Murine very small embryonic-like (VSEL) cells, defined by the Lin−Sca-1+CD45− phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin−Sca+CD45− population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin−Sca-1+CD45− population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit+KDR−, c-Kit−KDR+, and c-Kit−KDR− subpopulations could be distinguished, while c-Kit+KDR+ events were very rare. The c-Kit+KDR− subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit−KDR+ cells. The c-Kit−KDR−FSClow subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin−Sca-1+CD45−FSClow population, even by single-cell qRT-PCR. We also found that the Lin−Sca-1+CD45−c-Kit+ subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1+c-Kit+Lin− (SKL) CD34−CD45−CD105+ cells, expressing particular HSC markers. Co-culture of Lin−Sca-1+CD45−FSClow with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45−CD105+ subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin−Sca-1+CD45−FSClow cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin−Sca-1+CD45−c-Kit+KDR− subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.

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.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1 À/À ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1 À/À mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1 À/À animals have reduced quiescence and regenerative potential. Young HO-1 À/À HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1 +/+ HSCs transplanted into HO-1 À/À recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1 À/À HSCs to the HO-1 +/+ recipients recovers the regenerative potential of HO-1 À/À HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.EMBO Reports (2020) 21: e47895 Until now, the mechanisms found to contribute to the aging of HSCs have been mostly intrinsic to the HSCs [6,15]. These include age-related accumulation of mutations and cell-autonomous changes in the transcriptome and epigenome of HSCs [6,7,[16][17][18][19].Although HSC-extrinsic factors from the local bone marrow (BM) environment of HSCs-the HSC niche-or systemic factors are critical for HSC maintenance [20][21][22], little is known about their contribution to HSC aging.Recent findings indicate that HSCs occupy a perivascular niche and localize in direct proximity to endothelial cells (ECs) and mesenchymal stromal cells (MSCs) surrounding vessels [23,24]. Among the many cell types in the HSC niche, the ECs and MSCs constitute the main source of stromal cell-derived factor 1a (SDF-1a) and stem cell factor (SCF)-extrinsic factors critical for HSC maintenance [25][26][27][28]. Specific deletion of either Sdf1a or Scf in either ECs or MSCs causes hematopoietic collapse or triggers overactivation of HSCs and their release from the niche [22,[25][26][27]. J endomucin CD31 HO-1 Figure 1. ª 2019 The Authors EMBO reports 21: e47895 | 2020 ****P < 0.0001. Data are shown as mean AE SEM. IECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean AE SEM. *P < 0.05, ***P < 0.001, two-tailed unpaired t-test. JMiddle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean AE SEM. **P < 0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1 À/À bone marrow section is provided in Appendix Fig S1. 4 of 21 EMBO reports 21: e47895 | 2020 ª 2019 The Authors H Only 1 out of 267 DEGs identified in non-tran...

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.