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，皮肤细胞与眼细胞不同，眼细胞与血细胞不同。当然，每种细胞类型都有自己独特的作用。成熟细胞，无论它们在哪个组织中，都是我们所知道的分化细胞，因为它们已经适应了它们在组织中的生物学作用。但是，在胚胎发育过程中，我们的细胞不具有这种组织特异性。事实上，胚胎发生的早期细胞可以沿着不同的发育途径发展，成为不同的细胞类型。这些早期未分化的细胞被称为干细胞。长期以来，人们认为细胞一旦"选择了自己的道路"，分化成某种特定类型的细胞，就开始了一个不可逆的过程。但是，在2012年，两名科学家获得了诺贝尔生理学或医学奖（英国剑桥大学的约翰古尔登爵士和日本京都大学的山中伸弥教授），因为他们的研究表明，通过将四种遗传因子添加到细胞核中，可以对分化的成年细胞进行重新编程-这些因子现在被称为山中因子。这些重编程的细胞成为较早的、分化程度较低的细胞，能够分化成许多细胞类型。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坂大学的合作者对这一提议的发现，与我们以及附近京都大学的山中伸弥教授和他的团队合作。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细胞多区，其中不同区域的细胞类似于不同眼组织的细胞;例如，透镜，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层细胞-可以移植到眼睛上，在那里它们保持功能。这项研究对人类iPS细胞用于治疗眼部疾病具有强大的未来潜力。在我们计划的研究中，我们将研究已形成眼样组织的iPS细胞。为此，我们将使用强大的3D电子显微镜技术，可以以高分辨率和高放大率对细胞进行成像。我们还将使用针对特定分子的抗体来观察iPS细胞衍生的眼样细胞如何相互通信，以及我们如何使用某些化学物质来操纵这些通信途径以调节眼样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聚糖的分子如何帮助干细胞保持在组织中的利基区域或环境中。例如，在健康的角膜中，我们知道各种特殊类型的蛋白聚糖聚集在角膜的一个称为干细胞龛的区域。基本上，这是组织的一个区域，有助于支持和维持干细胞。我们现在将从角膜中提取和纯化蛋白聚糖，并使用酶对其进行化学修饰。然后，修饰的蛋白聚糖将用于帮助生长iPS细胞衍生的角膜上皮构建体，以了解什么类型的蛋白聚糖或切割的蛋白聚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iPS细胞衍生的角膜上皮细胞生产的潜力，用于未来的手术用途。最后，重要的是，我们将详细研究如何在我们的眼睛样多区的iPS细胞可以用来复制透镜细胞，这具有令人兴奋的潜力，帮助再生的晶状体在人类的眼睛。

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.