Gmp scgm serum free media

Human leukemia cell lines (K562, isogenic K562 with a CRISPR-HDR corrected TP53 allele^{38 (link)}), kindly provided by Benjamin Ebert (Dana Farber Cancer Institute) were maintained in RPMI media (Gibco, 11875-119) supplemented with 10% FBS, penicillin and streptomycin (Gibco). Primary cultures of bone marrow fibroblasts were established and maintained in Chang D media (Irvine Scientific, T105). Mobilized peripheral blood CD34+ and bone marrow mononuclear cells were maintained in GMP SCGM Serum-free Media (Cellgenix, 20802–0500) supplemented with 100 ng/mL hSCF (Peprotech, 300-07), hTPO (Peprotech, 300-18), hFLT3-L (Peprotech, 300-19), and for bone marrow mononuclear cell culture, 20 ng/mL of IL-3 (R&D systems 203-IL-010/CF) was added. All cells lines were cultured at 37 °C under 5% CO₂ and routinely screened for mycoplasma^{39 (link)}.

For methylcellulose colony formation assays, G-CSF mobilized peripheral blood CD34+ cells (Fred Hutch CCEH Core B) were resuspended in GMP SCGM Serum-free Media plus cytokines noted above (Cellgenix, 20802-0500) and allowed to recover for 36 h. Cells were transduced with indicated lentiviral vectors and for CD34+ cells were sorted using BD FACSAria (BD biosciences) to obtain double-positive population. Doublets were excluded using standard methods of FSC/FSC-A doublet exclusion and then distinctly positive RFP and GFP cells were selected based on comparison to untransduced CD34+ cells(gating shown in Supplementary Fig. 5A). Then 1750 CD34+ cells were added to 3.5 ml of methylcellulose (Stem Cell Technologies, H4434). One milliliter was plated in triplicate wells of six-well Smartdishes (Stem Cell Technologies, 27370). After 12 days of growth at 37 °C/5% CO₂, colonies were imaged, results blinded and then counted using STEMVision (Stem Cell Technologies). Counts were averaged for triplicate wells.