Single-cell RNAseq of differentiating human iPSCs during endothelial-to-hematopoietic transition with targeted CRISPR activation
Single-cell RNAseq of differentiating human iPSCs during endothelial-to-hematopoietic transition with targeted CRISPR activation
The cells were sorted by FACS as CD34+ live and singlet. hPSCs were differentiated in a xeno-free composition of SFD medium 14, BSA was substituted with human serum albumin, HSA, (Irvine-Scientific). Day 0 differentiation medium, containing 10 ng/ml BMP4 was added to the colonies prior cutting. Cut colonies were transferred to a bacteriological grade well to form embryoid bodies and cultured for two days. At day 2 media was changed and supplemented with 3 uM CHIR (StemMacs). At day 3, medium was changed and supplemented with 5 ng/ml bFGF and 15 ng/ml VEGF. At day 6 media was changed for final haematopoietic induction in SFD medium supplemented with 5 ng/ml bFGF, 15 ng/ml VEGF, 30 ng/ml IL3, 10 ng/ml IL6, 5 ng/ml IL11, 50 ng/ml SCF, 2 U/ml EPO, 30 ng/ml TPO, 10 ng/ml FLT3L and 25 ng/ml IGF1. From day 6 onward, cytokines were replaced every two days. According to 10x manufacturer protocol According to 10x manufacturer protocol