Bit serum replacement

Primary CD34⁺ HPCs were thawed and recovered overnight in stem cell media (IMDM containing 10% BIT serum replacement (Invitrogen), penicillin/streptomycin and stem cell cytokines (SCF, FLT3L, IL-3, IL-6). Following recovery, HPCs were transfected using the Amaxa 4D system and the Primary Cell P3 solution according to the manufacturer’s instructions (Lonza). HPCs were transfected with 1ug pSIREN plasmid DNA per 10⁶ cells using either program EH-100 or EO-100. HPCs were recovered in stem cell media for 48hrs, then isolated by FACS (BD FACS Aria equipped with 488, 633 and 405 lasers, run FACS DIVA software) for a pure population of viable, CD34⁺, GFP⁺ HPCs. Pure populations of sorted HPCs were plated either at 500 cells/mL in Methocult H4434 (Stem Cell Technologies) in 6 well plates in triplicate for myeloid colony assays, or at 10⁴ cells/mL in stem cell media, 200uL/well in 96 well plates for proliferation assays. Total and specific myeloid colony types were enumerated at 7 and 14 days using a standard microscope and standard protocols as previously described [63 ]. Proliferation was assessed at indicated times by Trypan Blue exclusion and manual counting.