Human Cord Blood-Derived CD34-Negative Hematopoietic Stem Cells (HSCs): A New Class of HSCs in the Human HSC Hierarchy

Background. We previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34^-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the CD34^- SRCs that we identified are a distinct class of primitive hematopoietic stem cells (HSCs), which show myeloid-biased long-term repopulating capacity, suggesting that they are at the apex of the human HSC hierarchy (Blood Cancer J 2015:5,e290). We recently developed a high-resolution purification method for primitive CD34^- SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34^- SRC at the 1/1,000 level (Exp Hematol 2011:39;203). We then identified CD133 as a positive marker of human CB-derived CD34^+/- SRCs. Moreover, limiting dilution analyses (LDAs) demonstrated that the frequencies of SRCs in the 18Lin^-CD34^+/-CD133⁺ fractions were 1/100 and 1/140, respectively (Leukemia 2014:28;1308). Since we aim to purify the CD34^- SRCs (HSCs) at the single cell level, it was necessary to identify other specific positive markers for CD34^- SRCs. We extensively analyzed the expression of candidate positive markers in the 18Lin^-CD34^- cell population by FACS. Finally, we identified glycosylphosphatidyl- inoshitol-anchored surface protein (GPI-80) as a useful marker of human CB-derived CD34^+/- SRCs and succeeded in highly purifying primitive human CD34^+/- SRCs to the level of 1/20 cells.

Aim. We herein attempted to purify CD34^+/- SRCs to the single cell level in order to precisely characterize the CD34^-SRCs (HSCs) in the human HSC hierarchy.

Materials and Methods. We first developed an ultra-high resolution purification method using two reliable markers for CD34^+/- SRCs, including CD133 and GPI-80. Namely, we sorted 18Lin^-CD34⁺CD38^-CD133⁺GPI-80⁺ (34⁺38^-133⁺80⁺) and 18Lin^-CD34^-CD133⁺GPI-80⁺ (34^-133⁺80⁺) cells by FACS. Thereafter, these two fractions of cells were transplanted by the IBMI technique into NOD/Shi-scid/IL-2Rγc^null (NOG) mice to investigate their long-term repopulating capacities. We ultimately performed single cell transplantations and analyzed their human hematopoietic cell reconstitution for up to 20 weeks. Finally, we analyzed their gene expression profiles, including the key genes for the self-renewal and maintenance of HSCs of single 34⁺38^-133⁺80⁺ and 34^-133⁺80⁺ cells using a BioMark System (Fluidigm).

Results. Approximately 15% of the 34⁺38^-133⁺ and 34^-133⁺ cells expressed GPI-80. These highly purified cells showed very immature blast-like morphologies. These two fractions of cells were then transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. In the results, all of the mice received 200 34⁺38^-133⁺80⁺ (n=25) and 34^-133⁺80⁺ (n=23) cells were repopulated with human CD45⁺ cells (Mean % of human CD45⁺ cells, 47% vs. 35%), including CD34⁺, CD19⁺ and CD33⁺ cells. Most of the secondary transplanted mice were also repopulated with human CD45⁺ cells with multi-lineage reconstitution (Mean % of human CD45⁺ cells, 0.4% vs. 10%). An LDA is currently underway; however, the frequencies of CD34^+/- SRCs in the 34⁺38^-133⁺80⁺ and 34^-133⁺80⁺ cells are estimated to be 1/5-1/10. Interestingly, significant numbers of the recipient mice that received single cells displayed multi-lineage human cell repopulation at 20 weeks after transplantation. In order to provide an independent line of evidence for characterizing our highly purified CD34^+/- SRCs, we analyzed the gene expression profiles of these two types of SRCs at the single cell level. The principle component analysis clearly demonstrated that the gene expression profiles of individual CD34⁺ and CD34^- SRCs were clearly different. Both SRCs expressed high levels of HSC maintenance genes, including RUNX1, TAL1, BMI1 and MYBC. Very interestingly, CD34⁺ SRCs expressed a high level of ETV6. In contrast, CD34^-SRCs expressed higher levels of EZH2 and RING1. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two SRCs.

Conclusion. We developed an ultra-high resolution purification method using two markers for CD34^+/- SRCs, including CD133 and GPI-80. The precise single cell-based analysis allows us to map CD34^- SRCs (HSCs) at the apex of the human HSC hierarchy.