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Cited by 479Pluripotent Stem Cells ResearchDevelopmental Biology and Gene RegulationSingle-cell and spatial transcriptomics

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Single-cell RNA-seq reveals novel regulators of human embryonic stem cell differentiation to definitive endoderm

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Chu, Li-Fang, Leng, Ning, Zhang, Jue, Hou, Zhonggang, Mamott, Daniel, Vereide, David T., Choi, Jeea, Kendziorski, Christina, Stewart, Ron, Thomson, James A.·2016·Genome Biology
DOI 10.1186/s13059-016-1033-xPMC PMC4989499
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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.

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Fig. 1 Snapshot scRNA-seq analysis of human ES-derived progenitors. a Schematics of experimental strategy. Human ES-derived lineage-specific progenitors were profiled at the single-cell resolution. b Principal component analysis (PCA) of all the cell types profiled. Shown are PC4 vs. PC1 and PC2 vs. PC4. c Hierarchical clustering analysis of progenitors differentiated from H1 cells with selected lineage-specific markers shown on the right. d PCA of all the cell types profiled, shown are PC5 vs. PC6. The light green shade highlights all the single DE cells. e Enrichment analysis of PC5 shown in (d). Bar graph shows the significant z scores of selected GO terms. Summary on collections of GO terms are shown on the right. Dashed line indicates statistical significant threshold at z score = 1.62 (one tailed p value <0.05). NPC neuronal progenitor cell, DEC definitive endoderm cell, EC endothelial cell, TB trophoblast-like cell, HFF human foreskin fibroblasts
Fig. 1 Snapshot scRNA-seq analysis of human ES-derived progenitors. a Schematics of experimental strategy. Human ES-derived lineage-specific progenitors were profiled at the single-cell resolution. b Principal component analysis (PCA) of all the cell types profiled. Shown are PC4 vs. PC1 and PC2 vs. PC4. c Hierarchical clustering analysis of progenitors differentiated from H1 cells with selected lineage-specific markers shown on the right. d PCA of all the cell types profiled, shown are PC5 vs. PC6. The light green shade highlights all the single DE cells. e Enrichment analysis of PC5 shown in (d). Bar graph shows the significant z scores of selected GO terms. Summary on collections of GO terms are shown on the right. Dashed line indicates statistical significant threshold at z score = 1.62 (one tailed p value <0.05). NPC neuronal progenitor cell, DEC definitive endoderm cell, EC endothelial cell, TB trophoblast-like cell, HFF human foreskin fibroblastsCC BY · PubMed Central
Fig. 2 Acute hypoxic treatment enhanced DE formation. a FACS analysis of anti-CXCR4 staining of H1 cells differentiated for three days towards (blue population) under various hypoxia conditions. Undifferentiated H1 cells were gated as negative controls (gray populations). The x-axis indicates the APC channel. b Summary of the percentages of CXCR4+ cells (with various oxygen concentrations) from H1 or H9 differentiation for three days toward DE cells. c QPCR analysis of experiments performed in (b). All expression levels were first normalized to endogenous GAPDH. For pluripotency markers (upper panels), samples were normalized to undifferentiated H1 or H9 samples, which were arbitrarily set to 1. For other markers (mid and lower panels), samples were normalized to the 20 % O2 samples, which were arbitrarily set to 1. d Left panel, schematics of various lengths of hypoxic treatment. Right panel, qPCR analysis at day four of differentiation for H1 or H9 cells. The x-axis indicates the number of days treated with 1.5 % O2 as indicated in the left panel. Samples were normalized to those from normoxia, which were arbitrarily set to 1. e Confocal images of OCT4, FOXA2, and SOX17 immunofluorescence staining at day two of differentiation under 20 % or 1.5 % O2. Scale bars = 50 μm. All data are mean ± S.D. ***p <0.001; **p <0.01; *p <0.05, by one-tailed t-test. n.s. no significant difference
Fig. 2 Acute hypoxic treatment enhanced DE formation. a FACS analysis of anti-CXCR4 staining of H1 cells differentiated for three days towards (blue population) under various hypoxia conditions. Undifferentiated H1 cells were gated as negative controls (gray populations). The x-axis indicates the APC channel. b Summary of the percentages of CXCR4+ cells (with various oxygen concentrations) from H1 or H9 differentiation for three days toward DE cells. c QPCR analysis of experiments performed in (b). All expression levels were first normalized to endogenous GAPDH. For pluripotency markers (upper panels), samples were normalized to undifferentiated H1 or H9 samples, which were arbitrarily set to 1. For other markers (mid and lower panels), samples were normalized to the 20 % O2 samples, which were arbitrarily set to 1. d Left panel, schematics of various lengths of hypoxic treatment. Right panel, qPCR analysis at day four of differentiation for H1 or H9 cells. The x-axis indicates the number of days treated with 1.5 % O2 as indicated in the left panel. Samples were normalized to those from normoxia, which were arbitrarily set to 1. e Confocal images of OCT4, FOXA2, and SOX17 immunofluorescence staining at day two of differentiation under 20 % or 1.5 % O2. Scale bars = 50 μm. All data are mean ± S.D. ***p <0.001; **p <0.01; *p <0.05, by one-tailed t-test. n.s. no significant differenceCC BY · PubMed Central
Fig. 3 Time course scRNA-seq data analysis and reconstructing DE differentiation trajectory. a Upper panel, schematics of experimental strategy illustrating time points of scRNA-seq sampling along the differentiation from pluripotent state though mesendoderm to DE cells. Lower panel, PCA of scRNA-seq data, shown is PC1 vs. PC2. b Violin plots of selected stage-specific markers identified by SCPattern analysis. The y-axis indicates normalized expression value, Log2(expected count + 1). The x-axis indicates time points of sampling. c Reconstructed single-cell order by Wave-Crest. Shown are four of the eight genes used for temporal reconstruction. The x-axis indicates cells following Wave-Crest recovered cell order. The y-axis indicates normalized expression value. Fitted lines of gene-specific expression are shown in black. d Top 25 upregulated or downregulated genes identified by Wave-Crest at 36 h of differentiation. The x-axis indicates gene symbol, the y-axis indicates the significance value, -Log (p value) of each gene
Fig. 3 Time course scRNA-seq data analysis and reconstructing DE differentiation trajectory. a Upper panel, schematics of experimental strategy illustrating time points of scRNA-seq sampling along the differentiation from pluripotent state though mesendoderm to DE cells. Lower panel, PCA of scRNA-seq data, shown is PC1 vs. PC2. b Violin plots of selected stage-specific markers identified by SCPattern analysis. The y-axis indicates normalized expression value, Log2(expected count + 1). The x-axis indicates time points of sampling. c Reconstructed single-cell order by Wave-Crest. Shown are four of the eight genes used for temporal reconstruction. The x-axis indicates cells following Wave-Crest recovered cell order. The y-axis indicates normalized expression value. Fitted lines of gene-specific expression are shown in black. d Top 25 upregulated or downregulated genes identified by Wave-Crest at 36 h of differentiation. The x-axis indicates gene symbol, the y-axis indicates the significance value, -Log (p value) of each geneCC BY · PubMed Central
Fig. 4 Generation of T-2A-EGFP knock-in reporter cell line. a Gene targeting strategy of knock-in T-2A-EGFP-PGK-Puro cassette to replace the endogenous T stop codon, facilitated by the CRISPR/Cas9. Gray boxes indicate the exons of the endogenous gene. The arrow indicates the position of the Cas9/sgRNA cut site. The position of the probe for southern blot is indicated. E EcoRI sites. The lengths for each EcoRI-digested genomic DNA fragments are indicated. b Southern blot shows the targeted allele shifted from 4.2 (wild type genomic fragment, WT) to 4.8 kb (knock-in genomic fragment, KI). c Confocal images of SOX2, T, and EGFP immunofluorescence staining over three days of differentiation. Hours of differentiation are indicated. Scale bars = 50 μm. d FACS analysis monitoring the dynamics of the percentage of CXCR4+ and T-2A-EGFP+ cells over three days of differentiation. The x-axis indicates GFP/FITC channel. The y-axis indicates APC channel
Fig. 4 Generation of T-2A-EGFP knock-in reporter cell line. a Gene targeting strategy of knock-in T-2A-EGFP-PGK-Puro cassette to replace the endogenous T stop codon, facilitated by the CRISPR/Cas9. Gray boxes indicate the exons of the endogenous gene. The arrow indicates the position of the Cas9/sgRNA cut site. The position of the probe for southern blot is indicated. E EcoRI sites. The lengths for each EcoRI-digested genomic DNA fragments are indicated. b Southern blot shows the targeted allele shifted from 4.2 (wild type genomic fragment, WT) to 4.8 kb (knock-in genomic fragment, KI). c Confocal images of SOX2, T, and EGFP immunofluorescence staining over three days of differentiation. Hours of differentiation are indicated. Scale bars = 50 μm. d FACS analysis monitoring the dynamics of the percentage of CXCR4+ and T-2A-EGFP+ cells over three days of differentiation. The x-axis indicates GFP/FITC channel. The y-axis indicates APC channelCC BY · PubMed Central
Fig. 5 KLF8 mediates mesendoderm to DE differentiation. a Schematics of siRNA knockdown strategy. FACS analysis of CXCR4 and T-2A-EGFP expression at day two of differentiation with representative gene-specific siRNA transfection results. b Summary Differentiation Scores of siRNA knockdown experiments of the top 25 genes tested. The dashed line indicates a Differentiation Score of 1, set by the non-target siRNA control. Genes in blue font indicate control experiments. c Upper panel, schematics of KLF8 overexpression strategy during differentiation. Lower panel, FACS analysis shows CXCR4 and T-2A-EGFP expression at day two of differentiation. DOX Doxycycline. d QPCR analysis of three independent KLF8 overexpression clones tested at day two of differentiation. All data are shown as mean ± S.D. ***p <0.001; **p <0.01; *p <0.05, all by one-tailed t-test. In all FACS plots, the x-axis indicates GFP/FITC channel, the y-axis indicates APC channel
Fig. 5 KLF8 mediates mesendoderm to DE differentiation. a Schematics of siRNA knockdown strategy. FACS analysis of CXCR4 and T-2A-EGFP expression at day two of differentiation with representative gene-specific siRNA transfection results. b Summary Differentiation Scores of siRNA knockdown experiments of the top 25 genes tested. The dashed line indicates a Differentiation Score of 1, set by the non-target siRNA control. Genes in blue font indicate control experiments. c Upper panel, schematics of KLF8 overexpression strategy during differentiation. Lower panel, FACS analysis shows CXCR4 and T-2A-EGFP expression at day two of differentiation. DOX Doxycycline. d QPCR analysis of three independent KLF8 overexpression clones tested at day two of differentiation. All data are shown as mean ± S.D. ***p <0.001; **p <0.01; *p <0.05, all by one-tailed t-test. In all FACS plots, the x-axis indicates GFP/FITC channel, the y-axis indicates APC channelCC BY · PubMed Central
Fig. 6 Snapshot and temporal scRNA-seq profiling on progenitor cell states. scRNA-seq from snapshots of lineage-specific progenitors revealed that hypoxia enhances DE differentiation in a time-sensitive manner, likely acting on the mesendodermal progenitors. The differentiation trajectory was reconstructed by Wave-Crest (blue arrow) using time course scRNA-seq along the differentiation towards DE cells. Results from loss-of-function and gain-of-function experiments demonstrated that KLF8 function as a positive regulator mediating cell states transitions from T + mesendoderm to CXCR4 + DE cells. We hypothesize that KLF8 can suppress T + state and in turn enhance the activation of CXCR4 + state. The molecular mechanisms of how KLF8 directly or indirectly suppress T + state require future investigation
Fig. 6 Snapshot and temporal scRNA-seq profiling on progenitor cell states. scRNA-seq from snapshots of lineage-specific progenitors revealed that hypoxia enhances DE differentiation in a time-sensitive manner, likely acting on the mesendodermal progenitors. The differentiation trajectory was reconstructed by Wave-Crest (blue arrow) using time course scRNA-seq along the differentiation towards DE cells. Results from loss-of-function and gain-of-function experiments demonstrated that KLF8 function as a positive regulator mediating cell states transitions from T + mesendoderm to CXCR4 + DE cells. We hypothesize that KLF8 can suppress T + state and in turn enhance the activation of CXCR4 + state. The molecular mechanisms of how KLF8 directly or indirectly suppress T + state require future investigationCC BY · PubMed Central

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Authors
Chu, L.-F., Leng, N., Zhang, J., Hou, Z., Mamott, D., Vereide, D. T., Choi, J., Kendziorski, C., Stewart, R., & Thomson, J. A.
Affiliations
  • Morgridge Institute for Research (US)
  • Genentech (US)
  • Harvard University (US)
  • University of Wisconsin–Madison (US)
Corresponding author
  • Chu, Li-Fang
  • Thomson, James A.
ORCID
Venue
Genome Biology
Publisher
Springer Science and Business Media LLC
Published
2016-08-17
Volume
17
Issue
1
Pages
173-173
ISSN
  • 1465-6906 (ISSN-L)
  • 1474-760X
DOI
10.1186/s13059-016-1033-x
PMC
PMC4989499
OpenAlex
W2511896561
Cited by
477
References
83
Citations by year
2026: 41 · 2025: 52 · 2024: 71 · 2023: 48 · 2022: 61 · 2021: 66 · 2020: 49 · 2019: 39 · 2018: 21 · 2017: 22 · 2016: 5
FWCI
15.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Genome biology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
Funders
National Institutes of Health (4UH3TR000506-03, 5U01HL099773-06, GM102756, U54 AI117924)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
definitive endoderm differentiation, human embryonic stem cells, mesendoderm, lineage specification, hypoxia, KLF8, Brachyury, CXCR4, cell fate determination, pluripotency exit
MeSH terms
Human Embryonic Stem Cells, Cell Differentiation, Endoderm, Humans, RNA, Gene Expression Regulation, Developmental, Cell Culture Techniques, Hepatocytes, Pluripotent Stem Cells, Single-Cell Analysis, High-Throughput Nucleotide Sequencing

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