Abstract 714: Mapping the Transcriptional Dynamics of Pluripotent Stem Cell-derived Endothelial Cell Differentiation by Single Cell RNA Sequencing

Abstract

Objectives: Human embryonic stem cell derived endothelial cell (hESC-EC) differentiation offers a model to study the process of endothelial development, commitment and maturation, thus potentially informing future regeneration strategies. We used high throughput single cell RNA sequencing (scRNA-seq) to characterise the cellular heterogeneity and associated transcriptional signatures throughout hESC-EC differentiation. Methods and Results: Using an established eight-day hESC-EC (H9) differentiation protocol yielding 66% CD31 + /CD144 + cells, we sampled cells for scRNA-seq (10X Chromium) at pluripotent (day 0), mesodermal (day 4), early (day 6) and late (day 8) endothelial stages (total 21,369 cells). The pluripotent and mesodermal population were largely homogeneous prior to the emergence of distinct endothelial and mesenchymal populations. Pseudotime analysis characterised the transcriptional signatures throughout differentiation, revealing a bifurcation point giving rise to both populations. Repeating the study with a second hESC line (RC11) produced equivalent populations and transcriptional signatures. In addition, scRNA-seq analysis of an alternative hESC-EC differentiation protocol revealed substantially more heterogeneity, including nephrogenic and haemogenic populations. However, pseudotime analysis revealed common expression of known and uncharacterised endothelial transcription factors across both protocols. Comparison of hESC-EC to foetal and mature EC revealed their transcriptional distinction, suggesting their non-specified nature. Conclusions: We sequenced over 105,000 cells allowing characterisation of hESC-EC differentiation across different hESC lines and protocols. We identified a common bifurcation point and remarkable concordance between the transcriptional signatures across different hESC lines and differentiation protocols. This included identifying novel transcription factors that may drive endothelial commitment and maturation.