Human erythroid progenitor cell culture and treatment

This study was performed in accordance with the Helsinki declaration and was approved by Mahidol University Institutional Review Board (approval number MU-CIRB 2013/022.1103). Written informed consents were obtained from all individual participants in this study. The total of 5 β-thalassemia/HbE patients were recruited. Mononuclear cells were isolated from peripheral blood by gradient centrifugation with Lymphoprep, density 1.077 g/ml (Axis-Shield, Oslo, Norway). The cells were then cultured in a two-phase culture as previously described³⁹. Briefly, the first phase, expansion phase, the mononuclear cells were cultured for 7 days in Iscove’s Modified Dulbecco’s Medium (IMDM, GIBCO-Invitrogen, Carlsbad, CA) containing 30% FBS (Sigma-Aldrich), 25 ng/ml interleukin-3 (IL-3; Cell Signaling Technology, Beverly, MA), 50 ng/ml stem cell factor (SCF; Cell Signaling Technology) and 0.1 U/ml erythropoietin (EPO; Janssen-Cilag, New Brunswick, NJ). The second phase, differentiation phase, progenitor cells from expansion phase were cultured in IMDM supplemented with 30% FBS, 0.1 ng/ml IL-3 and 3 U/ml EPO. On day 7 of the second phase, the erythroid progenitors were treated with curcuminoids and trienone compounds. Cells were harvested on day 11 of the second phase for HbF and mRNA expression and DNA methylation analysis. HU (100 μM) (Bristol-Myers Squibb, Rome, Italy) was used as positive control. DMSO (0.25%) was used as negative control. Untreated cells were used as analyzed baseline control.