Sodium valproate directly inhibits thrombopoietin induced megakaryocytopoiesis from human bone marrow CD34+ cells in vitro

Abstract Sodium valproate is a commonly used anticonvulsant in the management of complex seizure disorders. The primary reported hematologic side effect is thrombocytopenia. To investigate whether the effects observed in vivo are due to direct actions of sodium valproate on progenitor cell differentiation, we studied the in vitro formation of megakaryocytes in liquid cultures containing the drug. We then compared the ability of sodium valproate to suppress megakaryocytopoiesis with that of a second agent previously described to inhibit this process, TGFβ. Human mPBCD34 + cells were purified from apheresis samples by immunomagnetic separation techniques. These progenitor cells were cultured in serum-free media with 20 ng/ml SF, 20 ng/ml Tpo and increasing concentrations of the agents for 10 days. Differentiation was assessed by monitoring the expression of CD34 and CD41 on the cell surface using fluorescence activated cell sorting analysis. Addition of 50 μg/ml sodium valproate did not appear to be toxic to the mPBCD34 + cells as it did not inhibit cell growth. On day 7, the number of cells expressing CD34 in these cultures equaled that detected in cultures not containing the drug. However, in the presence of sodium valproate approximately 23% fewer cells expressed CD41. A 2-fold increase in the number of CD34 + CD41 + cells was observed in these cultures. Ten days of exposure to the drug produced a 3-fold increase in CD34 + cells over that detected in cultures without the drug. The majority of these CD34 + cells (72–75%) were single positives that did not express CD41. Increasing the concentration of sodium valproate to 100 μg/ml was toxic and by day 10 resulted in approximately 66% fewer cells than in control cultures. Thirty percent of these cells were CD34 + (23% CD34 + , 7% CD34 + CD41 + ) as compared to 6–7% CD34 + cells in cultures grown in the absence of the drug. TGFβ was added to mPBCD34 + cell cultures at concentrations of 5, 50 and 500 pg/ml. Toxicity was observed at 50 pg/ml on day 10 with the majority of the surviving cells expressing CD41 (approximately 90%) and few cells expressing CD34 (3–4%). These results suggest that the observed thrombocytopenia in patients receiving sodium valproate is likely to be due at least in part to direct suppression of megakaryocyte development from progenitor cells and that the mechanism by which it inhibits megakaryocytopoiesis differs from that of TGFβ.