Transcriptional Profiling of Human Hematopoiesis During In Vitro Lineage‐Specific Differentiation

To better understand the transcriptional program that a ccompanies orderly lineage‐specific hematopoietic differentiation, we performed serial oligonucleotide microarray analysis of human normal CD34+ bone marrow cells during lineage‐specific differentiation. CD34+ bone marrow cells isolated from healthy individuals were selectively stimulated in vitro with the cytokines erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony‐stimulating factor (G‐CSF), and granulocyte macrophage colony‐stimulating factor (GM‐CSF). Cells from each of the lineages were harvested after 4, 7, and 11 days of culture for expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG‐U133A; Affymetrix, Santa Clara, CA). Experiments were done in triplicates. We identified 258 genes that are consistently upregulated or downregulated during the course of lineage‐specific differentiation within each specific lineage (horizontal change). In addition, we identified 52 genes that contributed to a specific expression profile, yielding a genetic signature specific for successive stages of differentiation within each of the three lineages. Analysis of horizontal changes selected 21 continuously upregulated genes for EPO‐induced differentiation (including GTPase activator proteins RAP1GA1 and ARHGAP8, which regulate small Rho GTPases), 21 for G‐CSF–induced/GM‐CSF–induced differentiation, and 91 for TPO‐induced differentiation (including DLK1, of which the role in normal hematopoiesis is not defined). During the lineage‐specific differentiation, 58 (erythropoiesis), 30 (granulopoiesis), and 37 (thrombopoiesis) genes were significantly downregulated, respectively. The expression of selected genes was confirmed by real‐time polymerase chain reaction. Our data encompass the first extensive transcriptional profile of human hematopoiesis during in vitro lineage‐specific differentiation.

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