The Development of Eye Tissues via Human Induced Pluripotent Stem (iPS) Cells.

存在于我们身体组织中的细胞具有特定的功能，并适应它们帮助形成的特定组织。例如，皮肤细胞不同于眼睛细胞，而眼睛细胞不同于血细胞。当然，每种细胞类型都有其独特的作用。成熟细胞，无论它们在哪个组织中，都是我们所知的分化细胞，因为它们已经适应了它们在帮助形成的组织中的生物学角色。但是，在胚胎发育过程中，我们的细胞没有这种组织特异性。事实上，胚胎发育的早期细胞可以沿着不同的发育途径，变成不同的细胞类型。这些早期未分化的细胞被称为干细胞。很长一段时间以来，人们都认为，一旦细胞“选择了自己的道路”并分化成特定类型的细胞，它就走上了一个不可逆转的过程。但在2012年，两位科学家(英国剑桥大学的约翰·格登爵士和日本京都大学的山中伸弥教授)因他们的研究而获得诺贝尔生理学或医学奖。他们的研究表明，通过在细胞核中添加四种遗传因素，分化的成年细胞可以重新编程--这些因素现在被称为山中因素。这些重新编程的细胞成为较早、分化较少的细胞，能够分化为多种类型的细胞。这种细胞被称为诱导多能干细胞。我们新计划的研究是基于我们在日本大阪大学与我们以及附近京都大学的山中教授和他的团队在这一提议上的合作者所做的一项发现。它表明，某些类型的人类iPS细胞可以在实验室中生长并形成一个细胞多区，其中不同区域的细胞类似于不同眼睛组织的细胞，例如晶状体、视网膜、角膜。这一发现特别令人兴奋，因为与自然角膜上皮最相似的细胞--即眼睛上支持泪膜的前层细胞--可以移植到眼睛上，在那里它们仍然具有功能。这项研究在利用人类iPS细胞治疗眼病方面具有很大的潜力。在我们计划的研究中，我们将研究iPS细胞已经形成眼样组织的情况。为此，我们将使用强大的3D电子显微镜技术，以高分辨率和高倍率对细胞进行成像。我们还将使用针对特定分子的抗体来了解iPS细胞衍生的类眼睛细胞是如何相互通信的，以及我们如何使用某些化学物质来操纵这些通信途径来调节类眼睛iPS细胞的形成。我们还从之前的角膜和软骨实验中获得了专业知识，以了解一些称为蛋白多糖的分子是如何帮助干细胞保持干细胞留在组织中的利基区域或环境中的。例如，在健康的角膜中，我们知道各种特殊类型的蛋白多糖存在于角膜的一个被称为干细胞巢的区域。基本上，这是一个帮助支持和维持干细胞的组织区域。我们现在将从角膜中提取和纯化蛋白多糖，并使用酶对其进行化学修饰。然后，修饰后的蛋白多糖将被用于帮助培养iPS细胞来源的角膜上皮结构，以了解哪些类型的蛋白多糖或切割的蛋白多糖片段对于保持细胞的“干性”是重要的。这将有助于提高人类诱导性多能干细胞来源的角膜上皮细胞的生产潜力，供未来手术使用。最后，也是重要的一点，我们将详细研究如何使用我们眼睛状多区的iPS细胞来复制晶状体细胞，这具有帮助人眼晶状体再生的令人兴奋的潜力。

The cells that exist in the tissues of our body have specific functions and are adapted to suit the particular tissue that they help form. Skin cells, for example, are different to eye cells, which are different to blood cells. And, of course, each cell type has its own distinctive role. Mature cells, whichever tissue they are in, are what we know as differentiated cells, because they have become adapted to their biological role in the tissue in which they help form. But, during development in the embryo our cells do not have this tissue-specific identity. Indeed, the early cells in embryogenesis can go down different developmental pathways and become different cell types. These early undifferentiated cells are known as stem cells.For a long time, it was accepted that once a cell had "chosen its path" and differentiated into a particular type of cell, it had embarked on an irreversible process. But, in 2012 two scientists were awarded the Nobel Prize in Physiology or Medicine (Sir John Gurdon (Cambridge University, UK) and Prof Shinya Yamanaka (Kyoto University, Japan)) for their research, which showed that differentiated adult cells could be reprogrammed via a manipulation using four genetic factors added to a cell's nucleus - factors that are now known as Yamanaka factors. These reprogrammed cells became an earlier, less differentiated cell, capable of differentiation into a number of cell types. Such cells are called iPS cells -- induced pluripotent stem cells.Our new planned research is based on a discovery made by our collaborators on this proposal in Osaka University, Japan, working with us and with Prof Yamanaka and his team in nearby Kyoto University. It showed that some types of human iPS cells can grow in the laboratory and form a cellular multi-zone, in which cells in different areas resemble cells of different eye tissues; lens, retina, cornea, for example. This discovery is particularly exciting because the cells that most closely resemble a natural corneal epithelium - i.e. the front layer of cells on the eye that support the tear film - can be transplanted onto the eye where they remain functional. This research has a strong future potential for the use of human iPS cells for the treatment of eye disease.In our planned study we will investigate the iPS cells that have formed into eye-like tissue. To do so, we will use powerful 3D electron microscopy technologies that can image the cells at high resolution and at high magnification. We will also use antibodies to specific molecules to see how the iPS cell-derived eye-like cells are communicating with one another, and how we can manipulate these communication pathways using certain chemicals to modulate the formation of eye-like iPS cells. We also have expertise from our previous experiments on cornea and cartilage in understanding how some molecules, called proteoglycans, help keep stem cells to stay stem cells in their niche areas or environments in tissues. In the healthy cornea, for example, we know that various special types of proteoglycans populate an area of the cornea known as the stem cell niche. Basically, this is a region of a tissue that helps support and maintain the stem cells. We will now extract and purify proteoglycans from cornea and chemically modify them using enzymes. The modified proteoglycans will then be used to help grow iPS cell-derived corneal epithelial constructs to understand what types of proteoglycans or cleaved proteoglycan fragments are important to retain cellular "stemness". This will help improve the potential for human iPS cell-derived corneal epithelial cell production for future surgical use. Finally, and importantly, we will examine in detail how iPS cells in our eye-like multi-zone can be used to replicate lens cells, which has the exciting potential of helping the regeneration of lenses in the human eye.