Single-cell RNA-seq Reveals Novel Regulators of Human Embryonic Stem Cell Differentiation to Definitive Endoderm

Overview

Journal Genome Biol

Specialties Biology
Genetics

Date 2016 Aug 19

PMID 27534536

Citations 203

Authors

Li-Fang Chu

Ning Leng

Jue Zhang

Zhonggang Hou

Daniel Mamott

David T Vereide

Jeea Choi

Christina Kendziorski

Ron Stewart

James A Thomson

Affiliations

Soon will be listed here.

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Citing Articles

AcImpute: a constraint-enhancing smooth-based approach for imputing single-cell RNA sequencing data.

Zhang W, Liu T, Zhang H, Li Y Bioinformatics. 2025; 41(3).

PMID: 40037523 PMC: 11890269. DOI: 10.1093/bioinformatics/btae711.

GABA Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell-Derived Sensory Neurons by Baclofen and α-Conotoxin Vc1.1.

St Clair-Glover M, Yousuf A, Kaul D, Dottori M, Adams D J Neurochem. 2025; 169(1):e70004.

PMID: 39871624 PMC: 11773314. DOI: 10.1111/jnc.70004.

Proteomic Approach Using DIA-MS Identifies Morphogenesis-Associated Proteins during Cardiac Differentiation of Human iPS Cells.

Urasawa T, Kawasaki N ACS Omega. 2025; 10(1):344-357.

PMID: 39829588 PMC: 11740111. DOI: 10.1021/acsomega.4c06371.

Supervised analysis of alternative polyadenylation from single-cell and spatial transcriptomics data with spvAPA.

Zhang Q, Kang L, Yang H, Liu F, Wu X Brief Bioinform. 2025; 26(1.

PMID: 39799000 PMC: 11724721. DOI: 10.1093/bib/bbae720.

Analyzing scRNA-seq data by CCP-assisted UMAP and tSNE.

Hozumi Y, Wei G PLoS One. 2024; 19(12):e0311791.

PMID: 39671349 PMC: 11642954. DOI: 10.1371/journal.pone.0311791.

References

Burden C, Qureshi S, Wilson S . Error estimates for the analysis of differential expression from RNA-seq count data. PeerJ. 2014; 2:e576. PMC: 4179614. DOI: 10.7717/peerj.576. View

DAmour K, Agulnick A, Eliazer S, Kelly O, Kroon E, Baetge E . Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005; 23(12):1534-41. DOI: 10.1038/nbt1163. View

Stegle O, Teichmann S, Marioni J . Computational and analytical challenges in single-cell transcriptomics. Nat Rev Genet. 2015; 16(3):133-45. DOI: 10.1038/nrg3833. View

Rodaway A, Takeda H, Koshida S, Broadbent J, Price B, Smith J . Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF. Development. 1999; 126(14):3067-78. DOI: 10.1242/dev.126.14.3067. View

Kim J, Marioni J . Inferring the kinetics of stochastic gene expression from single-cell RNA-sequencing data. Genome Biol. 2013; 14(1):R7. PMC: 3663116. DOI: 10.1186/gb-2013-14-1-r7. View

Liang K, Liu T, Chu N, Kang J, Zhang R, Yu Y . KLF8 is required for bladder cancer cell proliferation and migration. Biotechnol Appl Biochem. 2014; 62(5):628-33. DOI: 10.1002/bab.1310. View

Leng N, Choi J, Chu L, Thomson J, Kendziorski C, Stewart R . OEFinder: a user interface to identify and visualize ordering effects in single-cell RNA-seq data. Bioinformatics. 2016; 32(9):1408-10. PMC: 4848403. DOI: 10.1093/bioinformatics/btw004. View

Bendall S, Davis K, Amir E, Tadmor M, Simonds E, Chen T . Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development. Cell. 2014; 157(3):714-25. PMC: 4045247. DOI: 10.1016/j.cell.2014.04.005. View

Yan Q, Zhang W, Wu Y, Wu M, Zhang M, Shi X . KLF8 promotes tumorigenesis, invasion and metastasis of colorectal cancer cells by transcriptional activation of FHL2. Oncotarget. 2015; 6(28):25402-17. PMC: 4694840. DOI: 10.18632/oncotarget.4517. View

10.

Robinson M, McCarthy D, Smyth G . edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009; 26(1):139-40. PMC: 2796818. DOI: 10.1093/bioinformatics/btp616. View

11.

Hockemeyer D, Wang H, Kiani S, Lai C, Gao Q, Cassady J . Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011; 29(8):731-4. PMC: 3152587. DOI: 10.1038/nbt.1927. View

12.

Tam P, Loebel D . Gene function in mouse embryogenesis: get set for gastrulation. Nat Rev Genet. 2007; 8(5):368-81. DOI: 10.1038/nrg2084. View

13.

Leng N, Dawson J, Thomson J, Ruotti V, Rissman A, Smits B . EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics. 2013; 29(8):1035-43. PMC: 3624807. DOI: 10.1093/bioinformatics/btt087. View

14.

Kharchenko P, Silberstein L, Scadden D . Bayesian approach to single-cell differential expression analysis. Nat Methods. 2014; 11(7):740-2. PMC: 4112276. DOI: 10.1038/nmeth.2967. View

15.

Li B, Dewey C . RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12:323. PMC: 3163565. DOI: 10.1186/1471-2105-12-323. View

16.

Hardcastle T, Kelly K . baySeq: empirical Bayesian methods for identifying differential expression in sequence count data. BMC Bioinformatics. 2010; 11:422. PMC: 2928208. DOI: 10.1186/1471-2105-11-422. View

17.

Vereide D, Vickerman V, Swanson S, Chu L, McIntosh B, Thomson J . An expandable, inducible hemangioblast state regulated by fibroblast growth factor. Stem Cell Reports. 2014; 3(6):1043-57. PMC: 4264065. DOI: 10.1016/j.stemcr.2014.10.003. View

18.

Trapnell C . Defining cell types and states with single-cell genomics. Genome Res. 2015; 25(10):1491-8. PMC: 4579334. DOI: 10.1101/gr.190595.115. View

19.

Fan J, Salathia N, Liu R, Kaeser G, Yung Y, Herman J . Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat Methods. 2016; 13(3):241-4. PMC: 4772672. DOI: 10.1038/nmeth.3734. View

20.

Xie W, Schultz M, Lister R, Hou Z, Rajagopal N, Ray P . Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell. 2013; 153(5):1134-48. PMC: 3786220. DOI: 10.1016/j.cell.2013.04.022. View