GSE104689
Maturation of hematopoietic stem cells from pre-hematopoietic stem cells is accompanied by upregulation of programmed death ligand 1
Hematopoietic stem cells (HSCs) in the fetal liver mature from pre-HSCs that originate in the major arteries of the embryo. To generate HSCs from other cell sources it will be necessary to reproduce the maturation of HSCs from pre-HSCs ex vivo. We refined the markers used to purify pre-HSCs and HSCs matured ex vivo, and compared the transcriptomes of pre-HSCs, ex vivo matured HSCs, and fetal liver HSCs. We found that maturation of pre-HSCs into HSCs in vivo or ex vivo is accompanied by the downregulation of genes involved in embryonic development and vasculogenesis, and upregulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that functional ex vivo-matured and fetal liver HSCs increase the expression of programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on functional HSCs after ex vivo culture. PD-L1 signaling, however, is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for HSC maturation. Overall design: Cells were sorted into Trizol-LS. Total RNA was prepared using RNeasy micro kit (Qiagen, Valencia, CA, USA). DNase treatment was performed either on-column (Qiagen) or using DNA-free DNA Removal Kit (Life Technologies). Total RNA was quantified with RNA HS Kit (Q32852) for Qubit fluorometer (Life Technologies) and analyzed for integrity using RNA 6000 Pico Kit (5067-1513) for 2100 Bioanalyzer (Agilent). mRNA was isolated from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, NEB). PolyA-selected mRNA was fragmented to average size of 300 nt, reverse transcribed to generate double-stranded cDNA, and converted to a paired end library using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB) according to manufacturer''s instructions including the optional double size selection procedure using Agencourt AMPure XP beads. Prepared libraries were quantified with dsDNA HS Kit (Q32851) for Qubit and the size distribution was assessed using High Sensitivity DNA Kit (5067-462) for Bioanalyzer. Libraries were sequenced on Illumina HiSeq2500 in paired-end mode with the read length of 75 nt.