Fig. 1: A transitionary substate between pluripotency and early mesendodermal differentiation is marked by MIXL1-GFP and SSEA-3 coexpression. | Nature Communications

Fig. 1: A transitionary substate between pluripotency and early mesendodermal differentiation is marked by MIXL1-GFP and SSEA-3 coexpression.

From: Generation and trapping of a mesoderm biased state of human pluripotency

Fig. 1

a Flow cytometry scatter plot of MIXL1-GFP and SSEA-3 expression by HES3-MIXL1 cells cultured in MEF/KOSR conditions. Coloured populations indicate the fractions isolated by FACS for bulk and single cell transcriptomic analysis, MIXL1-GFP(−)/SSEA-3(+) (Red) MIXL1-GFP(+)/SSEA-3(+) (Green), MIXL1-GFP(+)/SSEA-3(−) (Blue). Principal component analysis (PCA) plots generated from RNAseq data of the MIXL1-GFP(−)/SSEA-3(+), MIXL1-GFP(+)/SSEA-3(+) and MIXL1-GFP(+)/SSEA-3(−)fractions: b plot generated from all mRNA expression; plots generated from genes associated with c pluripotency and d mesoderm. e t-SNE plot of the expression of 45 genes (Supplementary Table 1) in individual cells measured by Fluidigm Biomark qPCR. All fractions are displayed in the same dimensional space: MIXL1-GFP(−)/SSEA-3(+) cells as red squares, MIXL1-GFP(+)/SSEA-3(+) cells as green circles and MIXL1-GFP(−)/SSEA-3(+) cells as blue triangles. f Heatmap analysis of 232 single cells analysed across 14 of 45 analysed genes using a Fluidigm BioMark system: hierarchical clustering was performed for the genes assessed. The heatmap visualises the individual gene expression after normalisation across genes. White coloured genes indicate undetected levels of expression. Single cells are labelled by colour with the fraction from which they were sorted. Cluster analysis split cells into two broad classification, Cluster A (Pluripotency, Pluri) and Cluster B (Differentiation, Diff). Cells co-expressing pluripotency and differentiation associated genes were readily detected in the MIXL1-GFP(+)/SSEA-3(+) fraction but not the other fractions. g Scatter plot of 1/Ct values for each single cell for SOX2 and T expression (MIXL1-GFP(−)/SSEA-3(+) cells as red squares, MIXL1-GFP(+)/SSEA-3(+) cells as green circles and MIXL1-GFP(−)/SSEA-3(+) cells as blue triangles). A large proportion of MIXL1-GFP(+)/SSEA-3(+) expressed both SOX2 and T. e–g Source data are provided as a Source Data file.

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