Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution

Abstract

Conventional human embryonic stem cells are considered to be primed pluripotent but can be induced to enter a naive state. However, the transcriptional features associated with naive and primed pluripotency are still not fully understood. Here we used single-cell RNA sequencing to characterize the differences between these conditions. We observed that both naive and primed populations were mostly homogeneous with no clear lineage-related structure and identified an intermediate subpopulation of naive cells with primed-like expression. We found that the naive-primed pluripotency axis is preserved across species, although the timing of the transition to a primed state is species specific. We also identified markers for distinguishing human naive and primed pluripotency as well as strong co-regulatory relationships between lineage markers and epigenetic regulators that were exclusive to naive cells. Our data provide valuable insights into the transcriptional landscape of human pluripotency at a cellular and genome-wide resolution.

Keywords: early embryonic development; evolution; heterogeneity; human embryonic stem cells; naive; pluripotency; primed; single-cell RNA-seq.

Graphical abstract

Figure 1

Naive and Primed Human ESCs Exhibit Strong Differences in Gene Expression (A) Naive and primed human ESCs were cultured in N2B27 supplemented with t2iL+Gö or in E8 medium, dissociated into single cells, and sorted into 96-well plates loaded with RLT lysis buffer and External RNA Controls Consortium (ERCC) spike-ins. RNA-seq libraries were prepared using the SmartSeq2 protocol and submitted for sequencing. (B) PCA plot of hESC expression profiles, constructed from batch-corrected and normalized log expression values of highly variable genes detected across the entire dataset. Cells are colored by their condition, and the percentage of variance explained by the first two principal components is shown. (C) Smear plot of log₂-fold changes in expression between the naive and primed conditions, where differential expression (DE) genes were detected using edgeR at a false discovery rate (FDR) of 5%. See also Figure S1 and Table S1.

Figure 2

The Naive Subpopulation Is Transcriptionally Distinct from the Other Naive and Primed Cells (A) Heatmap of the top 50 genes with the strongest differential expression between the naive and intermediate cells (top) or between the intermediate and primed cells (bottom). The box for each cell (column) and gene (row) is colored according to the log₂-fold change from the average expression for each gene. (B) Log₂ expression profiles of selected marker genes across cells in the naive, intermediate, and primed populations. Each point represents a cell in the corresponding population. (C) Normalized protein expression of DPPA5 against ABCG2 in naive and primed hESCs. Protein expression was determined using immunofluorescence staining of cytospin-fixed cells. (D) Representative immunofluorescence images of naive and primed hESCs using DPPA5 and ABCG2 antibodies. The scale bar represents 20 μm. See also Figure S2 and Table S2.

Figure 3

Naive and Primed Populations Do Not Exhibit Lineage-Associated Structure, but Correlations between Lineage Markers and Epigenetic Regulators Are Stronger under the Naive Condition (A and B) Gene expression of germ layer-specific marker genes in the (A) naive and (B) primed population, visualized using tSNE on the batch-corrected normalized log expression values. Each point in the scatterplot represents a cell, which is colored by the expression of respective mesoderm (SNAI1), ectoderm (ITGA6), or endoderm (PAF1) markers. (C and D) Heatmaps of the strongest correlation values between selected pluripotency and lineage markers (rows) and epigenetic markers (columns) for the naive (C) and the primed (D) population. The correlation values were bound at [−0.5, 0.5]. See also Figure S3 and Tables S3 and S4.

Figure 4

Cells Shift from a Naive-like to a Primed-like Expression Pattern during Early Embryonic Development Naive and primed markers were identified from the DE analysis of the hESC data. New cells are mapped onto the naive-primed axis based on the proportions of naive and primed markers that they express. This was performed for cells derived from mouse embryos (A), cynomolgus monkey embryos (B), and human pre-implantation embryos (C). For each plot, the density of cells is represented by the color of the pixels. Cells on the red line have equal proportions of expressed primed and naive markers. See also Figure S4.