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，并适应于它们帮助形成的特定组织。例如，皮肤细胞不同于眼细胞，眼细胞又不同于血细胞。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，都是我们所说的分化细胞，因为它们已经适应了它们在组织中帮助形成的生物角色。但是，在胚胎发育过程中，我们的细胞没有这种组织特异性身份。事实上，胚胎发生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，一旦一个细胞“选择了它的路径”并分化成一种特定类型的细胞，它就开始了一个不可逆转的过程。但是，2012年，两位科学家（英国剑桥大学的约翰·戈登爵士和日本京都大学的山中伸弥教授）因他们的研究获得了诺贝尔生理学或医学奖。他们的研究表明，分化的成年细胞可以通过向细胞核中添加四种遗传因子来重新编程，这些因子现在被称为山中因子。这些重新编程的细胞变成了较早的、分化程度较低的细胞，能够分化成多种细胞类型。这种细胞被称为iPS细胞——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的合作者的发现，他们与我们以及附近京都大学的山中教授和他的团队合作。结果表明，某些类型的人类iPS细胞可以在实验室中生长并形成细胞多区，其中不同区域的细胞类似于不同眼组织的细胞；比如晶状体，视网膜，角膜。这一发现特别令人兴奋，因为最接近天然角膜上皮的细胞——即眼睛上支持泪膜的前一层细胞——可以移植到眼睛上，在那里它们保持功能。这项研究在使用人类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，我们将研究已经形成眼样组织的iPS细胞。为此，我们将使用强大的3D电子显微镜技术，可以在高分辨率和高放大倍率下对细胞进行成像。我们还将使用针对特定分子的抗体来观察iPS细胞衍生的眼状细胞是如何相互交流的，以及我们如何使用某些化学物质来调节眼状iPS细胞的形成来操纵这些交流途径。我们还从之前的角膜和软骨实验中获得专业知识，了解一些被称为蛋白聚糖的分子如何帮助干细胞在组织中的生态位区域或环境中保持干细胞。例如，在健康的角膜中，我们知道各种特殊类型的蛋白聚糖填充在角膜的一个区域，称为干细胞壁龛。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聚糖，并用酶对其进行化学修饰。修饰的蛋白聚糖将用于帮助培养iPS细胞衍生的角膜上皮结构，以了解哪种类型的蛋白聚糖或裂解的蛋白聚糖片段对保持细胞“干性”很重要。这将有助于提高人类iPS细胞衍生的角膜上皮细胞生产的潜力，用于未来的外科应用。最后，重要的是，我们将详细研究如何在我们的眼睛样多区iPS细胞可以用来复制晶状体细胞，这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.