Comparative gene expression of CD34+ human hematopoietic progenitor cells upon knockdown of STAT5

The objective of the study is to identify the genes regulated by STAT5 in very primitive human hematopoietic stem progenitor cells (HSPCs). We used Affymetrix Human Genome U133 Plus 2.0 microarrays to investigate gene expression in human CD34+CD38- HSPC upon expression of STAT5 or control luciferase shRNA-encoding vectors. Human cord bloods were collected and CD34+ cells were isolated and immediately frozen. CD34+CD38- were sorted from thawed cells and cultured for one day in four-cytokine (IL3, 10 ng/ml; Flt3L, 100 ng/ml; SCF, 100 ng/ml; TPO, 20 nM) minimal cocktail medium before being transduced at multiplicity of infection of 20 with Ctl luciferase- or STAT5-shRNA encoding lentiviral vectors that co-express GFP marker. A few hours later, cells were seeded on MS5 stromal layer and allowed to grow for 5 days. CD34+/GFP+ cells were then sorted and RNA were prepared using Trizol reagent according to manufacturer’s instructions.