Human erythropoietin

FACS-isolated BFU-E enriched cells were cultured in progenitor culture media (PCM) consisting of serum free expansion media (SFEM II, Stem Cell Technologies) supplemented with 100 ng/mL murine SCF (Peprotech), 40 ng/mL murine insulin-like growth factor 1 (Peprotech), 2 U/mL human erythropoietin (Amgen), 1% penicillin/streptomycin (Gibco), and for indicated cultures 100 nM Dexamethasone (Sigma). Cells were seeded at known quantities and cultured at 37 degrees Celsius. Live cell numbers were counted at indicated days by both hemocytometer using Trypan Blue (Gibco) for dead cell exclusion, and by FACS on a FACS Fortessa cytometer (BD) normalizing to Precision Count Beads (Biolegend) and using Propidium Iodide (Sigma) for dead cell exclusion.

CD34+ cell were seeded at 0.5 – 1 × 10⁶/mL in a 6 well plate containing Alpha MEM (Sigma), FBS (Stem Cell Technologies), Glutamine (Gibco), BSA (Sigma), human erythropoietin (Amgen), β-mercaptoethanol (Gibco), dexamethasone (American Regent Laboratories), holo-transferrin (American Regent Laboratories) and human recombinant SCF (Sigma). Cells were counted and fed from day 4 to 10 with the same EC differentiation medium. This differentiation process was used for in vitro modeling of erythropoiesis and VCN determination. For CD34+ cells pre- and post-transduction, CD34+ cells were plated at 500 cells per plate in MethoCultH4435 Enriched (StemCell Technology). BFU-E, CFU-M, CFU-G and CFU-GM were distinguished and counted after 14 – 16 days as per as per manufacturers’ directions.

T cells, B cells, Mph and DCs were sorted by flow cytometry using markers TCRβ, CD4, CD8, CD11c, CD11b, CD19, CD45R, MHC II as described before (12 (link)). Purity of sorted cells was generally >98% and viability was >97% as determined by 7-AAD staining.
Erythrocytes, normoblasts, reticulocytes and red blood cells were generated using a mouse adapted protocol for the long-term ex vivo erythroid differentiation culture protocol described by Giarratana et al (17 (link)) and Konstantinidis et al (18 (link)). Briefly, low-density bone marrow cells were cultured in erythroblast growth medium (StemPro-34 with 2.6% StemPro-34 supplement; Invitrogen), 20% BIT 9500 (StemCell Technologies), 900 ng/mL ferrous sulfate, 90 ng/mL ferrous nitrate, 10⁻⁶M hydrocortisone, penicillin/streptomycin, L-glutamine), in 3 subsequent phases. For the proliferative phase (days 1–5) cells were expanded with 100 ng/mL SCF, 5 ng/mL IL-3, and 2 IU/mL human erythropoietin (Amgen). In the differentiation step (days 6–7), the cells were supplemented with only erythropoietin in fibronectin coated plates. For enucleation (day 8–9) cells were grown without cytokines. Cells were sorted based on size gating and nucleotide staining using different combinations of Syto-16, Draq5, Mitotracker Red and MitotrackerGreen (Invitrogen).