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.

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a subpopulation is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients using a selected panel of 96 primers. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). CITE-seq allows for unbiased, further in-depth transcriptome analysis as wells as immunophenotypic characterization by pre-staining cells with a panel of DNA-barcoded antibodies prior to sequencing. DNA-barcoded antibodies convert the protein expression into readable sequences through unique oligo-conjugates as identifiers. Using CITE-seq with a panel of 44 distinct surface markers designed to immunophenotypically differentiate between stem/progenitors cells and leukemic clones we simultaneously characterize the molecular and immunophenotypic heterogeneity within Lin-CD34+/Lin-CD34+CD38- CML stem/progenitor compartment at diagnosis. Additionally by comparing the LSCs transcriptome from patients with different therapeutic outcome after 12 months of therapy we describe how differences in heterogeneity and the presence of immunophenotypic therapy-insensitive LSCs at diagnosis (Lin-CD34+CD38-CD45RA-cKIT-CD26+) contribute to therapy response. Disclosures Richter: Novartis: Consultancy; Pfizer: Consultancy, Research Funding.

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