Komor Martina, Güller Saskia, Baldus Claudia D, de Vos Sven, Hoelzer Dieter, Ottmann Oliver G, Hofmann Wolf-K
Department of Hematology, Oncology and Transfusion Medicine, University Hospital Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany.
Stem Cells. 2005 Sep;23(8):1154-69. doi: 10.1634/stemcells.2004-0171. Epub 2005 Jun 13.
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.
为了更好地理解伴随有序的谱系特异性造血分化的转录程序，我们对人正常CD34+骨髓细胞在谱系特异性分化过程中进行了系列寡核苷酸微阵列分析。从健康个体分离的CD34+骨髓细胞在体外分别用细胞因子促红细胞生成素（EPO）、血小板生成素（TPO）、粒细胞集落刺激因子（G-CSF）和粒细胞巨噬细胞集落刺激因子（GM-CSF）刺激。培养4、7和11天后，收集每个谱系的细胞进行表达谱分析。通过寡核苷酸微阵列（HG-U133A；Affymetrix，加利福尼亚州圣克拉拉）分析基因表达。实验重复进行三次。我们鉴定出258个基因，它们在每个特定谱系的谱系特异性分化过程中持续上调或下调（水平变化）。此外，我们鉴定出52个基因，它们促成了特定的表达谱，产生了三个谱系中每个谱系内连续分化阶段特有的遗传特征。水平变化分析选择了21个在EPO诱导的分化中持续上调的基因（包括调节小Rho GTP酶的GTP酶激活蛋白RAP1GA1和ARHGAP8），21个在G-CSF诱导/GM-CSF诱导的分化中持续上调的基因，以及91个在TPO诱导的分化中持续上调的基因（包括DLK1，其在正常造血中的作用尚未明确）。在谱系特异性分化过程中，分别有58个（红细胞生成）、30个（粒细胞生成）和37个（血小板生成）基因显著下调。所选基因的表达通过实时聚合酶链反应得到证实。我们的数据涵盖了体外谱系特异性分化过程中人类造血的首个广泛转录谱。
