CA-initiating cells are expanded on AFT024. (A) A quantitative estimate of the number of 28-day cultured stem cell equivalents required to form a CA after replating on secondary AFT024 monolayers was determined. Four different AFT024 cocultures from separate fetal liver purifications were studied (A, B, C, and D). In limiting-dilution assay, the frequency of stem cell equivalents required to form a CA in another 7 days was 1 in 4 (3.56 ± 0.64, R² = .96). (B) CA maintenance in 1 of 4 cultures was followed for an additional 4 weeks. The frequency of stem cell equivalents maintaining CA was determined. At 37% negative wells, the frequencies were as follows: 2 weeks, 1 in 3; 3 weeks, 1 in 10; 4 weeks, 1 in 19; and 5 weeks, 1 in 29.