Extended Data Fig. 4: BM NO^Hi HSCs are frequently contained within C1q+CD200R^HiSSC^Hi HSCs.

a, Gating strategy to enrich NO^Hi HSCs without using DAF-FM. NO^Hi HSCs are enriched within C1q⁺CD200R⁺SSC^HiCD150⁺CD48⁻Lin⁻Sca1^HicKit⁺ HSCs. b, NO^Hi HSC frequencies. These were shown in C1q⁺CD200R⁺SSC^HiCD150⁺CD48⁻Lin⁻Sca1^HicKit⁺ HSCs and CD150⁺CD48⁻Lin⁻Sca1⁺cKit⁺ HSCs. N = 6. The results were pooled from three independent experiments. The results were analysed by two-sided Student’s t-test. c, CD45.1⁺ donor chimerism in the secondary recipients transplanted with C1q⁺CD200R⁺SSC^HiCD150⁺CD48⁻Lin⁻Sca1^HicKit⁺ HSCs. C1q⁺CD200R⁺SSC^HiCD150⁺CD48⁻Lin⁻Sca1^HicKit⁺ HSCs (15 cells/recipient; B6 SJL; CD45.1) were injected into tail veins of lethally irradiated B6 congenic recipient mice (CD45.2), together with competitor B6 BM cells (200,000 cells/recipient; CD45.2). The secondary (2nd) transplantation of the recipient BM cells (5,000,000 cells/recipient) to lethally irradiated B6 mice was performed six months after the first (1st) transplantation. The results were pooled from three independent experiments (three donors, ten recipients/group in total) and analyzed using the Mann–Whitney U test for non-parametric two-group comparison. d, Ratio of chimerism at four weeks after the secondary transplantation to chimerism at five weeks after the first transplantation of C1q⁺CD200R⁺SSC^HiCD150⁺CD48⁻Lin⁻Sca1^HicKit⁺ HSCs. The results were analysed by two-sided Mann–Whitney U test for non-parametric two-group comparison. e. NO^Hi HSCs reside predominantly in the BM. Right: representative flow cytometric plots of peripheral blood (PB) cells (upper) and NO^Hi HSCs (lower), demonstrating the staining levels of intravenously injected APC-conjugated CD45 antibodies and PE-conjugated CD45 antibodies stained ex vivo. The B6 mice received tail vein injections of APC-conjugated CD45 antibodies. BM cells were harvested 5 min after the injection and stained with PE-conjugated CD45 antibodies ex vivo. Left: frequencies of PB cells, NO^Hi HSCs and NO^Low HSCs stained with intravenously injected APC-CD45 antibodies. N = 5. The results were pooled from three independent experiments. The results were analysed by 1-way ANOVA with post hoc test with Bonferroni correction. f, Frequencies of G-CSF receptor- and CXCR4-expressing cells within NO^Hi HSCs and NO^Low HSCs. N = 5. The results were analysed by Student’s t-test. All data are presented as mean ± SD. Asterisks indicate significant differences (p-value < 0.05, *; <0.01, **; <0.001, ***; <0.0001, ****; N.S., >0.05).