Genetic and Chemical Screenings Identify HDAC3 as a Key Regulator inHepatic Differentiation of Human Pluripotent Stem Cells.

Shuang Li,Yongxu Zhao,Yan Qiu,Yanhao Chen,Zhuanghui Feng,Yuda Wei,Hao Ying,Yuanyuan Yang,Danjun Ma,Xiaojian Liu,Mushan Li,Kiran Musunuru,Zhen Shao,Jing Fang,Qiurong Ding,Tingting Zhou,Hui Wang

doi:10.1016/j.stemcr.2018.05.001

Abstract

SummaryHepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) offer a promising cell resource for disease modeling and transplantation. However, differentiated HLCs exhibit an immature phenotype and comprise a heterogeneous population. Thus, a better understanding of HLC differentiation will improve the likelihood of future application. Here, by taking advantage of CRISPR-Cas9-based genome-wide screening technology and a high-throughput hPSC screening platform with a reporter readout, we identified several potential genetic regulators of HLC differentiation. By using a chemical screening approach within our platform, we also identified compounds that can further promote HLC differentiation and preserve the characteristics of in vitro cultured primary hepatocytes. Remarkably, both screenings identified histone deacetylase 3 (HDAC3) as a key regulator in hepatic differentiation. Mechanistically, HDAC3 formed a complex with liver transcriptional factors, e.g., HNF4, and co-regulated the transcriptional program during hepatic differentiation. This study highlights a broadly useful approach for studying and optimizing hPSC differentiation.

Highlights

Human pluripotent stem cells hold great promise as an attractive resource of human somatic cells, due to their ability to self-renew and, theoretically, to differentiate into any of the myriad of somatic cell types in the human body
Generation of the ALB-Venus Reporter Line Albumin is regarded as a molecular marker of hepatocytes that can reflect cell maturation
To perform screenings for regulators in Hepatocyte-like cells (HLCs) differentiation, we developed a reporter

Summary

Introduction

Human pluripotent stem cells (hPSCs) hold great promise as an attractive resource of human somatic cells, due to their ability to self-renew and, theoretically, to differentiate into any of the myriad of somatic cell types in the human body. The ability to differentiate into a desired cell type often depends on the availability of an efficient protocol. While many of the improvements to the hepatic differentiation protocol are based on the knowledge acquired in understanding signal pathways that control embryonic lineage bifurcations, none of the existing studies tackled the differentiation problems through an unbiased genetic screening approach. With recent advances in genome editing technologies, especially the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, genome-wide genetic screening becomes a high-throughput, low-cost platform that enables comprehensive study of regulators in a biological process (Shalem et al, 2015). We devised a reporter system and applied a CRISPR/Cas9-based genetic screening approach to identify potential regulators in hepatic lineage determination. We have identified several regulators in hepatic differentiation and an efficient small molecule that can improve the differentiation efficiency. Our study demonstrated a broadly useful approach for studying hPSC differentiation

Results

Discussion

Conclusion