Human CD34-negative hematopoietic stem cells: The current understanding of their biological nature

Hematopoietic stem cell (HSC) heterogeneity and hierarchy are a current topic of interest, having major implications for clinical HSC transplantation and basic research on human HSCs. It was long believed that the most primitive HSCs in mammals, including mice and humans, were CD34 antigen positive (CD34⁺). However, 2 decades ago, it was reported that murine long-term multilineage reconstituting HSCs were lineage marker negative (Lin^-, i.e., c-kit⁺Sca-1⁺CD34^low/-), known as CD34^low/- KSL cells. In contrast, human CD34^- HSCs, a counterpart of murine CD34^low/- KSL cells, were hard to identify for a long time mainly because of their rarity. We previously identified very primitive human cord blood (CB)-derived CD34^- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method and proposed the new concept that CD34^- SRCs (HSCs) reside at the apex of the human HSC hierarchy. Through a series of studies, we identified two positive/enrichment markers: CD133 and GPI-80. The combination of these two markers enabled the development of an ultrahigh-resolution purification method for CD34^- as well as CD34⁺ HSCs and the successful purification of both HSCs at the single-cell level. Cell population purity is a crucial prerequisite for reliable biological and molecular analyses. Clonal analyses of highly purified human CD34^- HSCs have revealed their potent megakaryocyte/erythrocyte differentiation potential. Based on these observations, we propose a revised road map for the commitment of human CB-derived CD34^- HSCs. This review updates the current understanding of the stem cell nature of human CB-derived primitive CD34^- as well as CD34⁺ HSCs.