Immunophenotypic- and Molecular Analysis of Human Hematopoietic Stem and Progenitor Heterogeneity

Hematopoietic stem cells (HSCs) have the capacity to differentiate into all hematopoietic lineages and at the same time self-renew to maintain the HSC pool. HSCs have been thoroughly investigated using immunophenotypic-, molecular- and functional-analysis resulting in the development of protocols for high-purity prospective isolation of human HSCs. However, within the current state-of-the-art HSC populations, 90% of the cells lack stem cell activity, confounding molecular analysis of HSC function. Thus, identification of novel immunophenotypic markers to delineate the HSC population would improve our understanding of HSC biology. To identify cell-surface markers with the potential to discriminate between functionally different cells within the HSC population, we performed antibody screens measuring the expression of 340 markers on human cord blood (CB) and bone marrow (BM). Candidate markers that divide the HSC population were included in single-cell CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of immunophenotype and RNA sequencing. This allowed us to correlate the molecular signature of each single-cell with the expression of 40 cell-surface proteins in CD34+ and CD34+CD38- populations of fetal liver (FL), CB, young- and old BM. Following sequencing, the cells were clustered based on molecular signature. Fourteen distinct groups with HSC-, multipotent progenitor-, and early committed progenitor profiles were identified. To investigate how the molecularly defined groups correlate to established populations within CD34+ HSPCs, the surface marker expression from the CITE-seq experiment was included in the analysis. The immunophenotypically defined GMP, MEP and CMP populations showed high molecular heterogeneity with cells at different stages of differentiation. The immunophenotypic HSCs (CD38-CD90+CD45RA-) correlated with the molecularly defined HSC population with a 75.6% overlap. To find novel surface markers for prospective isolation of HSCs pseudo-time analysis was used, allowing for correlation of surface marker expression with differentiation status. Interestingly, both CD35 and CD11a correlated with differentiation, with CD35 expression decreasing and CD11a expression increasing with pseudo-time. These two novel HSC marker-candidates are currently being functionally validated by transplantation analysis. To compare the progenitor composition of CD34+ HSPCs at different stages of life, young BM was used as a baseline control. Interestingly, compared to young BM CB CD34+ cells contained a higher frequency of multipotent progenitor cells and a decreased proportion of committed progenitors. In contrast, old CD34+ BM was reduced in multipotent progenitor frequencies with a corresponding relative increase of committed progenitors. However, both CB and old BM showed similar proportions of molecularly defined HSCs as compared to young BM. These results indicate that ageing causes a depletion of the earliest hematopoietic progenitor populations while the HSC pool remains intact. Together, using single cell CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify two novel cell-surface marker candidates for prospective isolation of HSCs. Disclosures No relevant conflicts of interest to declare.

Prospective isolation of nonhematopoietic cells of the niche and their differential molecular interactions with HSCs

The bone marrow niche environment is essential for the control and maintenance of hematopoietic stem cells (HSCs). The investigators present the first global analysis of the communication between distinct niche cell types and HSCs.

Neogenin-1 distinguishes between myeloid-biased and balancedHoxb5+mouse long-term hematopoietic stem cells

ABSTRACTHematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single-cell transplants (1–3) and lineage tracing (4, 5) 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 withHoxb5, a specific reporter of long-term HSCs (LT-HSCs) (6). We show that NEO1+Hoxb5+LT-HSCs expand with age and respond to myeloablative stress, while NEO1−Hoxb5+LT-HSCs exhibit no significant change in number. 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 comparison reveals increased expression of cell cycle genes and evidence of lineage-priming in the NEO1+fraction. 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.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) are rare cells that have the unique ability to regenerate themselves and produce all blood cells throughout life. However, HSCs are functionally heterogeneous and several studies have shown that HSCs can differ in their contribution to major blood lineages. In this study, we discovered that the surface marker, Neogenin-1, can divide mouse HSCs into two subpopulations—one that is more active but biased towards producing myeloid cells and another that is more dormant and capable of equally producing all blood lineages. Neogenin-1 reveals the diversity and hierarchical relationship of HSCs in the mouse bone marrow, enables the prospective isolation of myeloid-biased and balanced HSCs, and opens opportunities to do the same in humans.