Resolving Human Hematopoietic Stem Cell Functional Heterogeneity with ATP2B1 Surface Expression
Bibliographic record
Abstract
A pool of human long-term hematopoietic stem cells (LT-HSC) maintains life-long hematopoiesis by generating shorter-living progenitors that ultimately replenish mature cells. Molecular (transcriptional, epigenetic) and functional analysis have revealed that highly purified CD49f+LT-HSC are heterogeneous, with inflammation response as one prominent driver of heterogeneity. To uncover putative surface markers to help untangle CD49f+ LT-HSC heterogeneity, we leveraged our recent discovery of two human HSC subsets following recovery from inflammatory challenge; one that does not respond and the other subset that retains memory of prior inflammation (Zeng et al, in revision). Among transcriptional candidates encoding surface proteins that were enriched in the HSC inflammatory memory subset, we identified ATPase plasma membrane Ca2+ transporting 1 (ATP2B1/PMCA1). ATP2B1was intriguing, because calcium flux was reported to impact embryonic stem cell and HSC function (MacDougall et al.,Cell Stem Cell 2019; Luchsinger et al., Cell Stem Cell, 2019). 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".