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

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

Researchers at the Morgridge Institute for Research analyzed the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). They identified a definitive endoderm (DE) transcriptomic signature that lead them 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, they demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation.

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

The researchers 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. Their 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.

Availability – Both SCPattern and its graphical interface implementation are freely available at https://github.com/lengning/SCPattern. The software is licensed under the terms of the Apache License 2.0.

Wave-Crest is available as an R package, freely available at (https://github.com/lengning/WaveCrest). The softwares are licensed under the terms of the Apache License 2.0.