Alpha mem

BG was synthesized by Robert Moschel at the Frederick Cancer Research Institute (Frederick, MD). BCNU was obtained from the Drug Synthesis and Chemistry Branch of the National Cancer Institute (NCI; Bethesda, MD). The in vitro drug treatment incubations were carried out in serum free media at 37 °C as previously described^{35 (link)}. Briefly, cells were pretreated with BG for 1 h, then BCNU for 2 h. K562 cells were treated in Iscove’s media and then allowed to expand for 7 d. For in vitro selections, murine BMCs or LSK cells were treated with 25 μM BG and 0, 5, 15, or 25 μM BCNU in alpha-MEM containing 1.2% spleen cell conditioned media (Stem Cell Technologies, Vancouver, BC, Canada). Drug treated murine BMCs or LSK cells were expanded in liquid culture using the same media formulation as described for transduction without polybrene for 10 or 15 d, respectively. Progenitor (CFU) drug treatment survival assays were performed as previously described^{39 (link)}. For in vivo selection, three rounds of selection, starting at 5 weeks post-transplant, were carried out allowing 3 weeks recovery between each treatment. Each round, mice received i.p. injection with 30 mg/kg BG (dissolved to 3 mg/mL in 40% polyethylene glycol (Union Carbide Corp., Danbury, CT) and 60% PBS, pH 8.0), followed by i.p. injection of 10 mg/kg BCNU (dissolved in ethanol and diluted to 1 mg/mL in PBS) an hour later.

Immediately after euthanasia, rMSCs were isolated from the bone marrow of the humerus of each rat. Briefly, the humerii from each rat was removed, the ends of the bone cut, and the bone marrow flushed with growth media and plated directly onto tissue culture plates. rMSCs were cultured in alpha modified Eagle medium (alpha-MEM, Stem Cell Technologies) with nucleosides supplemented with 50 μg/mL penicillin (Gibco), 50 μg/mL streptomycin (Gibco), and 1 μg/mL fungizone (Gibco) containing 10% (vol/vol) fetal bovine serum (Gibco). Media was changed every 2-3 days. After 10 days (70-80% confluency), rMSCs from each individual rat were frozen down in a solution of 40% growth media, 40% FBS and 20% DMSO. Passage 1 (P1) rMSCs from individual rats were pooled, expanded for an additional 7-10 days, and frozen at P2 in Cell Freezing Medium (ThermoFisher). All experiments used P3 pooled SHAM and OVX rMSCs.