(A) The representative FACS profiles of CB-derived 18Lin⁻CD34⁺CD38⁻GPI-80^+/− and 18Lin⁻CD34⁻GPI-80^+/− cells (i to vi) and (B) a comparison of the frequency of SRCs in CB-derived 18Lin⁻CD34⁺CD38⁻GPI-80^+/− and 18Lin⁻CD34⁻GPI-80^+/− cells (i to iv). (A) (i) The forward scatter/side scatter (FSC/SSC) profile of immunomagnetically separated Lin⁻ cells. The R1 gate was set on the blast-lymphocyte window. (ii) The R2 gate was set on the 18Lin⁻ living cells. (iii) The 18Lin⁻ living cells (R2) were subdivided into 2 fractions: 18Lin⁻CD45⁺CD34⁺ (R3) and 18Lin⁻CD45⁺CD34⁻ (R4) cells, according to their CD34 expression levels. CD34^+/− cells were defined as follows: the CD34⁺ fraction contains cells expressing maximum antigen-presenting cell fluorescence intensity (FI) to the 5% level of FI. The CD34⁻ level of FI was determined based on Fluorescence Minus One controls. (iv) The 18Lin⁻CD45⁺CD34⁺ cells residing in the R3 gate were further subdivided into CD38⁻ (R5) cells, according to their CD38 expression levels. The CD38⁻ level was defined as <15% of the maximum PE-Cy7 FI. (v) The 18Lin⁻CD45⁺CD34⁺CD38⁻ cells residing in the R5 gate were further subdivided into 2 fractions: 18Lin⁻CD45⁺CD34⁺CD38⁻GPI-80⁺ (R6) and GPI-80⁻ (R7) cells, according to their GPI-80 expression levels. GPI-80^+/− cells were defined as follows: the GPI-80⁻ level of FI was determined based on Fluorescence Minus One controls, and remaining cells were defined as GPI-80⁺ cells. (vi) The R4-gated cells were further subdivided into 2 fractions: 18Lin⁻CD45⁺CD34⁻GPI-80⁺ (R8) and GPI-80⁻ (R9) cells. The definitions of GPI-80^+/− cells are the same as abovementioned. As shown in (v) and (vi), the percentages of GPI-80⁺ cells in the CD34⁺ (R6) and CD34⁻ (R8) fractions ranged from 5.4% to 19.9% (mean 11.8%, n = 6) and 5.4% to 29.9% (mean 14.2%, n = 6), respectively. (B) Various numbers of 18Lin⁻CD34⁺CD38⁻GPI-80⁺ cells (1, 2, and 8, n = 32) (i), 18Lin⁻CD34⁺CD38⁻GPI-80⁻ cells (40, 80, and 160, n = 11) (ii), 18Lin⁻CD34⁻GPI-80⁺ cells (10, 20, 50, 100, 200, and 400, n = 26) (iii), and 18Lin⁻CD34⁻GPI-80⁻ cells (500, 1000, and 2000, n = 15) (iv) were transplanted into NOG mice. The human CD45⁺ cell repopulation in the mouse BM was analyzed by FCM at 8 weeks after transplantation. The frequency of SRC was 1 per 20.7 in 18Lin⁻CD34⁺CD38⁻GPI-80⁺ cells, 1 per 35.4 in 18Lin⁻CD34⁺CD38⁻GPI-80⁻ cells, 1 per 27.5 in 18Lin⁻CD34⁻GPI-80⁺ cells, and 1 per 874 in 18Lin⁻CD34⁻GPI-80⁻ cells. The middle solid line represents the estimated weighted mean frequency (f_WM). The lower and upper dotted lines represent the 95% confidence interval of f_WM. The detailed LDA data are presented in supplemental Table 2.