No abstract

Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single cell transplants and lineage tracing suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). We show that NEO1+Hoxb5+ LT-HSCs expand with age and respond to myeloablative stress in young mice while NEO1−Hoxb5+ LT-HSCs exhibit no significant change in number. Furthermore, NEO1+Hoxb5+ LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1−Hoxb5+ LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+ LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution while NEO1−Hoxb5+ LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression analysis reveals erythroid and myeloid priming in the NEO1+ fraction and association of quiescence and self-renewal–related transcription factors with NEO1− LT-HSCs. Finally, transplanted NEO1+Hoxb5+ LT-HSCs rarely generate NEO1−Hoxb5+ LT-HSCs while NEO1−Hoxb5+ LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced NEO1−Hoxb5+ LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+ LT-HSCs.

The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.

27Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with 28 single-cell transplants and lineage tracing suggest that adult HSCs are diverse in their 29 reconstitution and lineage potentials. However, prospective isolation of these subpopulations 30 has remained challenging. Here, we identify Neogenin-1 (NEO1) as a novel surface marker on a 31 fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). 32We show that NEO1 + Hoxb5 + LT-HSCs are more proliferative and expand with age, while NEO1 -33 Hoxb5 + LT-HSCs remain largely quiescent with no significant increase in number. Upon serial 34 transplantation, NEO1 + Hoxb5 + LT-HSCs exhibit myeloid-biased differentiation and reduced 35 reconstitution, while NEO1 -Hoxb5 + LT-HSCs are lineage-balanced and stably reconstitute 36 recipients. Gene expression comparison further reveals evidence of lineage-priming in the 37 NEO1 + fraction. Finally, transplanted NEO1 + Hoxb5 + LT-HSCs rarely generate NEO1 -Hoxb5 + LT- 38HSCs, while NEO1 -Hoxb5 + LT-HSCs repopulate both LT-HSC fractions, supporting that NEO1 - 39Hoxb5 + LT-HSCs can hierarchically precede NEO1 + Hoxb5 + LT-HSCs. neuronal survival 30 , skeletal myofiber differentiation 31 , intracellular iron homeostasis 32 , mammary 80 epithelial development 33 , and endothelial migration 34 , as of yet, little is known about its role in 81 bone marrow hematopoiesis. We find that NEO1 + Hoxb5 + LT-HSCs represent a myeloid-biased 82 subset of LT-HSCs that responds to myeloablative stress and expands with age. Contrastingly, 83 NEO1 -Hoxb5 + LT-HSCs exhibit greater reconstitution potential, balanced lineage output, and a 84 more quiescent cell cycle status compared to NEO1 + Hoxb5 + LT-HSCs. After transplant, NEO1 - 85Hoxb5 + LT-HSCs give rise to NEO1 + Hoxb5 + LT-HSCs, but the reverse transition is rarely 86 observed. We, therefore, propose a model of early long-term hematopoiesis in which balanced, 87 quiescent LT-HSCs self-renew and generate long-term myeloid-biased LT-HSCs in response to 88 stress and during the course of aging. 89 RESULTS 91 Neogenin-1 (NEO1) marks a subpopulation of mouse Hoxb5 + LT-HSCs and human HSCs 92Functional heterogeneity within Hoxb5 + LT-HSCs is poorly understood. To identify surface 93 candidates that fractionate Hoxb5 + LT-HSCs, we first pattern-searched 64 microarray 94 expression profiles of 23 distinct mouse hematopoietic cell types 35 for (1) genes annotated to 95 code for cell surface proteins (GO Biological Process: 0009986) and (2) genes specifically 96 expressed in HSCs compared to downstream cell types ( Fig. 1a). We found several known 97 HSC-specific markers, including Robo4 36 , Slamf1 5 , Ly6a 2 , Vwf 26 , TEK 37 , and a member of the 98 Gpcr5 family 38 , validating the utility of our approach. We also identified several novel markers of 99 HSCs that have not been previously reported (Fig. 1a). Among the top 3 most enriched surface 100 markers on HSCs, Neogenin-1 (Neo1) was more highly expressed on HSCs compared...