title: Single-cell RNA-seq reveals novel regulators of human embryonic stem cell differentiation to definitive endoderm.
abstract: Human pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states.
  
  
  
  Here, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T) (+) mesendoderm toward a CXCR4 (+) DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation.
  
  
  
  We report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems.
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA. lchu@morgridge.org.
authors.forename: Li-Fang
authors.initials: LF
authors.lastname: Chu
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA.|Present address: Genentech, Inc., South San Francisco, CA, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Leng
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA.
authors.forename: Jue
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA.|Present address: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Zhonggang
authors.initials: Z
authors.lastname: Hou
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mamott
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Vereide
authors.affiliation: Department of Statistics, University of Wisconsin, Madison, WI, USA.
authors.forename: Jeea
authors.initials: J
authors.lastname: Choi
authors.affiliation: Department of Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Kendziorski
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA.
authors.forename: Ron
authors.initials: R
authors.lastname: Stewart
authors.affiliation: Regenerative Biology, Morgridge Institute for Research, Madison, WI, 53715, USA. jthomson@morgridge.org.|Department of Cell & Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA. jthomson@morgridge.org.|Department of Molecular, Cellular, & Developmental Biology, University of California, Santa Barbara, CA, USA. jthomson@morgridge.org.
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics*; D004707:Endoderm / Q000254:growth & development / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D022781:Hepatocytes / Q000166:cytology; D059014:High-Throughput Nucleotide Sequencing; D000066449:Human Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology; D012313:RNA / Q000235:genetics*; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D012313:RNA
keywords: Brachyury; CRISPR/Cas9; Definitive endoderm; Embryonic stem cells; KLF8; Mesendoderm; SCPattern; Single-cell RNA-seq; Wave-Crest