Circulating Hematopoietic Stem/Progenitor Cell subsets contribute to human hematopoietic homeostasis

In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPC) are present in peripheral blood (PB) and their role remain unsolved. By integrating advanced immunophenotyping, high-throughput transcriptome profiling and functional assays we characterized the biological properties of human cHSPC. We found distinct composition but comparable bone marrow (BM) homing and differentiation potential after xenotransplantation of cHSPC with respect to their BM counterpart. Trafficking HSPC are in a low-cycling pre-activated state with primitive and committed progenitors being transcriptionally and functionally skewed towards erythroid differentiation. Moreover, a subset of circulating lymphoid progenitors are primed for seeding lymphoid organs and can actively contribute to T-cell production in patients treated with gene therapy. In case of hematopoietic impairment, cHSPC content and composition correlate with BM-HSPC state. Altogether, our data unveiled the cHSPC contribution to steady-state hematopoiesis, with a key function in sustaining extramedullary lympho- and erythro-poiesis, and supported their clinical exploitation as biomarker for BM-HSPC. Overall design: To compare the intrinsic molecular properties of HSPC subsets derived from BM and PB, we analyzed their transcriptional profiles by CITE-seq, which allows unbiased single-cell transcriptome and immunophenotypic analysis. To this aim, we exploited antibody derived tag (ADT)-labeled antibodies directed toward HSPC phenotypic markers. Human BM CD34+ (n=5) or PB CD34+ cells (n=4) or total PBMC (n=1) samples were thawed and stained with a mix of FACS antibodies and ADT-barcoded antibodies directed toward surface antigens used for the phenotypic classification of human HSPC. Then, we FACS sorted primitive (Lin- CD34+ CD38-) and/or bulk (Lin- CD34+) or committed (Lin- CD34+ CD38+) HSPC fractions, depending on the sample. Sorted fractions were processed according to Chromium 10x protocol, generating for each sample separated sequencing libraries from cDNA or ADT enriched fractions. Libraries were subsequently sequenced and resulting data were analyzed and integrated by bioinformatic approaches.