Extended Data Fig. 3: Functional modeling of normal flow-sorted populations.
a) Two hundred cells from the indicated populations were sorted and cultured on MS-5 stroma cells for 3, 7 or 14 days (see Fig. 3g for 7 and 14 days) in Ly-My or LyP promoting media and resulting colonies analyzed by flow cytometry. The proportion of each population is represented in the pie charts. b) Representative FACS plots from individual wells summarized in (a) at day 14 of culture. c) Total number of cells from (a) at day 14 of culture. N = 2 cord blood pools for all groups (a, b, c). The dots in the plots represent technical replicates (Ly-My: LMPP, n = 4; MLP, n = 4; CLP, n = 4; pre-pro-B, n = 3; pro-B, n = 8. LyP: LMPP, n = 4; MLP, n = 4; CLP, n = 4; pre-pro-B, n = 3; pro-B, n = 3). d) Engraftment analysis of indicated cell populations 2 weeks after intra-femoral injection into NSG mice. Injected cells were (number/mouse): LMPP: 3,000; MLP: 4,000; CLP: 6,000; pre-pro-B: 6,000; pro-B: 10,000. The percentage of human CD45 positive (CD45 + ) cells and relative engraftment normalized by the number of cells injected in the right femur (injected bone) are shown. The dots in the plots represent different injected mice from 3 human cord blood donors (n = 23 for LMPP and MLP; n = 14 for CLP; n = 3 for pre-pro-B; n = 9 for pro-B). Two-tailed P values are from unpaired t test. e) Engraftment levels in the left femur. The dots in the plot represent different injected mice from 3 human cord blood donors (n = 13 for LMPP and MLP; n = 9 for CLP; n = 3 for pre-pro-B; n = 9 for pro-B). Two-tailed P values are from unpaired t test. f) Percentage of CD33 + , CD19 + , CD56+ and CD1a+ cells in the right femur from panel (d). The dots in the plots represent different injected mice from 3 human cord blood donors (n = 23 for LMPP and MLP; n = 14 for CLP; n = 3 for pre-pro-B; n = 9 for pro-B). g) Human CB (n = 5), adult bone marrow (BM)(n = 8) and mobilized peripheral blood (MPB)(n = 7) were stained and analyzed by flow cytometry with the indicated cell surface markers. The percentage of CD33+ and CD33- cells in each population is represented. Each histogram bar represents an individual donor. h) Single cells from indicated populations were sorted into MS-5 stroma cells and cultured for 16-17 days with Ly-My or LyP media (Supplementary Table 14). Colonies were scored under the microscope and analyzed by flow cytometry for differentiation markers. The percentage of each colony type resulting from each starting population as labels on the x axis is shown. i) From (h) the clonogenic potential (wells with colonies/seeded wells multiplied by 100; bar plots represent the mean with SD) and the number of B, Myeloid (M) or NK cells/colony are shown for both media (LMPP CD33 + , n = 4 cord blood pools; MLP CD33 + , n = 4 cord blood pools; MLP CD33-, n = 3 cord blood pools; CLP CD33 + , n = 5 cord blood pools; CLP CD33-, n = 5 cord blood pools; pre-pro-B CD33 + , n = 2 cord blood pools; pre-pro-B CD33-, n = 3 cord blood pools; pro-B, n = 3 cord blood pools).
