In vitro proliferative potential of CD34⁺ Lin⁻ cells further fractionated by huHCA expression and Rh123 staining. CD34⁺ Lin⁻ ABM cells were sorted first (A). The sorted cells were stained with Rh123 as described and then for huHCA expression, based on isotope control. HCA⁺ Rh123^lo (•), HCA⁺ Rh123^mid (▴), HCA⁺ Rh123^hi (▪), and HCA⁻ Rh123^hi (♦) (see sorting gates on [B]) were deposited into 96-well plates preseeded with SyS-1 stromal cells. CAFC growth was scored between 3 to 8 weeks of culture in two independent experiments (C). No cobblestone areas were detected in wells plated with HCA⁻ Rh123^hi cells. CAFC frequency (with lower 95% to upper 95% confidence interval) was determined by linear regression analysis as described in the Materials and Methods. CAFC frequency at 3 weeks in the HCA⁺ Rh123^lo, HCA⁺ Rh123^mid, and HCA⁺ Rh123^hi cell subsets was 1/78 (50 to 174), 1/37 (26 to 67), and 1/266 (154 to 422), respectively. CAFC frequency at 4 weeks in the HCA⁺ Rh123^lo, HCA⁺ Rh123^mid, and HCA⁺ Rh123^hi cell subsets was 1/18 (14 to 25), 1/16 (12 to 23), and 1/121 (89 to 188), respectively. CAFC frequency at 5 weeks in the HCA⁺ Rh123^lo, HCA⁺ Rh123^mid, and HCA⁺ Rh123^hi cell subsets was 1/12 (9 to 15), 1/21 (16 to 31), and 1/185 (130 to 321), respectively. CAFC frequency at 6 to 8 weeks in the HCA⁺ Rh123^lo, HCA⁺ Rh123^mid, and HCA⁺ Rh123^hi cell subsets was 1/11 (9 to 14), 1/24 (17 to 37), and 1/133 (98 to 208), respectively. On some occasions, proliferation of multilineage CD34⁺, CD33⁺, or CD19⁺ cell populations was detected; the example shown represents cells derived from HCA⁺ Rh123^lo single cells deposited in culture 7 weeks earlier (D and E).