Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Basic and Developmental
Hematology Disease Topics & Pathways:
Research, Fundamental Science, hematopoiesis, Biological Processes
In the present study, we phenotypically characterized cHSPC composition by applying multi-parametric flow cytometry on 110 PB and, as control, 48 BM samples of healthy donors (HD) of different age groups. These analyses were combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and ad hoc designed in vitro and in vivo assays to investigate the transcriptional and functional properties of steady-state cHSPC subpopulations with respect to BM counterpart. Moreover, to study circulating vs. resident HSPC differentiation potential in vivo in humans, we exploited integration site (IS) clonal tracking of cHSPC, BM HSPC and mature PB lineages isolated from patients treated with autologous HSPC-gene therapy (GT) at late follow-ups post-treatment (>2 years), once steady-state hematopoiesis is established.
We observed that cHSPC show a progressive reduction in number during aging and a different composition than BM counterpart, with Multi Lymphoid Progenitors (MLP) displaying the highest PB circulation propensity. cHSPC were endowed with BM homing capability and multilineage differentiation potential both in vitro and in vivo, showing an early skewing toward erythroid differentiation after transplantation. Moreover, we detected a reduced long-term human cell engraftment in cHSPC- than BM HSPC-transplanted mice. This latter finding might be explained by the low primitive Hematopoietic Stem Cell (HSC) content and the transcriptional pre-activated state observed in steady-state PB HSC, as assessed in our single-cell RNA-sequencing analyses.
Indeed, applying CITE-seq on PB- and BM-derived HSPC, we identified a unique transcriptional profile of both primitive and lineage-committed cHSPC subpopulations, characterized by lower replicative, metabolic and transcriptional activity, but increased differentiation-, adhesion- and immune response-priming than BM counterpart. Furthermore, we observed that PB phenotypic primitive HSC and Multipotent Progenitors (MPP) and Committed Myeloid Progenitors (CMP) were transcriptionally and functionally skewed toward erythroid lineages as compared to BM-resident counterpart.
Finally, since upon gene correction each HSPC and its progeny became univocally marked by distinct IS, we exploited IS analyses from 5 patients treated with HSPC-GT at >2 years after GT to investigate the steady-state hematopoietic output of PB and BM HSPC in vivo in humans. In line with the enrichment of lymphoid transcriptional signature observed in trafficking HSPC, we found a higher IS sharing between PB mature lymphoid compartment and steady-state PB-derived HSPC. Moreover, a substantial fraction of trafficking HSPC displayed an enriched expression of the gene signature associated with thymus seeding progenitor type 1 (TSP1), a group of low-cycling immature lymphoid progenitors with thymus-emigrant properties. Although at lower level with respect to PB cells, we detected a TSP1 signature also in BM dataset, thus suggesting that few thymus-emigrant lymphoid progenitors originate in the BM and preferentially egress into the PB in order to seed lymphoid organs.
Altogether, our findings indicate PB trafficking HSPC as a peculiar steady-state reservoir of low-cycling, pre-activated hematopoietic progenitors, which are endowed with BM homing and repopulation potential. The enrichment of phenotypic and functional erythroid-committed progenitors in cHSPC suggest a supportive role of these cells in sustaining extramedullary erythropoiesis. Furthermore, our results of IS analyses combined with the higher expression of TSP1 signature in PB vs. BM HSPC indicate the key function of steady-state trafficking HSPC in the seeding of the thymus, with the aim of locally differentiating into lymphoid progeny. Overall, our work represents one of the most comprehensive studies on cHSPC, unveiling fundamental insights on their biological role in humans.
Disclosures: Gentner: ExCellThera: Consultancy; Genenta Science: Consultancy, Current holder of stock options in a privately-held company, Other: Founder, Research Funding.
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