Generation of Megakaryocytes and Platelets from Human Endometrium

Abstract

<p>In vitro culture systems that produce a large number of megakaryocytes (MKs) and platelets from human stem cells have the potential of revolutionizing transfusion medicine as a treatment for a variety of bleeding disorders. In previous studies, MKs and platelets were generated in vitro from hematopoietic and embryonic stem cells with some success. However, the isolation of human endometrial stromal stem/progenitor cells is a burgeoning field of investigation and the in vitro generation of MKs and platelets from these cells can lead to the treatment of heavy menstrual bleeding disorders using the patient's own stem cells. Here, we have reported a novel in vitro culture system that generates MKs and platelets from human endometrial stromal cells (ESCs). Method: Endometium samples were collected from a premenopausal woman undergoing a hysterectomy for uterine leiomyomata. The ESCs were isolated from the benign proliferative endometrium and cultured for 4 passages. The cells were then used for two differentiation strategies. The first strategy involved incomplete differentiation into adipocytes followed by subsequent culturing in MK differentiation buffer, which contained 50 ng/ml of thrombopoietin, for 18 days. The second strategy involved culturing the ESCs directly in the MK differentiation buffer for 18 days. Confirmation of megakaryocyte and platelet production was conducted by using immunocytochemistry straining for MK-specific markers (CD41a, CD42a, and CD42b) with propidium iodide and subsequent examination under fluorescent and confocal microscopy. Positive identification of the megakaryocytes and platelets were obtained by their morphological characteristics, size, immunophenotypic expression of the specific markers, and nuclear staining patterns. Results: After confirming the megakaryocyte and platelet production it was determined that both strategies have a similar efficacy (35±8/field and 26±6/field, n=5/group, strategy one vs. strategy two, p>0.05). Conclusion: Human ESCs can serve as a useful source for in vitro generation of megakaryoctes and platelets for potential clinical use.</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 16, 2010</p>