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• W2605540679 abstract "Abstract Background. We 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). Recently, we identified CD133 as a positive marker of human CB-derived CD34+/- SRCs. 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). We then identified glycosylphosphatidylinositol-anchored surface protein (GPI-80) as a useful enrichment 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 attempted to purify CD34+/- SRCs to the single-cell level using CD133 and GPI-80 in order to precisely characterize the CD34- HSCs in comparison to CD34+ HSCs. Materials and Methods. We first developed an ultra-high-resolution purification method using two positive/enrichment markers for CD34+/- SRCs, including CD133 and GPI-80. 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 NOG/NSG mice to investigate their long-term repopulating capacities. Finally, we performed single-cell transplantations. We then analyzed the gene expression profiles of single 34+38-133+80+ and 34-133+80+ cells using a BioMark System (Fluidigm). In parallel, we performed microarray experiments to analyze their gene expression profiles. Results. Approximately 14% of 34+38-133+ cells and 8% of 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/NSG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. All of the mice that received 200 34+38-133+80+ (n=25)and 34-133+80+ (n=23) cells were repopulated with human CD45+ cells, including CD34+, CD19+ and CD33+ cells. Almost all of the secondary transplanted mice showed comparable human CD45+ cell reconstitution with multi-lineage differentiation. An LDA demonstrated that the frequencies of CD34+/- SRCs in the 34+38-133+80+ and 34-133+80+ cells were 1/5 and 1/8, respectively. Interestingly, 13.2% (7/53) of the recipient NSG mice that received single 34+38-133+80+ cells displayed distinct human CD45+ cell reconstitution (0.5%-44.2%) with multi-lineage human cell repopulation at 20 weeks after transplantation. In contrast, 6.6% (5/76) of the recipient NSG mice that received single 34-133+80+ cells displayed distinct human CD45+ cell reconstitution (0.6%-30.1%) with multi-lineage human cell repopulation at 20 weeks after transplantation. Both groups of reconstituted mice showed secondary reconstitution with multi-lineage differentiation. These results indicated that individual 34+38-133+80+ and 34-133+80+ cells extensively self-renew. We then analyzed the gene expression profiles of these two types of SRCs at the single-cell level. A principle component analysis showed 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 and BMI1. Very interestingly, CD34+ SRCs expressed a high level of IFITM1, IKZF1 and ETV6. In contrast, CD34- SRCs expressed higher levels of EZH2 than CD34+ SRCs. Furthermore, a gene set enrichment analysis demonstrated that 34+38-133+80+ cells expressed higher levels of genes related to cell adhesion, chemokine signaling, and trans-endothelial migration, whereas 34-133+80+ cells expressed higher levels of genes related to developmental maturation. Very interestingly, 34-133+80+ cells expressed lower levels of interferon signal-related genes, including STAT1, IFTM1, IFTM3, Ddx58, IFI44, CCL5 and CXCL10, than 34+38-133+80+ cells. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two types of CD34+/- SRCs. Conclusion. Wedeveloped an ultra-high-resolution purification method using two markers for CD34+/- SRCs, including CD133 and GPI-80. This precise single cell-based analysis allows us to map CD34- SRCs (HSCs) at the apex of the human HSC hierarchy. Disclosures No relevant conflicts of interest to declare." @default.
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• W2605540679 date "2016-12-02" @default.
• W2605540679 modified "2023-09-28" @default.
• W2605540679 title "Isolation of Single Human Cord Blood-Derived CD34-Negative Hematopoietic Stem Cells (HSCs) Residing at the Apex of the Human HSC Hierarchy" @default.
• W2605540679 doi "https://doi.org/10.1182/blood.v128.22.367.367" @default.
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