Impact of OB, SC, and OB + SC cocultures on stem and progenitor cell function. (A) Cells were cultured in different combinations as indicated, and all wells received recombinant murine stem cell factor and interleukin-3 (10 ng/mL), insulin-like growth factor 1 and thrombopoietin (20 ng/mL), interleukin-6 and Fms-like tyrosine kinase 3 (25 ng/mL), and OPN (50 ng/mL). Cells were harvested on day 7 and counted. Fold increase in total cell number from the original 1000 LSK cells was calculated relative to day 0; n = 6 to 8 independent experiments. (B) LSK progeny cells harvested on day 7 were plated in methylcellulose-based clonogenic assays, and colony formation was assessed 7 days later. CFU fold increase was calculated relative to that obtained from 250 freshly isolated LSK cells assayed on day 0; n = 4 or 5 independent experiments. (C) LSK progeny harvested on day 7 were stained and analyzed for the Lin⁻Sca1⁺ content; n = 3 independent experiments. (D) Lin⁻Sca1⁺ cells were sorted from each group and analyzed for cell-cycle status with propidium iodide; n = 5 independent experiments. (E) BM repopulating potential of freshly isolated and in vitro expanded LSK cells for 10 days in cocultures of OBs, SCs, or OBs + SCs or on plastic. LSK cells from C57Bl/6 (CD45.2) mice were cotransplanted with 100 000 BoyJ (CD45.1) competitor cells in lethally irradiated (1100 cGy, split dose) CD45.2 × CD45.1 F1 recipients. Control mice (Fresh) received 1000 freshly isolated LSK cells and 100 000 competitor cells. At monthly intervals, chimerism was assessed as [CD45.2/(CD45.2 + CD45.1)] × 100, thus eliminating the contribution of residual host-derived HSCs. Data are from 1 experiment, 4 or 5 mice per group, except for the LSK cells cultured on plastic where only 1 mouse survived. (F) Secondary transplantations from primary recipients. At 4 months after primary transplantation, the BM content of 1 femur from each primary recipient was transplanted into a lethally irradiated secondary recipient without competitor cells, and engraftment was assessed at monthly intervals. Each group contained 4 mice, except the LSK cells group, which had 2 mice transplanted with cells from a single primary recipient. *Significant at P < .01 compared with OB + LSK group for panels A, B, C, and D and at P < .05 for panels E and F. Differences between fresh and OB + LSK groups for primary and secondary transplantations were not significant.