Next Generation Sequencing Identifies the Heterogeneity of Human Pluripotent Stem Cell-Derived AGM-like Cells

Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for different cell lineage formation from human pluripotent stem cells (hPSCs). We report here the application of single cell RNA-sequencing (scRNA-seq) technology for transcriptome profile of hPSC-derived aorta-gonad-mesonephros (AGM)-like endothelial and hematopoietic cells, and compare to those of primary AGM cells. Day 8 and day 18 samples were collected and performed in IIIumina NovaSeq6000. The resulting sequence reads (day 8: 90,000 reads/cells and day 18: 75,000 reads/cell) were mapped to human genome (hg19) using Cell Ranger, and the RefSeq transcript levels (RPKMs) were quantified using the Seurat R package version 3.2. Our scRNA-seq data confirmed the stable expression of key definitive hematopoietic cell markers including SOX17, RUNX1, LMO4 and CD44, and no detectable expression of primitive hematopoietic cell markers, such as GYPA (CD235a), were observed. This study represents the first detailed analysis of AGM-like cell transcriptomes generated by scRNA-seq technology, providing insight into the mechanisms underlying the differentiation of hPSCs into AGM-like cells. Overall design: Endothelial and hematopoietic transcriptome profiles of hPSC-derived AGM-like cells were generated by scRNA-seq technology using IIIumina NovaSeq6000