pluripotent Stem Cells / drug effects
Pluripotent Stem Cells / metabolism
Regulatory Factor X Transcription Factors / metabolism
Time Factors
Trans-Activators / metabolism

Substances

Basic Helix-Loop-Helix Transcription Factors
Homeodomain Proteins
NEUROG3 protein, human
Nerve Tissue Proteins
Regulatory Factor X Transcription Factors
Rfx6 protein, human
Trans-Activators
pancreatic and duodenal homeobox 1 protein
Glucose

Abstract

Publication types

MeSH terms

Substances

Grants and funding