Rhuepo

De-identified peripheral blood samples from adults and cord blood were purchased from Research Blood Components, LLC (Boston, MA 02135) and New York Blood Center, Inc. (New York, NY 10065). Erythroid progenitors were cultured in a two-phase system, as previously described.^{29 –31 (link)} Briefly, mononuclear cells from adult peripheral blood (PBMC) or cold blood were isolated on Ficoll-Paque (GE Healthcare, Piscataway, NJ), and cultured in a two-phase culture system.²⁹ The first phase media utilized HyClone™ IMDM (Thermo Scientific, Waltham, MA) containing 30% charcoal treated fetal bovine serum (Life Technology, New York, NY) and rHuIL-3 (25 ng/mL), rHuSCF (50 ng/mL), and rHuEPO at (0.1 u/mL) (GenScript, Piscataway, NJ) for the first 7 days, followed by replacement second phase differentiation media, with 30% charcoal treated fetal bovine serum, rHuEPO at 3 U/mL, and rHuIL-3 (0.1 ng/mL). Test compounds SRT2104 (APExBIO, Houston, TX) and SRT1720 (EMD Millipore, Billerica, MA) were added 4 days after initiation of the Phase 2 erythroid differentiation cultures. Erythroid progenitors were confirmed by Giemsa stain.²⁹ Cells were enumerated by hemocytometry and harvested on day 12 of phase 2 for mRNA and ChIP analysis, and on day 14 for F-cell analysis by flow cytometry and for γ-globin analysis by immunoblotting.