Extended Data Fig. 8: NO^Hi HSCs localize at CD200^Hi capillaries, while NO^Low HSCs are at metaphyseal Type H vessels or sinusoids.

a, Histological images illustrating the localization of NO^Hi and NO^Low CD150⁺HSCs. NO^HiCD150⁺CD48⁻Lin⁻ HSCs (yellowish) localize at CD200^Hi capillaries (magenta), both at the metaphysis and diaphysis. NO^LowCD150⁺CD48⁻Lin⁻ HSCs (red) localize at EMCN⁺ Type H vessels (cyan) in the metaphysis and at sinusoids (blue) in the diaphysis. Green: DAF-FM. Red: Alexa 568-CD150. Grey: BV421-Lin/CD48. Cyan: APC-R700-EMCN. Magenta: Q655-CD200. Blue: Alexa 647-IB4. The scale bar indicates 20 µm. Refer to Supplementary Videos 5–8. b, Histological images illustrating the localization of NO^Hi and NO^Low Pdzk1ip1^creER × ROSA26TdTomato HSCs in tamoxifen-treated Pdzk1ip1^creER × ROSA26TdTomato mice. NO^HiTdTomato⁺CD48⁻Lin⁻ HSCs (yellowish) localize at CD200^Hi capillaries (magenta), both at the metaphysis and diaphysis. NO^LowTdTomato⁺CD48⁻Lin⁻ HSCs (red) localize at type H vessels (cyan) in the metaphysis and at sinusoids (blue) at the diaphysis. Long bones were harvested three days after tamoxifen treatment (oral gavage, 1 mg) in Pdzk1ip1^creER × ROSA26TdTomato mice. Green: DAF-FM. Red: Pdzk1ip1^creER × ROSA26TdTomato. Grey: BV421-Lin/CD48. Cyan: IB4-Alexa 647 (metaphysis). Blue: IB4-Alexa 647 (diaphysis). Magenta: Q655-CD200. The scale bar indicates 20 µm. Refer to Supplementary Videos 9–12. c, Histological images illustrating the localization of NO^Hi and NO^Low CD150⁺HSCs. NO^HiCD150⁺CD48⁻Lin⁻ HSCs (yellowish) localize at metaphysis CD200^Hi capillaries (magenta), and NO^LowCD150⁺CD48⁻Lin⁻ HSCs (red) localize at sinusoids (cyan) in the diaphysis. Green: DAF-FM. Red: CD150-Alexa 568. Blue: BV421-Lin/CD48. Grey: Hoechst 33342. Cyan: Alexa 647-CD31. Magenta: Q655-CD200. The scale bar indicates 20 µm. d. Distances of NO^Hi and NO^Low HSCs concerning CD200^Hi capillaries (CD200Hi), Type H vessels (Type H), and sinusoids. Top two rows: NO^Hi and NO^Low CD150⁺CD48⁻Lin⁻ HSCs. Bottom two rows: NO^Hi and NO^Low TdTomato⁺CD48⁻Lin⁻ HSCs in tamoxifen-treated Pdzk1ip1^creER × ROSA26TdTomato mice. Automated analysis was performed using Imaris software (Bitplane). Refer to Fig. 2h. The results were pooled from the experimental results using six long bones from five mice for CD150 staining, and five long bones from five mice for Pdzk1ip1^creER × ROSA26TdTomato mice and analysed by Kruskal-Wallis with Dunn’s post hoc test for nonparametric multi-group comparison. Pooled from six long bones isolated from 5–6 mice for each analysis. The total numbers of analysed HSCs are shown in each graph. All data are presented as mean. Asterisks indicate significant differences (p-value < 0.05, *; <0.01, **; <0.001, ***; <0.0001, ****; N.S., >0.05).

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