Transcriptome Analysis of Human Hematopoietic Progenitor Cells during In Vitro Erythroid Differentiation.

The hematopoietic progenitor cell (HPC) has the potential to differentiate into multiple cell types. Deciphering transcriptional regulatory networks necessary for HPCs to undergo lineage commitment is critical. Toward this goal, we established a serum-free in vitro differentiation system for human HPCs using CD34−positive cells isolated from peripheral blood of normal donors. Cells were cultured in the presence of Epo, SCF and IL-3 for two-weeks, and assessed for total hemoglobin and percent fetal hemoglobin. Adult-derived HPCs express large amounts of Hb A after approximately one week of culture with little expression of Hb F. Transcriptome analysis, using Affymetrix Human Genome U133 Plus 2.0 Arrays on RNA from days 1, 3, 5, 7, 9, 11 and 13 of culture shows more than 1500 differentially-expressed (DE)(> 2-fold up or down) genes, a subset of which were validated independently by real-time RT-PCR. Up-regulated erythroid-specific genes included those for blood group antigens (Kell, Duffy, Rhesus), membrane proteins (ankryin 1, α- and β-spectrin, Band 4.1, Band 4.2, Band 4.9, glycophorin A), receptors (transferrin and erythropoietin receptors), heme biosynthesis enzymes (ALAS, ALAD, UROS, UROD, CPOX, PPOX, FECH), and transcription factors important in globin-gene expression (EKLF, BKLF, NFE2, GATA1) as well as α-, β- and γ-globin mRNAs. Down-regulated genes included those expressed in hematopoietic stem cells (GATA2, CD34) and non-erythroid hematopoietic lineages (MHC class I and II genes, CD33, CD53). The DE genes were clustered into a limited number of expression patterns, suggesting coordinate regulation of genes within each cluster. The DE gene set was examined in silico for co-regulation using transcriptional regulatory network analysis employing PAINT v3.2 software (