Abstract
The steps that lead to platelet production are poorly understood. Current theories suggest that megakaryocytes mature under the influence of contact with sinusoidal endothelium, and release platelets either in the sinusoids or in the lungs. We hypothesized that platelet release would be accentuated following hematopoietic stem cell transplant, and that sites of platelet release would be apparent during the period of platelet recovery. We transplanted highly purified hematopoietic stem cells based on lack of expression of markers for mature lineages (Linneg) and expression of Sca-1, c-kit, and Thy-1.1 (KTSL cells), and subfractionated these cells based on low expression of Rhodamine 1-2-3, into lethally irradiated hosts expressing an allelic version of glucose phosphate isomerase to identify donor and host-derived platelets. We collected bones, lungs, livers and spleens on day 7, 14, 21, and 28 post-tranplant, and stained formalin/fixed tissue with anti-Von Willebrand Factor antibody to identify megakaryocytes (5–10 animals per cohort, 2 separate experiments). We scored megakaryocytes based on their location relative to endothelial cells, and whether they were releasing platelets based on extension of proplatelet processes into the vascular spaces. Almost every megakaryocyte was associated with the endothelium during the period of platelet recovery, and we did not identify megakaryocytes that were migrating to the endothelium. We saw numerous megakaryocyte releasing platelets in both the bone marrow and the spleen during the time of platelet recovery, which occurred on days 13–28 following transplant of purified stem cells. Some of these megakaryocytes had disrupted the endothelium and were incorporated into the sinusoidal wall. Others were completely within the sinusoidal spaces. Between 30 and 50% of megakaryocytes were releasing platelets in the spleen and bone marrow at any given time following transplant, and platelet release did not correlate with the platelet counts. These levels were similar to levels of platelet release seen in healthy control mice. In contrast, we saw no identifiable megakaryocytes in the liver and lung during the period of platelet recovery. Our results suggest that in the mouse, the bone marrow and spleen, and not the lung, are major sites of platelet release following stem cell transplant.
Are we underutilizing bone marrow and cord blood? Review of their role and potential in the era of cellular therapies
