Abstract

Megakaryopoiesis is the process by which committed bone marrow progenitors develop into mature megakaryocytes. Differentiation of megakaryocytes is associated with the expression of a wide range of specific proteins but is also characterised by endoreplication of nuclear DNA and a phase of cytoplasmic maturation with the formation of demarcation membranes and granules prior to the release of platelets. In spite of a lot of interest, the molecular regulation underlying megakaryocytopoiesis remains poorly understood. Experimental advances have been hindered by the availability of systems and materials, not least of the difficulties being the paucity of bone marrow megakaryocytes and their precursors. Advances in genetic manipulation means that the study of megakaryocytopoiesis will focus on murine model systems, however the problems associated with obtaining and differentiating committed megakaryocyte progenitors are particularly acute for work involving the mouse.

Various culture systems are currently used to study megakaryocytopoiesis starting with CD34+CD41+ stem cells as a source of progenitors, however the low purity of these cultures limits their application. Here we describe a methodology for the expansion and purification of committed megakaryocyte progenitors from mouse bone marrow. Bone marrow cells are depleted of all mature haemopoietic cells and committed progenitors and are cultured in a “two step” serum-free system. Megakaryocyte precursors are first expanded before a population of progenitor cells is purified by immunomagnetic depletion. Cells isolated in this way are homogenous for the expression of surface markers characteristic of megakaryocyte progenitors (CD41+, c-Kit+, CD34low, CD9+, Fc_RII/IIIlow, CD45+, ERMP20, ERMP12+) and can be shown to be committed to the megakaryocyte lineage using a single-cell culture assay. Most importantly, the purified progenitor population can be induced to undergo normal megakaryocyte differentiation, achieving polyploidy of up to 128N and ultimately giving rise to platelets. Furthermore, the differentiating population exhibits a strict correlation between cell size and the extent of ploidy and a complete absence of differentiation along other lineages.

Ets family transcription factors are believed to be crucial for specific regulation of gene expression during megakaryocytopoiesis. This view derives largely from the observation that most genes specifically expressed in megakaryocytes have functional Ets binding sites in their promoter regions. Which, from amongst the large number of family members, are the relevant Ets factors is still a major question. The data on expression patterns is sketchy, although it is clear, mainly from analysis of human megakaryoblastic cell lines, that several Ets factors are likely to be expressed in megakaryocytic cells and that their expression may change during induced differentiation. We have used the purified primary megakaryocyte progenitors and cells undergoing TPO-induced differentiation to monitor which Ets factors are expressed and how their expression varies throughout megakaryocytopoiesis. This analysis revealed that at least 15 Ets sequences are expressed and that their levels vary in relation to the stage of differentiation. The utility of the primary cells, and in particular how they may help unravel the individual contributions of members of a complex family of proteins such as the Ets factors, is illustrated by the selective ablation of Tel RNA using siRNA.

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