Megakaryocytes generated from human CD34+ cells in vitro exhibit increased platelet production with 15d-PGJ2 treatment. Generating megakaryocytes and platelets from human cord blood–derived CD34+ cells in vitro. CD34+ cells cultured in the presence of rhTPO for 14 days are 92% positive for CD61 (data not shown). CD61 expressing cells generated from cord blood were treated with vehicle or 15d-PGJ2 (10 μM) for 24 hours, and cells were photographed in culture and platelet production was analyzed by flow cytometry. (A) Bar graph showing that 15d-PGJ2 increases the number of platelets derived from primary human megakaryocytes. Results are presented as mean plus or minus SD (P < .01). (B) Platelets were isolated by gradient centrifugation from CD61+ cell cultures treated with 15d-PGJ2. Left plot shows the forward and side scatter of untreated platelets. Right plot shows the forward and side scatter of platelets treated with collagen (10 μg/mL for 15 minutes). Note the decrease in size and increase in granularity. (C) Histogram showing the up-regulation of surface CD61 with collagen treatment. Values are a measure of geometric mean fluorescence intensity. (D) SEM showing a culture-derived human platelet spread on a fibrinogen-coated slide. (E) Left picture shows microscopy of a human megakaryocyte cultured in the absence of 15d-PGJ2. Middle picture shows microscopy of a human megakaryocyte cultured in the presence of 15d-PGJ2. Arrows indicate proplatelets. Far right picture shows magnification of proplatelet extensions.
