IMMUNOPHENOTYPIC AND MOLECULAR HETEROGENEITY ANALYSIS OF HUMAN HEMATOPOIETIC STEM AND PROGENITORS

Abstract

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 are not bona fide HSCs, confounding molecular analysis of HSCs. 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 CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of single-cell immunophenotype-/RNA sequencing analysis. This allowed us to correlate the molecular signature of each single-cell with expression of 40 cell-surface proteins, in CD34+ and CD34+CD38- populations of CB and BM. Following sequencing the cells were clustered based on molecular signature. Distinct groups with HSC, progenitor and early committed progenitor profiles were identified. Interestingly, comparison between CB and BM showed differences in progenitor proportions while the fraction of cells with HSC signature remained intact. Additionally, three novel HSC marker-candidates were discovered which are being validated. Together, using CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify three novel cell-surface marker candidates for prospective isolation of HSCs.