Transcriptional profiling of early haematopoietic mesoderm and endoderm in differentiating human pluripotent stem cells.

The aim of this project was to compare the transcriptional profiles of populations of cells derived from human pluripotent stem cells under haematopoietic and endoderm differentiation conditions. We used a reporter line (denoted SOX-RUNX) that carries mCHERRY inserted into the SOX17 locus and GFP inserted into the RUNX1C locus to mark haematopoietic progenitors (Ng et al., Nature Biotechnology, Nov 2016). SOX17 marks both endothelium and early endoderm. SOX17 is absolutely required at all stages of endoderm formation and the gene is also required for normal endothelial formation and subsequent haematopoieisis. We have also shown that RUNX1 is required for all definitive blood cell formation (Bruveris et al., manuscript submitted). Overall design: (i) Haematopoietic mesoderm differentiation and analysis. This compared the parental SOX-RUNX reporter line and on a derivative in which the DNA binding domain of the RUNX1 gene was deleted using CRISPR mediated gene editing (RUNX KO). These experiments will demonstrate key transcriptional changes occurring during the course of differentiation and will identify RUNX1 dependent transcripts. Samples differentiated for 0, 2, 4, 7 and 12 days were collected from each cell line. The protocol used biased differentiation towards an intra embryonic, AGM-like fate. The samples at d0, d2 and d4 were not flow sorted, but samples at d7 and d12 are sorted into 2 and 3 fractions respectively. Samples from d7 time points represent SOX17 expressing and non expressing CD34+CD43- endothelial cells and the d12 time points have been sorted on SOX17, CD34 and CD43 to generate endothelial and haematopoietic fractions. (ii) Endoderm differentiation. These samples provided comparison with mesodermal differentiation of the SOX-RUNX line for days 0, 2, 4 and 7 of differentiation. Given that the phenotype by day 7 is virtually 100% SOX17+EPCAM+CXCR4+KIT+ endoderm, there was no need to sort cells.