Effects of TPO, activin (ACT), BMP2, and BMP4 on the proliferation, viability, and cell surface marker expression of primary CD34⁺ progenitors, on primitive, total, and MK progenitors. Bone marrow CD34⁺ cells (6 × 10⁵/mL) were incubated in 4GF serum-free medium for 3 to 7 days in the presence or not (None) of 50 ng/mL TPO, activin A (ACT), BMP2, or BMP4. (A) After 3 days of culture, cell proliferation and viability were evaluated by trypan blue counting. Fold proliferation was determined by reference to the number of input viable cells. Results represent the mean plus or minus SEM of 24 experiments for addition of TPO or BMP4 and 12 experiments for activin or BMP2. (B) Phenotypic analysis of the hematopoietic progenitor CD34, the erythroid-specific marker glycophorin A (GPA), early MK-specific markers CD41 and CD61, and late MK markers CD42a and CD42b was performed by flow cytometric analysis using a Facscalibur cell analyzer (Beckman Coulter) and gating on viable cells. Results are presented as percentages of positive viable cells plus or minus SEM of 14 to 22 experiments for addition of TPO or BMP4 and 8 experiments for activin or BMP2. (C) The CFC or CFU-MK content of treated cells was analyzed, and results, expressed as CFC/1000 or CFU-MK/1000 seeded cells, represent the mean value on day 3 plus or minus SEM of, respectively, 14, 8, or 5 experiments for TPO/BMP4 alone, TPO + BMP4, and ACT or BMP2, and the mean value on day 7 plus or minus SEM of 8 experiments for all conditions. (D) Further sorted CD34⁺CD38⁻ cells were incubated (2 × 10³/mL) in 96-well round-bottom plates in strict serum-free IMDM containing 15% BIT without any cytokines (0) or with SCF (100 ng/mL), IL-3 (20 ng/mL), Flt3L (100 ng/mL), or TPO (50 ng/mL) alone () or in combination with BMP4 (50 ng/mL; ■). After 10 days, the cells were placed in an LTC-IC assay. Results are presented on a log scale graph as week 5-CFC/1000 input cells plus or minus SEM of 5 experiments. * indicates statistically significant difference from those in nontreated cells (P < .05).