Extended Data Fig. 3: Use of the higher dose of competitor cells frequently induced late-rising reconstitution from NO^Hi HSCs.

a-e, Competitive transplantation assays. NO^Hi or NO^Low HSCs (10 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 (300,000 cells/recipient; CD45.2). The secondary (2^nd) transplantation of the recipient BM cells (5,000,000 cells/recipient) to lethally irradiated B6 mice was performed four months after the first (1^st) transplantation. Data are pooled from four independent experiments (four donors in total; 20 recipients per group). a, Blood reconstitution patterns derived from NO^Hi or NO^Low HSCs. The colours of the trend lines correspond to those of the differential reconstitution patterns shown in Extended Data Fig. 3d,e. b, CD45.1⁺ donor blood chimerism following the 1st and 2nd transplantation. Data are presented as mean ± SD and analysed by two-sided Student’s t-test. c, Extent of chimerism at 12 weeks after the 2nd transplantation relative to that at five weeks after the 1st transplantation. Used 0.1% for the undetectable chimerism at five weeks after the 1st transplantation. Data were analyzed by two-sided Student’s t-test. d, CD45.1⁺ donor chimerism over the 1st and 2nd transplantation. Late-rising after the 2nd transplantation (red): Chimerism <1% from two to five months after the 1st transplantation but >2% following the 2nd transplantation. Come-back (pink): Chimerism initially >1% at five weeks after the 1st transplantation but <1% from two to three months after the 2nd transplantation; while returned to be >2% three months after the 2nd transplantation. Late-rising prior to the 2nd transplantation (orange): Chimerism <1% for two to three months after the 1st transplantation but >2% from three to four months following the 1st transplantation. Increasing pattern (dark blue): Chimerism >1% throughout the 1st and 2nd transplantation but exhibited at least a five-fold increase, both at four months after the 1st transplant and at three months after the 2nd transplant, as compared to chimerism at three weeks after the 1st transplant. Persisting pattern (light blue): Chimerism >1% throughout the 1st and 2nd transplant but demonstrated less than a two-fold increase at four months after the 2nd transplant, as compared to chimerism at three weeks after the 1st transplant. Exhausting pattern (brown): Chimerism initially >1% at three or five weeks after the 1st transplant but decreases three months after the 1st transplant and eventually <1% after the 2nd transplant. Not engrafted (grey): Chimerism that remained less than 1% throughout the 1st and 2nd transplant. e, Differential patterns of donor reconstitutions among recipients of NO^Hi or NO^Low HSCs. 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).

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