E13.5 fetal liver HSCs express the endothelial marker CD144 (VE-cadherin), but CD144 expression by HSCs declines over time and is no longer detected on adult bone marrow HSCs. (A) The frequency of CD144⁺ cells in the fetal liver declines over time. CD144^– cells (red boxes) and CD144⁺ cells (blue boxes) were fractionated for functional studies. (B) Irradiated mice were competitively reconstituted with 1350 CD144⁺ cells or 48 650 CD144^– cells from E13.5 liver, 500 CD144⁺ cells or 49 500 CD144^– cells from E16.5 liver, and 2000 CD144⁺ cells or 198 000 CD144^– cells from adult bone marrow. These cell doses were based on the proportion of CD144⁺ versus CD144^– cells in 50 000 fetal liver cells, or 200 000 bone marrow cells as done in previous studies of marker expression by HSCs.18,21 The percentage of donor-derived myeloid (panels in B), B, and T (not shown) cells was analyzed in mice that underwent transplantation with CD144⁺ (blue lines) or CD144^– (red lines) cells. Each line represents the level of donor myeloid cells in a mouse from 4 to 16 weeks after transplantation. The black line at 0.3% in each panel represents the background threshold observed in negative control mice, such that donor cell reconstitution could not be detected below this line. Mice that were long-term reconstituted by donor myeloid cells were always long-term multilineage reconstituted. The proportions of mice that became long-term multilineage reconstituted are indicated under each panel. (C) CD144 expression was analyzed in Sca-⁺⁺Lineage^–Mac-1⁺ HSCs from E13.5 and E16.5 fetal liver and in Sca-1⁺Lineage^–c-kit⁺ HSCs from adult bone marrow.^18,21