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. 2018 Mar 22;9:247–256. doi: 10.1016/j.omtm.2018.03.007

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

High-Level β-Globin Production with Efficient Lentiviral Transduction by Using IMDM-Based Serum-free Erythroid Differentiation Media with KSR Supplementation

(A) We evaluated higher concentration of SCF (5×) and/or IL-3 (10×) in IMDM-based serum-free differentiation media with 20% KSR in human CD34⁺ cells and PBMCs. (B and C) We evaluated erythroid differentiation (GPA and CD71 expression), transduction (GFP positive), and cell counts 13 and 14 days after differentiation of (B) CD34⁺ cells and (C) PBMCs, respectively. (D and E) Hb production was evaluated by Hb electrophoresis in (D) CD34⁺ cell- and (E) PBMC-derived erythroid cells. (F and G) Both β-globin (adult Hb) and γ-globin (fetal Hb) production was analyzed at the protein level by reverse-phase high-performance liquid chromatography (RP-HPLC). The globin amounts were quantified by areas of the peaks in RP-HPLC in (F) CD34⁺ cell- and (G) PBMC-derived erythroid cells. The bars represent mean ± SEM. All experiments except Hb electrophoresis (single run) were performed in triplicate.