High cMPL expression identifies adult human long-term hematopoietic stem cells with superior multilineage and clonally balanced reconstitution potential

Abstract

Abstract Thrombopoietin (TPO) and its receptor cMPL are key regulators of long-term hematopoietic stem cell (LT-HSC) maintenance. Clinical responses to the synthetic c-MPL agonist eltrombopag in immune-mediated bone-marrow failure implicate cMPL signaling in activating residual multipotent LT-HSCs (Townsley, NEJM 2017). We recently showed that high surface expression of cMPL alone robustly enriches functional LT-HSCs from adult human CD34+ cells in both bone marrow (BM) and mobilized peripheral blood (MPB) (Araki, Blood 2025). However, how cMPL compares to CD90, the canonical marker used to enrich human LT-HSCs within the immunophenotypic (CD34+CD38-CD45RA-) HSC/multipotent progenitor (MPP) compartment, remains undefined. Moreover, given cMPL's role in megakaryopoiesis and the emerging recognition of lineage-biased HSCs, including megakaryocyte-biased subsets with durable self-renewal, it remains unclear whether cMPL identifies broadly multipotent or lineage-restricted HSCs within the CD34+CD38-CD45RA- population. To compare high surface expression of cMPL and CD90 as defining markers for the enrichment of functional human LT-HSCs, we flow-sorted MPB CD34+ cells from a healthy adult donor into CD34+CD38-CD45RA- subsets expressing either high levels of cMPL (top 20%; cMPLhigh group) or high levels of CD90 (top 20%; CD90high group). Equal numbers of sorted cells were transplanted into NBSGW mice (n = 10 per group). While PB chimerism at 6 weeks was comparable between groups, the cMPLhigh group showed significantly higher engraftment at 12 weeks (3.0-fold, p = 0.0074) and 16 weeks (5.1-fold, p = 0.0111). At 16 weeks, BM chimerism was 2.9-fold higher (p < 0.005), with a 7.7-fold increase in CD34+ cell numbers in the cMPLhigh group relative to the CD90high group, without evidence of lineage skewing in both cohorts. These data support cMPL as a more functionally discriminating surface marker than CD90 for enriching LT-HSCs within the CD34+CD38-CD45RA- compartment. To determine whether cMPLhigh cells represent broadly multipotent or lineage-biased HSCs, we performed clonal tracking. Equal numbers of flow-sorted cMPLhigh and CD90high HSC/MPP cells were transduced with high-complexity, DNA-barcoded lentiviral library (MOI = 20) encoding GFP or YFP, then co-infused at a 1:1 ratio into NBSGW mice (n = 6 per group). At four months post-transplantation, BM was harvested and sorted into 5 major human hematopoietic lineages: CD13+ (myeloid), CD20+ (B cells), CD3+ (T cells), CD41+ (megakaryocytes), and CD71+ (erythroid). Barcode sequencing enabled assessment of clonal diversity, lineage contributions, and GFP/YFP clone frequencies. Consistent with our prior findings, the cMPLhigh group exhibited a significantly higher frequency of barcoded clones across all lineages. Among the top 10 contributing clones, a greater proportion were multilineage in the cMPLhigh group, defined as clones contributing to ≥ 4 distinct lineages, compared to the CD90high group (32.9% vs. 8.2%, p = 0.0001). Conversely, the CD90high group was enriched for uni- or bi-lineage clones (88% vs. 55%, p < 0.0001), with 24.9% of those demonstrating myeloid or myeloid-megakaryocyte bias. Neither group showed evidence of megakaryocyte-restricted output. Among multilineage clones, contribution evenness, as measured by the Shannon H index and Pielou's evenness score, was significantly higher in the cMPLhigh group (1.673 vs. 0.533, p = 0.0002; 0.899 vs. 0.536, p = 0.0378). Furthermore, 4 of 6 mice receiving CD90high cells exhibited dominance by a single multilineage clone (> 60% contribution within 3-4 lineages), whereas cMPLhigh recipients showed more balanced and polyclonal reconstitution. In summary, high cMPL expression marks a rare subset of human CD34+CD38-CD45RA- cells with superior, durable, and balanced multilineage reconstitution, supported by greater clonal diversity than CD90-selected cells. These findings position cMPLhigh LT-HSCs as an optimal population for advancing fundamental insights into human HSC biology, and as a strategic target for next-generation, non-genotoxic conditioning and in vivo gene-editing approaches directed at the most primitive HSCs.