CD27, CD62L and ESAM define a multipotent progenitor population with megakaryocytic potential Free

Background:How hematopoietic lineage specification occurs is a long-standing question, with major implications for disease development and aging. Recent studies using single cell and lineage-tracing techniques suggest a continuous differentiation model of hematopoiesis. In this model, hematopoietic stem cells (HSPCs) are a heterogeneous population with lineage commitment occurring at early multipotent stages, and blood cells are formed through a continuous differentiation process. However, surface markers used for phenotypic analysis of HSCs, multipotent progenitors (MPPs), and oligopotent progenitors were defined based on traditional hierarchical models. Thus, conventional HSPC subsets isolated using these markers remain heterogeneous and limit our ability to study their biology. Here, we investigated myeloid-biased MPP3 and common myeloid progenitor (CMP) populations that sit at the interface of megakaryocyte-erythroid (Mk/E) and granulocyte-monocyte (G/M) multilineage potential, identifying markers to more fully resolve their functional heterogeneity.

Methods: Candidate surface markers, identified from public bulk RNA-seq data, included genes down- or upregulated differentially during HSC differentiation toward MK/E or G/M lineages. Eighteen with commercially available antibodies were validated on adult murine bone marrow, revealing CD27, CD62L and ESAM as differentially expressed within MPP and myeloid progenitors when incorporated in a 24-color spectral flow panel. Populations of interest were FACS sorted and subject to phenotypic, transcriptomic (RNAseq), and functional analysis (liquid culture, methylcellulose culture, transplantation). Using publicly available scRNAseq and CITE-seq data for re-analysis, we further defined their global and subset-specific gene-expression.

Results: Co-staining with CD27 and CD62L within a rigorously gated pre-GM compartment (LKS^–CD135^–CD115^–CD16/32^–CD41^–CD150^–CD105^–) resolved three populations: C1 (CD27⁺ CD62L^–; enriched CMP), C2 (CD27⁺CD62L⁺; monocytic biased pre-GM) and C3 (CD27^–CD62L^–; pre-MEP). Principal-component analysis of bulk RNA-seq positioned C1 between C2 and C3, with C2 clustering proximal to GMPs and C3 to MEPs. Differential-expression and gene-set enrichment analyses showed that C3 up-regulated MK/E-associated genes and pathways (e.g., Klf1, Gata1, Samd14; GO: erythrocyte development), whereas C2 up-regulated myeloid genes (e.g., Emb, Mpo, Ctsg) mirroring GMP profiles.

In liquid culture, c-Kit down-regulation was slower in C1 and C3 than in C2. C1 generated both C2- and C3-like progenies; C2 yielded only GMPs and GPs/MPs, and C3 predominantly produced large number megakaryocytes and low granulocyte/monocyte output. Colony assays confirmed that C1 generated CFU-GM, CFU-Mk/E and significant numbers of CFU-GEMM colonies with the highest overall clonogenicity, C2 formed CFU-G/M/GM only, and C3 produced mainly CFU-Mk/E/MkE with small numbers of CFU-GM. Transplantation (2.5x10⁴ cells/mouse) using tdTomato⁺ cells corroborated lineage potential and demonstrated that C1 engrafted all mature lineages; C2 were restricted to myeloid cells; C3 produced platelets, red cells and limited monocytes. Additional attempts to resolve C1 heterogeneity found ESAM separated C1 into ESAM⁺ and ESAM^– subfractions. ESAM⁺ C1 produced >5-fold megakaryocytes than ESAM^– C1.

Upstream of CMPs, ESAM separated MPP3 into ESAM⁺ and ESAM^– subsets. ESAM⁺ MPP3s over-expressed MK/E regulators (Meis1, Mpl, Car2); ESAM^– MPP3s show higher myeloid gene expression (Mpo, Ctsg, Irf8). Re-analysis of CITE-seq LSK data (GSE145491) and scRNA-seq of purified MPP3s (GSE181902) reproduced these signatures: unbiased clustering identified three co-localized Esam⁺ clusters enriched for MK/E programs and seven Esam^– clusters enriched for innate-myeloid programs. Functionally, compared to ESAM^– MPP3s, ESAM⁺ MPP3s (i) retained a larger Lin^-Kit⁺ fraction at 72 hrs of culture, containing both CD27⁺CD16/32^– and CD27^–CD16/32^– cells (Mk/E producing progenitors); (ii) produced ⁓5-fold more megakaryocytes in culture, and (iii) demonstrated higher engraftment ability with sustained multilineage reconstitution for >5 weeks (1x10⁴ cells/mouse), reaching peak platelet chimerism of 75% by week 2 compared to 25% in ESAM^– MPP3 recipients.Conclusions: In addition to HSCs,ESAM⁺ marks a subset of MPP3s with Mk-potential, true CMPs with all 4 myeloid lineage potential, and Mk-biased progenitors with reduced CD27 expression.