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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHuman 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. In practice, the ability to differentiate into a desired cell type often depends on the availability of an efficient protocol. Stepwise protocols using defined factors have been established years ago for the differentiation from hPSCs toward human hepatocytes (, , ). However, the resulting hepatocytes exhibit an immature hepatic phenotype (e.g., express fetal markers such as alpha fetoprotein) and remain a heterogeneous population. In addition, substantial variation in differentiation efficiencies has been observed among different hPSC lines. Considerable effort has later been devoted to studying the directed differentiation process or screening for compounds for enhanced maturation of HLCs (, , , ). 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 (). In the present study, we devised a reporter system and applied a CRISPR/Cas9-based genetic screening approach to identify potential regulators in hepatic lineage determination. Based on the results from the genetic screening, we also performed a targeted, small-scale chemical screening. We have identified several regulators in hepatic differentiation and an efficient small molecule that can improve the differentiation efficiency. Our study also demonstrated a broadly useful approach for studying hPSC differentiation.