Characterization of transcriptomic changes induced by a gene therapy cell culture protocol to single human bone marrow and mobilized peripheral blood hematopoietic stem and progenitor cells [BM_MPB]

Here we present the single cell transcriptomes of human adult mobilized peripheral blood (mPB) and bone marrow (BM) hematopoietic stem and progenitor cell (HSPC) subsets,: long-term haematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs) and CD34+ cells before (0 h) and after 62 hours (62 h) of culture in a conditions mimicking gene therapy protocols. The culture protocol includes two hits of lentiviral transduction (lentiviral vector contains a GFP construct) and media as previously published . The objectives of this study are to investigate the effects of lentiviral transduction and cell culture in gene therapy protocol conditions on human mPB and BM haematopoietic stem and progenitor cell (HSPC) subsets. In the present study we report that 62 h of culture in gene therapy media significantly alters the transcriptome of BM and mPB subsets. In contrast, lentiviral transduction alone has a minimal effect on the transcriptome of all tested subsets. T=transduced GFP+; NT=non transduced; GFP-= transduced GFP- 4 independent healthy male mPB donors (aged 25-28) and 3 independent healthy male BM donors (aged 27-32) were used for the present study. CD34+ enriched cells were single cell sorted for scRNA-Seq at the 0 h timepoint or bulk sorted from the following populations: LT-HSCs (CD19-/CD34+/CD38-/CD45RA-/CD90+/CD49f+), ST-HSCs (CD19-/CD34+/CD38-/CD45RA-/CD90-/CD49f-) and CD34+ (CD19-/CD34). Bulk subsets were cultured in a 62 h ex vivo gene therapy protocol and either exposed to a lentiviral vector containing a GFP construct (GFP+ or GFP-) or non transduced (NT). A separate condition was also included where mPB LT-HSCs were transduced and cultured for 62 h in the presence of 200nM Palbociclib (PD033299). Palbociclib is a CDK6 inhibitor which reversibly arrests cell cycle progression past early G1 in tested culture conditions. At 62 h, Live cells (isolated as Zombie Aqua negative) from LT-HSC, ST-HSC and CD34+ subsets were single cell sorted for scRNA-Seq. scRNA-Seq plates were processed and libraries generated using an adapted version of the Smart-Seq2 protocol. Paired-end sequenced of 50 bp fragments was performed by Illumina HiSeq 4000. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.