A novel culture system of total BM cells dramatically expands HSCs. (A) Total BM cells (106) were initiated in serum-free medium with SCF, TPO, IGF-2, and FGF-1 as described in “Materials and methods,” and total cell numbers were counted at days 7, 10, 14, 21, and 28. Results from 3 independent cultures were plotted. (B) Comparison of the long-term repopulation potential of 10-day cultured and freshly isolated BM cells. We mixed 5 × 104, 1 × 104, 5000, 2500, or 1250 freshly isolated CD45.2 BM cells or 3.5 × 105, 7 × 104, 3.5 × 104, 1.75 × 104, or 9000 10-day cultured BM cells (the product of 5 × 104, 1 × 104, 5000, 2500, or 1250 initially plated CD45.2 cells, respectively) with 105 CD45.1 competitor BM cells and transplanted them into lethally irradiated recipients (n = 6 mice). In 1 case IGF-2 was not added, but the remainder of the culture conditions was unchanged. Peripheral blood cells were analyzed for the presence of CD45.2+ cells at 4 months after transplantation. Three independent experiments were performed that gave similar results. (C) Multilineage contribution of 3.5 × 105 cultured cells (derived from 5 × 104 input cells) at 4 months after transplantation (n = 6). (D) Multilineage contribution of the 3.5 × 105 cultured cells (equivalent to 5 × 104 input cells) at 4 months after transplantation of mice receiving a secondary transplant (n = 4). (E) Limiting dilution analysis of the repopulating ability of total BM cells before and after culture. Irradiated CD45.1 congenic mice were injected with 105 CD45.1 BM competitor cells and the indicated numbers of freshly isolated CD45.2 BM cells (▪ and —) or their progeny after 10 days of culture in serum-free medium with SCF, TPO, IGF-2, and FGF-1 (▿ and —). Plotted is the percentage of recipient mice containing less than 1% CD45.2 lymphoid and myeloid subpopulations in nucleated peripheral blood cells 4 months after transplantation versus the number of injected cells. The curve was anchored by the 0 cells/100% negative mice point. Error bars indicate SEM.
