THE DEVELOPMENT AND OPTIMIZATION OF A HUMAN MEGAKARYOCYTE CULTURE FROM HEMATOPOIETIC PROGENITOR CELLS ISOLATED FROM NORMAL PERIPHERAL BLOOD FOR IN VITRO INVESTIGATION OF PLATELET DISORDERS

Abstract

<p>Megakaryocyte cultures are a strong tool for the in vitro investigation of platelet production in platelet disorders. Peripheral blood derived hematopoietic progenitor cells (PB-HPCs) are the most accessible source of HPCs with high potential to produce mature megakaryocytes in vitro; however, they are present in low numbers making peripheral blood an inefficient source. Additionally, a megakaryocyte culture with an optimized thrombopoietin (TPO) concentration is required which can reliably allow the investigation of suppressive effects of antibodies/plasma from immune thrombocytopenia (ITP) patients. In this study, we developed a megakaryocyte culture with the utilization of human PB-HPCs in an efficient fashion resulting in the production of high purity megakaryocytes in a TPO-dependent manner.</p> <p>The mononuclear fraction was collected from 180 mL of peripheral whole blood and CD34+ cells were isolated by a positive selection yielding the average of 5.5 x 105 ± 2.5 x 105 CD34+ cells (n = 18). Using 96-well tissue-culture plates and seeding 10,000 CD34+ cells/well, the average of 13 experiments in triplicate can be set up utilizing isolated CD34+ in an efficient manner. Capitalizing on a TPO dose-dependent megakaryocyte production experiment, 20 ng/mL was established as the TPO concentration which resulted in the production of mature megakaryocytes without reaching the plateau in megakaryopoiesis response. On day 11 of culture, the expression of megakaryocytic lineage (CD41/61+) and maturation (CD41/61+CD42+) markers peaked at 90.65% and 76.10%. In conclusion, this culture system has broad application for investigation of platelet disorders and drug discovery which can be accessible to all researchers.</p>