Comparison of Human Bone Marrow CD34+ Cell Cultures Treated With TPO or Small Molecule TPO Mimetics

Small molecular TPO mimetics, LGD-4665 and eltrombopag, were efficacious in stimulating the formation of CD41+ cells from human bone marrow CD34+ cells. To better understand the mechanism of action of TPO mimetics, a microarray study was performed to compare global gene expression in CD34+ cells induced by small molecular TPO mimetics eltrombopag and LGD4665, to changes in response to recombinant human thrombopoietin (TPO). Keywords: Drug Treatment CD34+ human bone marrow cells were obtained from five independent healthy donors. Cells from each donor population were seeded at 500,000 cells/well in six-well culture dishes, and treated with vehicle control,100 ng/ml rhTPO, 1 uM eltrombopag, or 1 uM GD-4665. A timepoint of five days was chosen to capture early events since CD41 mRNA expression peaks at this time. Following five days of treatment, each culture well was harvested for RNA isolation. Two wells of vehicle-treated cells from each donor were pooled to yield sufficient RNA. This resulted in a total of 20 RNA samples representing five independent donors (or biological replicates) and four independent treatments. Each RNA sample was used to create a probe which was hybridized to a single microarray. In sum, there were five biological replicates, each subjected to three parallel treatments, with no technical replicates.