Conditioned medium for LTC-IC assay

For collection of mouse stroma cell-conditioned medium, bone marrow cells were isolated from tibiae, femurs, pelvic bones, and vertebrae of 6- to 12-week-old mice by gentle crushing in sterile PBS. Total bone marrow cells were cultured overnight in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin [penicillin (100 U/ml) and streptomycin (100 μg/ml); Mediatech Inc.]. Nonadherent cells were removed, and the remaining adherent cells were cultured until 80 to 90% confluency and subsequently irradiated (20 Gy) to suspend cell proliferation. Four to 5 hours after irradiation, adherent stromal cells were cultured in MyeloCult M5300 (STEMCELL Technologies) supplemented with Primocin (200 μg/ml; InvivoGen), and medium was collected, sterile-filtered every 48 hours, and then stored at −20°C in aliquots until use.

For collection of human stroma cell–conditioned medium, HS-5 (CRL-11882, American Type Culture Collection) bone marrow stromal cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Once at 80 to 90% confluency, cells were irradiated at 20 Gy and cultured in MyeloCult H5100 (STEMCELL Technologies) supplemented with Primocin (200 μg/ml; InvivoGen). Conditioned medium was collected, sterile-filtered every 48 hours, and then stored at −20°C in aliquots until use.