Understanding the molecular and cellular complexity of human cornea through single cell analyses

通过单细胞分析了解人类角膜的分子和细胞复杂性

基本信息

批准号：
MR/S035826/1
负责人：
Majlinda Lako
金额：
$ 58.46万
依托单位：
Newcastle University
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FS035826%2F1
关键词：
Understanding molecular cellular complexity human

项目摘要

The cornea is the clear window at the front of the eye that allows light to enter and be focused on the back of the eye. To see a clear image, the cornea needs to be transparent, regular and smooth. Presence of cataracts, corneal damage and corneal diseases are the largest cause of corneal blindness which accounts for 23 million people worldwide adding a huge burden to patients and health care resources. Often the only treatment option is surgical transplantation of donor cornea, a therapeutic option that has been unchanged for more than 50 years. In Europe, over 40,000 blind people are waiting for corneal transplant every year. This shortage results in about 10 million untreated patients globally and 1.5 million new cases of blindness annually.The outer layer of the cornea (the epithelium) is continuously replaced due to normal cell shedding in response to blinking and physical and environmental damage. Limbal stem cells (LSCs) endlessly produce new epithelial cells in the cornea and prevent conjunctival epithelium (a covering of the white part of the eye) from migrating over the cornea. If these LSC are damaged or diseased, a condition called limbal stem cell deficiency (LSCD) occurs, causing discomfort and reduced vision. In patients with LSCD in one eye, LSCs can be removed from their other healthy eye and grown in a laboratory and then transplanted back into the diseased eye to restore the stem cells and their vision. Using this technique, we have been able to successfully treat 33 patients with LSCD during the last 14 years. Despite this success, we do not know yet how to identify and purify individual LSCs. Moreover, the growth of LSCs under laboratory conditions is significantly limited and they rapidly lose their ability to grow continuously. For this reason, cells used for transplantation are a mixture of LSCs and other cell types which can vary from patient to patient and from one research group to another depending on the technique used for their expansion. The number of LSCs is crucially important for the success of clinical transplantation; for example patients transplanted with laboratory expanded cultures which contain less than 3% of LSCs had a successful transplantation in only 11% of the patients, while those with more than 3% had a successful transplant in 76% of the patients. It is essential to assess the fraction of LSCs before transplantation so unsuccessful transplants can be avoided. To achieve this, we need to know more about LSCs themselves.Stem cells have also been found in other parts of the cornea, for example the middle part called stroma, and the endothelium which keeps the cornea hydrated. We don't know if stem cells in each of these layers behave the same way as each other or whether there are several types which respond in different ways to corneal damage. Great advances in technologies that allow single cells to be studied individually have enabled development of the Human Cell Atlas, which is currently focusing on tissues such as skin and blood. In this project, we propose to focus on three different regions of adult cadaveric human cornea (central, peripheral and limbal) which have been shown to differ in cell composition and density. The single cell analysis will enable us to know which genes are expressed in every cell and why and to understand how many cell types are in every region and every layer. Since our eyes develop mostly before we are born, we also propose to look at samples from aborted specimens which are donated for research with mother's consent. By understanding the complexity of cell types from development to adulthood, we will better understand how cornea is formed, how stem cells maintain corneal homeostasis and how we can increase the success of the clinical transplantations. The ultimate impact of this project will be to contribute towards safer and more efficacious treatments of a significant proportion of world blindness caused by corneal damage and disease.

角膜是眼睛前面的透明窗口，允许光线进入并聚焦在眼睛后部。为了看到清晰的图像，角膜需要透明、规则且光滑。白内障、角膜损伤和角膜疾病是导致角膜失明的最大原因，全世界有2300万人患有角膜失明，给患者和医疗资源增加了巨大的负担。通常唯一的治疗选择是手术移植供体角膜，这种治疗选择50多年来一直没有改变。在欧洲，每年有超过 40,000 名盲人等待角膜移植。这种短缺导致全球约有 1000 万未经治疗的患者，每年新增 150 万例失明病例。由于眨眼以及物理和环境损伤导致的正常细胞脱落，角膜外层（上皮）不断被替换。角膜缘干细胞（LSC）在角膜中不断产生新的上皮细胞，并防止结膜上皮（眼睛白色部分的覆盖物）在角膜上迁移。如果这些 LSC 受损或患病，就会出现一种称为角膜缘干细胞缺乏症 (LSCD) 的病症，导致不适和视力下降。对于一只眼睛患有 LSCD 的患者，可以从另一只健康的眼睛中取出 LSC，在实验室中培养，然后移植回患病的眼睛中，以恢复干细胞和视力。在过去 14 年里，我们利用这项技术成功治疗了 33 名 LSCD 患者。尽管取得了这一成功，我们还不知道如何识别和纯化单个 LSC。此外，LSC在实验室条件下的生长受到显着限制，并且它们很快失去持续生长的能力。因此，用于移植的细胞是 LSC 和其他细胞类型的混合物，根据用于扩增的技术，这些细胞可能因患者和一个研究组的不同而异。 LSCs的数量对于临床移植的成功至关重要；例如，接受 LSC 含量低于 3% 的实验室扩增培养物移植的患者中，仅有 11% 的患者移植成功，而 LSC 含量超过 3% 的患者中，76% 的患者移植成功。在移植前评估 LSC 的比例至关重要，以避免移植不成功。为了实现这一目标，我们需要更多地了解LSC本身。在角膜的其他部分也发现了干细胞，例如称为基质的中间部分，以及保持角膜水分的内皮细胞。我们不知道每一层中的干细胞是否具有相同的行为方式，或者是否有几种类型以不同的方式对角膜损伤做出反应。允许对单个细胞进行单独研究的技术取得了巨大进步，从而促进了人类细胞图谱的开发，该图谱目前专注于皮肤和血液等组织。在这个项目中，我们建议重点关注成人尸体角膜的三个不同区域（中央、周边和角膜缘），这些区域的细胞组成和密度有所不同。单细胞分析将使我们能够了解哪些基因在每个细胞中表达以及为什么表达，并了解每个区域和每一层有多少种细胞类型。由于我们的眼睛主要在出生前发育，因此我们还建议查看来自流产标本的样本，这些样本是在母亲同意的情况下捐赠用于研究的。通过了解细胞类型从发育到成年的复杂性，我们将更好地了解角膜是如何形成的、干细胞如何维持角膜稳态以及如何提高临床移植的成功率。该项目的最终影响将是为更安全、更有效地治疗世界上很大一部分由角膜损伤和疾病引起的失明做出贡献。