Illumina HiSeq 4000 paired end sequencing; Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs
Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs
The sorting strategy for each sample is indicated in the file name and available in the processed metadata files. 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 M CHIR (StemMacs). At day 3, embryoid bodies were collected and dissociated in Accutase (Gibco) to single cell solution, cells were plated on tissue culture grade wells in SFD medium supplemented with 5 ng/ml bFGF and 15 ng/ml VEGF (2x105 cells/well on 6 well plates or 1x105 cells/well on 12 well plate). 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's protocol. According to 10x manufacturer's protocol.
Experimental Factor: time
13
Experimental Factor: immunophenotype
CD31+