Publication Date:
2009-11-20
Description:
Abstract 4584 Megakaryocyte (Mk) development and proplatelet formation are regulated at multiple levels although terminal stage molecular signals are incompletely delineated. We applied a customized, platelet-restricted oligonucleotide gene chip comprised of 432 genes to characterize genetic changes that occur in CD34+ cells differentiated along the Mk lineage, using a cytokine cocktail containing IL-6 (25 ng/mL), IL-11 (25 ng/mL), FLT3 (50 ng/mL), IL-1β (10 ng/mL), thrombopoietin (TPO, 50 ng/mL), and stem cell factor (SCF, 25 ng/mL). After day (D) 5, 〉60% of cells remained CD41 (αIIB)-positive by flow cytometric analysis, with 90% CD41-positivity at D20. Positive-selection using a β3 (glycoprotein IIIa) anti-CD61 antibody and magnetic beads was completed daily, and allowed for physical separation and comparative genetic profiles of the CD61+ and CD61- cell fractions. Gene-wise progressive correlation analysis of aggregate CD61+ to CD61- cell fractions demonstrated a correlation distribution centered between -0.2 to 0, confirming the distinct genetic profiles of CD61+ and CD61- cells. Unsupervised hierarchical clustering of the CD61+ cells – in conjunction with 41 normal platelet control profiles – confirmed the presence of two discriminatory dendograms that separated D1-15 profiles from D16-21 and normal platelet profiles, establishing that a genetic switch towards late Mk/proplatelet development occurred around D15/D16. Aggregate gene expression between D1-15 and D16-21 subsets identified 252 differentially-expressed genes (p 〈 0.05), only 12 of which were upregulated in D16-21 samples (11 of these 12 genes were differentially expressed between day-matched CD61- cell fractions, confirming the specificity of these changes). The conjunction of this 12-gene subset with a common 5-member pathway cluster of progressively induced genes identified three transcripts [transforming growth factor β-2 (TGFB2), CD99 antigen (CD99), and thromboxane A2 receptor (TBXA2R)] that were specifically probed to elucidate transcript and protein expression patterns in circulating platelets. Dual-color quantitative flow cytometry for these three proteins (along with ITGB3, β3, as control for a non-induced gene) was completed in both permeabilized (intracytoplasmic) and non-permeabilized (cell-surface) platelets, in parallel with thiazole orange (TO) as a measure of platelet RNA content; for all analyses, RNA and protein content in individual platelets were normalized by size and volume to adjust for size/volume differences. Despite a wide distribution of platelet size, RNA content, and protein expression, both the RNA and protein concentrations remained relatively constant. Furthermore, the RNA concentration remained tightly restricted within a narrow window, establishing that this parameter is critically maintained for all platelets unrelated to age. For all platelets, there was poor correlation (r
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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