OBJECTIVE: The differentiation of megakaryocytes is characterized by polyploidization and cytoplasmic maturation leading to platelet production. Studying these processes is hindered by the paucity of bone marrow megakaryocytes and their precursors. We describe a method for the expansion and purification of committed megakaryocyte progenitors and demonstrate their usefulness by studying changes in the expression of Ets and GATA family transcription factors throughout megakaryocytopoiesis. METHODS: A two-step serum-free method was developed. Cells isolated using this method were analyzed for surface marker expression by flow cytometry, and for their ability to differentiate using single-cell culture. Purified progenitors were induced to differentiate and analyzed with respect to their ploidy by flow cytometry and expression of specific genes by RT-PCR. RESULTS: A population of Lin- c-kit+ CD45+ CD41+ CD31+ CD34low CD9low FcgammaRII/IIIlow Sca-1med/low committed megakaryocyte progenitors was purified. These cells could be differentiated efficiently, achieving ploidy of up to 128N. Analysis of RNA demonstrated the expected increases in expression of key megakaryocyte-associated genes. RT-PCR analysis also revealed that a range of Ets and GATA factors are expressed, their individual levels and patterns of expression varying widely. Surprisingly, we find that GATA-6 is specifically expressed in late differentiated megakaryocytes and has the potential to regulate megakaryocyte-expressed genes in cooperation with Ets factors. CONCLUSION: Purified primary megakaryocytic progenitors are able to differentiate as a cohort into fully mature megakaryocytes. The number of cells obtainable, and the synchrony of the differentiation process, facilitates analysis of the dynamics of molecular processes involved in megakaryocytopoiesis. The expression pattern of Ets and GATA family transcription factors reveals the complexity of the involvement of these key megakaryocytic regulators. The finding of GATA-6 expression and demonstration of its functional activity suggests a novel mechanism for the regulation of certain genes late in megakaryocytopoiesis.
OBJECTIVE: The differentiation of megakaryocytes is characterized by polyploidization and cytoplasmic maturation leading to platelet production. Studying these processes is hindered by the paucity of bone marrow megakaryocytes and their precursors. We describe a method for the expansion and purification of committed megakaryocyte progenitors and demonstrate their usefulness by studying changes in the expression of Ets and GATA family transcription factors throughout megakaryocytopoiesis. METHODS: A two-step serum-free method was developed. Cells isolated using this method were analyzed for surface marker expression by flow cytometry, and for their ability to differentiate using single-cell culture. Purified progenitors were induced to differentiate and analyzed with respect to their ploidy by flow cytometry and expression of specific genes by RT-PCR. RESULTS: A population of Lin- c-kit+ CD45+ CD41+ CD31+ CD34low CD9low FcgammaRII/IIIlow Sca-1med/low committed megakaryocyte progenitors was purified. These cells could be differentiated efficiently, achieving ploidy of up to 128N. Analysis of RNA demonstrated the expected increases in expression of key megakaryocyte-associated genes. RT-PCR analysis also revealed that a range of Ets and GATA factors are expressed, their individual levels and patterns of expression varying widely. Surprisingly, we find that GATA-6 is specifically expressed in late differentiated megakaryocytes and has the potential to regulate megakaryocyte-expressed genes in cooperation with Ets factors. CONCLUSION: Purified primary megakaryocytic progenitors are able to differentiate as a cohort into fully mature megakaryocytes. The number of cells obtainable, and the synchrony of the differentiation process, facilitates analysis of the dynamics of molecular processes involved in megakaryocytopoiesis. The expression pattern of Ets and GATA family transcription factors reveals the complexity of the involvement of these key megakaryocytic regulators. The finding of GATA-6 expression and demonstration of its functional activity suggests a novel mechanism for the regulation of certain genes late in megakaryocytopoiesis.
Authors: Timo Vögtle; Sumana Sharma; Jun Mori; Zoltan Nagy; Daniela Semeniak; Cyril Scandola; Mitchell J Geer; Christopher W Smith; Jordan Lane; Scott Pollack; Riitta Lassila; Annukka Jouppila; Alastair J Barr; Derek J Ogg; Tina D Howard; Helen J McMiken; Juli Warwicker; Catherine Geh; Rachel Rowlinson; W Mark Abbott; Anita Eckly; Harald Schulze; Gavin J Wright; Alexandra Mazharian; Klaus Fütterer; Sundaresan Rajesh; Michael R Douglas; Yotis A Senis Journal: Elife Date: 2019-08-22 Impact factor: 8.140
Authors: Elizabeth J Haining; Jing Yang; Rebecca L Bailey; Kabir Khan; Richard Collier; Schickwann Tsai; Steve P Watson; Jon Frampton; Paloma Garcia; Michael G Tomlinson Journal: J Biol Chem Date: 2012-10-03 Impact factor: 5.157
Authors: Yotis A Senis; Michael G Tomlinson; Angel García; Stephanie Dumon; Victoria L Heath; John Herbert; Stephen P Cobbold; Jennifer C Spalton; Sinem Ayman; Robin Antrobus; Nicole Zitzmann; Roy Bicknell; Jon Frampton; Kalwant S Authi; Ashley Martin; Michael J O Wakelam; Stephen P Watson Journal: Mol Cell Proteomics Date: 2006-12-23 Impact factor: 5.911
Authors: Alexandra Mazharian; Ying-Jie Wang; Jun Mori; Danai Bem; Brenda Finney; Silke Heising; Paul Gissen; James G White; Michael C Berndt; Elizabeth E Gardiner; Bernhard Nieswandt; Michael R Douglas; Robert D Campbell; Steve P Watson; Yotis A Senis Journal: Sci Signal Date: 2012-10-30 Impact factor: 8.192
Authors: Catherine M Hobbs; Harriet Manning; Cavan Bennett; Louella Vasquez; Sonia Severin; Lauren Brain; Alexandra Mazharian; Jose A Guerrero; Juan Li; Nicole Soranzo; Anthony R Green; Steve P Watson; Cedric Ghevaert Journal: Blood Date: 2013-10-01 Impact factor: 22.113
Authors: Stephanie Dumon; David S Walton; Giacomo Volpe; Nicola Wilson; Emilie Dassé; Walter Del Pozzo; Josette-Renee Landry; Bryan Turner; Laura P O'Neill; Berthold Göttgens; Jon Frampton Journal: PLoS One Date: 2012-08-28 Impact factor: 3.240