Stemsep kit

Our method to produce MPSC from frozen samples of UCB is described in detail in other publications 12, 21, 25 and summarized here. We used either the Miltenyi‐MACS CD34+ selection kit, Bergisch, Germany or the Stem Cell Technologies Stem‐Sep kit, Vancouver, Canada to isolate CD34+ cells. CD34+ content was assessed using flow cytometry. The dead cell removal kit was used prior to CD34+ selection. Only frozen UCB units were used. Prior to the processing with the dead cell removal kit and selection, frozen units were filtered through a 70 micron mesh after thawing to remove clumps of dead cells that may have accumulated during the freeze/thaw process. Post column cells were seeded at 1 × 10⁵ cells/ml in FSFl medium (StemSpan media [Stem Cell Technologies] containing IMDM, 1% bovine serum albumin (BSA), 10 mg/ml insulin, 200 mg/ml human transferrin, 10⁻⁴ M 2‐mercaptoethanol, and 2 mM L‐glutamine. The media was supplemented with 25 ng/ml SCF [R&D Systems, Minneapolis, MN], 25 ng/ml Flt‐3 ligand [FL; R&D Systems, Minneapolis, MN] and 50 ng/ml Fibroblast Growth Factor‐4 [FGF‐4; R&D Systems, Minneapolis, MN], 50 ng/ml heparin and 10mg/ml low density lipoprotein [Sigma, Markham, Canada]). Fifty percent medium replacement occurred every 48 hours. For all animal experiments described here, the cells were used directly after 7–8 day culture in FSFl medium.

Bone marrow was extracted from the femur and tibia of 10–12 week old BALB/c female mice 48 hours after intraperitoneal (i.p.) injection of 100 µl 5-fluorouracil (30 mg/ml) (Mayne Pharma Pic, Warwickshire, UK) to expand the stem cell compartment. Bone marrow cells were enriched for hematopoietic progenitors and stem cells (HSCs) by negative selection using the StemSep kit (13309, StemCell Technologies) according to the manufacturer’s specifications. Cells were cultured for 24 hours in OPTIMEM (31985070, Invitrogen) supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 mg/ml streptomycin, 1% IL-6, 3% IL-3 and 3% SCF containing supernatants [40 (link)]. Next, HSCs were co-transduced by two rounds of spin infection with combinations of BCL-X_L-GFP and MYC-YFP retroviral particles in the presence of 10 mg/ml polybrene (TR-1003, Sigma-Aldrich). This procedure was repeated for three consecutive days and the cells were then cultured for an additional three days. The percentage of GFP and YFP positive cells was measured by flow cytometry prior to transplantation.