Transcriptome analysis of human embryonic stem cell differentiation to endothelial cells

Deep RNA sequencing was performed during human embryonic stem cell (hESC) differentiation to endothelial cells (ECs), via mesodermal precursors. Our work focused on long non-coding RNAs (lncRNAs) regulated during EC differentiation as lncRNA are known regulators of cell differentiation, physiology and disease. EC-enriched lncRNAs were screened for binding sites for the transcription factor ERG, important for endothelial cell identity and function. The LINC00961 gene, first annotated as a lncRNA but now reassigned as a protein coding gene for the SPAAR micropeptide, was potently activated during differentiation. Using primary ECs, we studied the role of LINC00961 locus after depletion and gain of function experiments and showed its effect on EC adhesion, tube formation, migration and proliferation. We showed that LINC00961 functions as a bona fide non-coding RNA in ECs, at least in part, through binding to TMSB4X. We used an embryoid body based directed differentiation protocol developed in our laboratory to generate ECs directly via mesodermal precursors (MP) (Boulberdaa et al, 2016 Mol Ther). To ensure the purity of specific cell lineages, we included purification steps based on mesodermal (CD326low/CD56+) and endothelial specific cell surface (CD31+/CD144+) markers to enrich target populations. Our RNAseq analysis includes the following populations: the d0 H9 human embryonic stem cells (ESC - 4 replicates), the day3 mesoderm population (CD326lowCD56+ purified from embryoid bodies) (MP - 4 replicates) and the day 3 remaining cell population (non-MP - 4 replicates), the day 7 selected endothelial cells (CD144+CD31+) (EC - 2 replicates) and the day 7 remaining population (non-EC - 2 replicates). We also included in our analysis Human Saphenous Vein Endothelial Cells (HSVEC - 4 replicates) as a control of mature endothelial cell population. RNA-seq was performed (45 million paired end reads per sample) on ribosomal RNAs depleted libraries.