Characterization of the functional properties of murine HSC fractionated with CD166. (A) C57BL/6 low-density BM cells were analyzed for the expression of lineage markers and CD48 (left dot plot). Lineage-CD48⁻ (Lin-48⁻) cells were analyzed for the expression of Sca-1 and cKit (middle dot plot) to identify Lin-Sca1⁺cKit⁺CD48⁻ (LSK48⁻) cells (group 1 in middle dot plot). LSK48⁻ cells were analyzed with CD150 and CD166 (right dot plot) to define groups 2, 3, and 4. Percentages next to each group represent the relative size of each population within the dot plot. A total of 100, 50, and 25 cells from groups 1 through 4 were transplanted into F1 mice with 10e5 BoyJ competitor cells (5 mice/group). (B) Chimerism 4-months PT for the 25-cells/mouse group. *P < .05 vs group 3. (C) Engraftment kinetics for groups 1, 2, 3, and 4 over 4 months measured in the PB of recipients of 100-cells/mouse group. *P < .05 vs corresponding values from LSK48⁻CD166⁺CD150⁺ cells. These data are from 1 of 3 independent experiments with similar results. (D) LDA analysis from BM engraftment at 4-months PT in mice receiving 25, 50, and 100 cells (5 mice/group). P = .01 for the overall test for differences in HSC frequencies between any of the groups. Table shows the estimated frequencies and range of repopulating cells within each group tested. (E) Chimerism at 4-months PT in primary recipients after the competitive transplantation of 10 cells/mouse from groups 1 to 4 identified in (A), along with 10e5 BoyJ competitor cells. (F) Chimerism at 4-months PT in secondary recipients of the equivalent of one half femur from primary recipients (BM cells were collected 4-months postprimary transplant) shown in (E) without competitor cells [4-5 mice/group in (E) and (F)]. (G) RU per 10 cells calculated from data shown in (E) as described in Harrison and Astle.¹⁷  *P < .05 vs group 3. (H) Multilineage differentiation 4-months PT of groups 1 through 4 in mice receiving 100 cells/animal. *P < .05 between LSK48⁻CD166⁺CD150⁺ cells and LSK48⁻, CD166⁺CD150⁻, and CD166⁻CD150⁺ cells within each group. (I) Expression of CD166 on LSK48⁻CD150⁺CD9⁺ cells (in the 3 dot plots from L to R). (J) Chimerism at 4-months PT in mice receiving 10 cells per mouse from groups I, II, and III transplanted competitively into F1 mice with 10e5 BoyJ competitor cells (5 mice/group). *P < .05 vs group III. (K) Expression of CD166 on the low (L), medium (M), and high (H) fractions of murine BM side population (SP) cells. Data shown in (K) are from 1 representative experiment from 4 independent analyses with similar results.