Dumon Stephanie, Heath Victoria L, Tomlinson Michael G, Göttgens Berthold, Frampton Jon
Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK.
Exp Hematol. 2006 May;34(5):654-63. doi: 10.1016/j.exphem.2006.01.014.
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.
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.
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.
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.
巨核细胞的分化特征为多倍体化和细胞质成熟，最终产生血小板。由于骨髓巨核细胞及其前体细胞数量稀少，对这些过程的研究受到阻碍。我们描述了一种用于扩增和纯化定向巨核细胞祖细胞的方法，并通过研究整个巨核细胞生成过程中Ets和GATA家族转录因子表达的变化来证明其用途。
开发了一种两步无血清方法。使用该方法分离的细胞通过流式细胞术分析表面标志物表达，并通过单细胞培养分析其分化能力。纯化的祖细胞被诱导分化，并通过流式细胞术分析其倍性，通过逆转录聚合酶链反应（RT-PCR）分析特定基因的表达。
纯化出一群Lin- c-kit+ CD45+ CD41+ CD31+ CD34low CD9low FcγRII/IIIlow Sca-1med/low定向巨核细胞祖细胞。这些细胞能够高效分化，实现高达128N的倍性。RNA分析表明，关键的巨核细胞相关基因表达预期增加。RT-PCR分析还显示，一系列Ets和GATA因子表达，它们各自的水平和表达模式差异很大。令人惊讶的是，我们发现GATA-6在晚期分化的巨核细胞中特异性表达，并有可能与Ets因子协同调节巨核细胞表达的基因。
纯化的原代巨核细胞祖细胞能够作为一个群体分化为完全成熟的巨核细胞。可获得的细胞数量以及分化过程中的同步性，便于分析巨核细胞生成中涉及的分子过程的动态变化。Ets和GATA家族转录因子的表达模式揭示了这些关键巨核细胞调节因子参与的复杂性。GATA-6表达的发现及其功能活性的证明提示了巨核细胞生成后期某些基因调控的新机制。
