Stem Cell Microvesicles: Potential Tools for Retinal Regeneration

描述(申请人提供)：低等脊椎动物的视网膜显示出显著的再生能力，而哺乳动物则失去了这种能力。从哺乳动物眼睛的睫状缘和视网膜分离出多能祖细胞，能够分化为各种类型的视网膜细胞。尽管存在这些祖细胞，但它们通常处于静止状态，无法再生受损的视网膜。这项提议将研究从小鼠胚胎干细胞释放的微囊重新激活小鼠视网膜前体细胞的能力。该项目的长期目标是发现重新激活人眼中静止的祖细胞群体的新方法，从而使受损视网膜的再生成为可能。 微囊是释放到细胞外环境中的质膜颗粒。最近，在体外培养的胚胎干细胞中发现了微囊泡。我们的初步结果表明，这些胚胎干细胞微囊含有RNA和蛋白质。最有趣的是，它们含有一类特殊的RNA分子，称为microRNAs，它是翻译的有效调节因素。微囊在细胞间通讯中可能以多种方式中的一种发挥作用。它们可以将microRNAs、mRNAs或蛋白质转移到细胞中。或者，它们可以通过微泡上发现的表面蛋白向细胞发出信号。 我们的第一个目标是表征小鼠胚胎干细胞微泡的RNA和蛋白质含量，以寻找可能改变干细胞编程的候选者。我们将使用微阵列分析和qRT-PCR来分析mRNA和microRNA，并使用质谱学、Western印迹分析和免疫细胞化学来分析蛋白质。我们还将在Aim I中探索这些微囊在体外将RNA或蛋白质直接转移到细胞的能力。我们的第二个目标是确定这些微泡是否能够激活在小鼠眼睛的睫状缘和视网膜中发现的静止的干细胞群体。我们将把微泡注射到小鼠的房水、玻璃体和视网膜下空间，并通过BrDU标记寻找促进干细胞增殖的因素。我们的最终目标是寻找房水和玻璃体中的内源性微囊，并使用微阵列分析、qRT-PCR、质谱学、Western印迹分析、免疫细胞化学和电子显微镜对其进行表征。这一建议中产生的信息不仅将使人们更好地理解外在mRNA、microRNAs和蛋白质在决定干细胞命运中所起的作用，而且还可能确定微泡是眼睛中新的内源性信号因子，并可能对干细胞生态位做出贡献。

DESCRIPTION (provided by applicant): The retina in lower vertebrates shows a remarkable regenerative ability that is lost in mammals. Multipotent progenitor cells, that are capable of differentiating into a variety of retinal cell types, have been isolated from the ciliary margin and retina of mammalian eyes. Despite their presence, these progenitor cells are normally quiescent and unable to regenerate damaged retina. This proposal will investigate the ability of microvesicles, released from mouse embryonic stem cells, to reactivate mouse retinal progenitor cells. The long term goal of this project is to discover novel ways of reactivating quiescent progenitor cell populations in the human eye, so that regeneration of damaged retina may be possible. Microvesicles are plasma-membrane particles that are released into the extracellular environment. Very recently, microvesicles have been reported from embryonic stem cells cultured in vitro. Our preliminary results show that these embryonic stem cell microvesicles contain RNA and protein. Most interestingly, they contain a specific class of RNA molecules called microRNAs, which are potent regulators of translation. Microvesicles may serve a role in intercellular communication in one of several manners. They may transfer microRNAs, mRNAs, or proteins to cells. Alternatively they can signal cells through surface proteins found on microvesicles. Our first aim is to characterize the RNA and protein contents of mouse embryonic stem cell microvesicles to look for candidates that might alter stem cell programming. We will use microarray analysis and qRT-PCR for mRNA and microRNA profiling, and mass spectrometry, Western blot analysis, and immunocytochemistry for protein profiling. We will also explore in Aim I the ability of these microvesicles to directly transfer RNA or protein to cells in vitro. Our second aim is to determine if these microvesicles can activate the quiescent stem cell population found in the ciliary margin and retina of mouse eyes. We will inject microvesicles into the aqueous, vitreous, and subretinal space of mice and look for increased proliferation of stem cells with BrDU- labeling. Our final aim is to look for endogenous microvesicles in the aqueous and vitreous and also to characterize them using microarray profiling, qRT-PCR, mass spectrometry, Western blot analysis, immunocytochemistry, and electron microscopy. The information generated in this proposal will not only lead to an increased understanding of the role that extrinsic mRNA, microRNAs, and proteins play in determining stem cell fate, but may also identify microvesicles as novel endogenous signaling factors in the eye, and possibly contributing to the stem cell niche.