Diverse and Distinct Human Hematopoietic Differentiation Programs Generate By Bone Marrow and G-CSF Mobilized Hematopoietic Stem and Progenitors Cells

Abstract Several studies have highlighted murine hematopoietic stem cell (HSC) heterogeneity using single cell transplantation, clonal tracking barcoding analysis as well as RNAseq single cell analysis. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome(WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPCs) and is consequently transmitted to all its progeny. Each integration sites (IS) corresponds to a particular stem/progenitor cell clone, for which we can quantify its contribution in each cell lineage. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique IS in fractionated blood cell populations. We concentrated on four WAS patients treated by gene therapy with two distinct sources of autologous HSPC : bone marrow (BM) or mobilized peripheral blood (MPB) (collected by apheresis following administration of granulocyte colony-stimulating factor (G-CSF)). In these patients, we have sorted 5 cell types from peripheral blood at two timepoints of follow up (1 year and 3 years): myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analyzed their IS profile (using our new optimized pipeline, INSPIIRED, Berry et al Mol. Ther. Methods Clin. Dev. 2017 and Sherman et al Mol. Ther. Methods Clin. Dev. 2017). Using this approach, we have characterized up to tens of thousands IS per patients. We used statistical methods to account for sparse sampling and imperfect cell purifications. Using clustering algorithms, we identified different groups of IS clones corresponding to different human hematopoietic differentiation programs. In patients grafted with BM, we showed that at least a third of IS clones correspond to multipotent HSPCs, contributing to all 5 lineages, while other IS clones are mostly oligopotent or unipotent HSPCs with either myeloid or lymphoid potential. In patients treated with MPB, we also observed a diversity of IS clones but corresponding mainly to unipotent or oligopotent HSPCs and very rare multipotent clones. Longitudinal analysis of clonal dynamics, still ongoing, suggests the maintenance of this heterogeneity of HSPC over time. This study demonstrates the heterogeneity of human HSPC, with a diversity of inferred hematopoietic programs contributing to human blood homeostasis. Importantly these data also highlight distinct hematopoietic differentiation programs depending on the source of HSPC , G-CSF mobilized HSPC or BM resident HSPC. These new findings and approaches provide unique data on human hematopoiesis and should help defining the best source of HSPC and mobilizing agent for future cell and gene therapy clinical trials. Disclosures Thrasher: Orchard Therapeutics: Consultancy; Rocket Pharmaceuticals Ltd: Consultancy; 4bio Ventures Management Ltd: Other: Advisory Board; Torus Therapeutics, Inc: Other: Advisory Board.