Identification of a development-restricted, megakaryocyte-primed population of hematopoietic progenitor cells during fetal and early postnatal development

Abstract During development, hematopoiesis unfolds in successive, partially overlapping waves. Primitive and pre-definitive progenitors arise during embryonic development, but are replaced by definitive hematopoietic stem and progenitor cells (HSPCs) that seed the fetal liver (FL), migrate to bone marrow (BM), and sustain lifelong blood production. Fetal-derived HSPCs persist several weeks after birth in mice, coexisting with adult-type cells and possessing distinct, age-restricted features. Previously indistinguishable due to overlapping surface marker profiles, developmental mapping using fluorescent reporter mice was conducted for lymphoid and myeloid lineages; however, how megakaryocyte-erythroid (MK/E) progenitors shift across the fetal-to-adult transition is poorly understood. This study sought to characterize postnatal progenitors to better understand how the fetal program dynamically changes in the Mk branch to better explain early occurrence of MK leukemias. We used the α-catulin-GFP reporter mouse, which labels adult HSCs via the Ctnnal1 promoter, to dissect the HSPC hierarchy. GFP expression is highly restricted to phenotypic HSCs (~35% GFP+) in adult (8-12 weeks) BM, whereas ⁓90-100% of HSCs in the FL are GFP+ on embryonic days 14.5-16.5 and decline during postnatal day 1 (P1) to P28. Additionally, multipotent progenitor 1 (MPP1), MPP2, and megakaryocyte progenitors (MkP) in the FL are GFP+ during P1-P28, but GFP disappears by adulthood. This coincides with the proliferative-to-quiescent transition of HSCs, suggesting a development-restricted phenotype. We FACS-isolated GFP+ HSCs and GFP- MPP1, MPP2, and MkPs from adult mice and GFP+ HSCs, GFP+ and GFP- MPP1, MPP2, and MkPs from P14 mice and performed bulk RNA-sequencing. Principal component analysis showed GFP+ or GFP- MPP1, MPP2 and MkPs cluster closer than adult counterparts and exhibit unique gene profiles. Gene set enrichment analysis showed GFP+ and adult MkPs or MPP2s are more enriched for GO pathways related to platelet activation and blood coagulation than GFP- MkPs or MPP2s, respectively. Direct comparison of GFP+ vs GFP- MPP2s or MkPs showed higher MK-related genes (Pf4, Vwf, Mpl) and pathways, while GFP- were enriched for myeloid genes (Mpo, Ctsg, Sting1) and pathways (defense response, myeloid leukocyte migration), suggesting GFP- populations may be myeloid-primed. To assess functionality, cells were cultured for short-term (24 & 48 hr) and longer-term (5-7 day) timepoints. GFP+ MkPs and GFP+ MPP2s produced more large MKs with fewer nuclear lobes than GFP- or adult populations. GFP+ MPP2s also retained more Lin-Sca1+cKit+ MPP2s by 48 hours, suggesting slower differentiation compared to GFP- or adult MPP2s. FACS analysis showed that almost all GFP+ MkP cultures become CD41high megakaryocytes, whereas GFP- and adult MkPs cultures produce many CD41-/low non-megakaryocytes. Compared to GFP+ and adult MPP2s, GFP- MPP2s produce significantly more myeloid cells. This myeloid cell production could be enhanced with M-CSF or GM-CSF stimulation, where FACS or Wright-Giemsa-stained cultures showed GFP- and adult cultures containing higher numbers or small myeloid cells. Finally, MK ploidy was determined using an Amnis Imagestream and showed GFP+ and adult MkPs and MPP2s produce a higher proportion of high ploidy MKs (16N/32N). These in vitro results suggest an MK lineage bias of GFP+ cells, with potential functional differences related to response to cytokines and MK maturation status. To trace differentiation in vivo, we crossed α-catulin-GFP and Rosa26tdTomato mice and transplanted 1,000 tdTomato+GFP+ 2-week, tdTomato+GFP- 2-week, or tdTomato+GFP+ adult MPP2s into lethally irradiated wild-type B6 mice. Both populations reconstituted erythroid/myeloid lineages, but GFP+ MPP2s show significantly higher reconstitution compared to GFP- by day 14. Adult MPP2s show similar engraftment of myeloid, platelet, and erythroid reconstitution for the first 14 days but subsequently increased in all but lymphoid lineages. BM and spleen analysis on day 56 post transplantation showed significantly higher tdTomato+CD45+ cells in adult MPP2 vs 2-week recipients, as well as GFP+ vs GFP- MPP2 recipients in all but lymphoid lineages.Thus, α-catulin-GFP+ MPP2 and MkPs represent novel, development-restricted, Mk-primed HSPC populations that decline postnatally. These populations may underlie unique platelet or leukemia biology during early development and merit further investigation.