Type: Oral
Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Epigenetic and Metabolic Control of Hematopoiesis
Hematology Disease Topics & Pathways:
Research, Fundamental Science
We analyzed ATP2B1 immunophenotypic expression in the human CD34+CD38–CD90+ HSC compartment and performed in vitro single cell differentiation assays and in vivo serial xenotransplantation assays to determine if CD49f LT-HSC could be functionally stratified. ATP2B1 and CD49f surface expression in human neonatal cord blood (CB) and adult mobilized peripheral blood (mPB) sources revealed three immunophenotypic subpopulations: CD49f+ATP2B1+ (P1 LT-HSC), CD49f+ATP2B1– (P2 LT-HSC), and CD49f– ATP2B1– (P3 HSC). The proportion of ATP2B1-expressing P1 LT-HSC is higher in neonatal than adult sources (10% in CB vs 3.5% in mPB) in the CD34+CD38–CD90+ compartment. Single-cell erythroid-myeloid differentiation assays on MS5-stroma following index sorting for ATP2B1 and CD49f expression in CB CD34+CD38–CD90+ HSC revealed increased total clonogenicity and CD34+ output from P1 LT-HSC relative to the other 2 subsets. Moreover, serial replating clonogenic assays showed increasing output from P1 LT-HSC>P2 LT-HSC>P3 HSC and increased maintenance of immunophenotypic CD34+ cells. Of note, P3 HSC and CD90–CD49f– ST-HSC showed similar limited serial clonogenic potential confirming previous reports that these subpopulations have limited self-renewal. We transplanted the three CD34+CD38–CD90+ HSC subsets into immunodeficient mice and found repopulating potential was similar at 4 weeks post-transplant. However, at 20 weeks post-transplantation, human CD45 repopulation as well as the proportion of CD34+CD19– cells was highest to lowest in mice transplanted with P1 LT-HSC>P2 LT-HSC>P3 HSC. As a surrogate for self-renewing LT-HSC (Garcia-Prat et al., Cell Stem Cell 2021), we undertook lysotracker flow cytometry analysis on P1 LT-HSC and P2 LT-HSC cultured for 16 hours. This assay showed that CB and mPB ATP2B1-expressing LT-HSC are significantly enriched for lysosomes, further supporting the notion that P1 LT-HSC may have superior self-renewal. Direct measurement of self-renewal by quantitative secondary limiting dilution transplantation of 20 week primary transplants of P1 LT-HSC and P2 LT-HSC demonstrated 6.9-fold increased stem cell frequency for P1 LT-HSC. Analysis of the combined single-cell transcriptional and epigenetic landscape from 10x Genomics Multiome profiling within P1 and P2 LT-HSC are ongoing. In conclusion, ATP2B1 surface protein expression bifurcates CD49f+ LT-HSC into two discrete functional LT-HSC subpopulations which will enable unprecedented mechanistic resolution into human HSC heterogeneity.
Disclosures: Dick: Pfizer/Trillium Therapeutics: Patents & Royalties: IP interest in SIRP-a therapeutics; Bristol-Myers Squibb/Celgene: Research Funding.