Erythroid differentiation of human CD34+ stem cells

Human primary erythroid cells were generated from adult CD34⁺ stem cells (STEMCELL Technologies, Vancouver, BC, USA) in a two‐phase culture system as previously published (Li et al, 2014). During phase I, stem cells were grown in minimum essential medium‐α (αMEM) containing AB serum, interleukin‐3 (10 ng/ml), stem cell factor (10 ng/ml), and erythropoietin (2 iu/ml). On day 7, cells transitioned to Phase II media where they remained under erythropoietin (2 iu/ml) stimulation. Erythroid progenitors were transfected on day 8 with human mature MIR29B or control scramble (Scr) mimic (Applied Biosystems, Foster City, CA, USA) by nucleofection using the Amaxa^® Human CD34⁺ Cell Nucleofector^® Kit. For drug studies, cells were treated with 0·5 μmol/l Dec alone or pretreatment with 100 nmol/l MIR29B on day 8 followed by drug treatment. After 48 h, cells were harvested for reverse transcription‐quantitative PCR (RT‐qPCR), Western blot, and flow cytometry analysis. Giemsa staining was used to monitor cell morphology and cell counts; viability was monitored using 0·4% trypan blue exclusion assay (Gibco, Carlsbad CA, USA).