Different stages of hematopoiesis in stroma-free LTC of CB CD34⁺ cells. (A) Expansion of hematopoietic cells in stroma-free cultures stimulated by TPO (10 U/mL) alone (•), by FL (50 ng/mL) alone (○), and by the combination of TPO (10 U/mL) + FL (50 ng/mL) (▪). The starting population was represented by 2 × 10³ CD34⁺ CB cells in 1 mL in 24-well plates. Fresh growth factors were added twice a week. Cells were counted weekly from quadruplicate wells and the fold increase was obtained by dividing the number of cells of each well counted each week by the number of the starting population. Represented here is the mean ± SEM of four different experiments (each performed in quadruplicate). (B) Expansion of the CD34⁺ (▪) and the CD34⁺ CD38⁻ (•) cell populations during LTC with FL + TPO. CB CD34⁺ cells were grown as described in Fig 1 for 25 weeks. Every 2 to 3 weeks the cells harvested were stained with FITC- and PE-conjugated anti-CD34 and anti-CD38 MoAb. The total number of each subpopulation was obtained by multiplying the percentage of each subset by the number of cells contained in each well. Results are expressed as the fold increase (compared with the starting subpopulation) and represent the mean ± SEM of analyses performed on quadruplicate wells in four separate experiments. (□) CD34⁺ cells in LTC grown in the presence of FL alone. (○) CD34⁺ cells in LTC grown in the presence of TPO alone. (C) Production of hematopoietic progenitors in LTC in the presence of FL + TPO. CD34⁺ CB cells were grown for 25 weeks as described in Fig 1. CFU-GM (▪), CFU-MK (○), BFU-E (•), and CFU-GEMM (□) were cultured as described in Fig 1. Results represent the fold increase of each class of progenitors present in each well in determined periods of LTC, compared with the number of progenitors of the beginning of the cultures. Mean ± SEM of four wells per point in four separate experiments.