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

角膜是眼睛前部的透明窗口，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，规则和光滑。白内障、角膜损伤和角膜疾病的存在是角膜失明的最大原因，全世界有2300万人患有角膜失明，这给患者和卫生保健资源增加了巨大负担。通常唯一的治疗选择是供体角膜的手术移植，这是一种50多年来一直没有改变的治疗选择。在欧洲，每年有超过4万名盲人等待角膜移植。这种短缺导致全球每年约有1000万患者未经治疗，150万新失明病例。由于眨眼以及身体和环境损害引起的正常细胞脱落，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）迁移到角膜上。如果这些LSC受损或患病，就会发生角膜缘干细胞缺乏症（LSCD），导致不适和视力下降。在一只眼睛患有LSCD的患者中，LSC可以从另一只健康的眼睛中取出并在实验室中生长，然后移植回患病的眼睛以恢复干细胞和视力。在过去的14年中，我们已经成功地使用这种技术治疗了33例LSCD患者。尽管取得了这一成功，但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著限制，并且它们迅速失去连续生长的能力。出于这个原因，用于移植的细胞是LSC和其他细胞类型的混合物，这些细胞类型可能因患者而异，也可能因研究组而异，这取决于用于扩增的技术。LSC的数量对于临床移植的成功至关重要;例如，移植了含有少于3% LSC的实验室扩增培养物的患者仅在11%的患者中成功移植，而那些超过3%的患者在76%的患者中成功移植。在移植前评估LSC的分数是至关重要的，因此可以避免不成功的移植。为了实现这一目标，我们需要更多地了解LSC本身。干细胞也在角膜的其他部分被发现，例如称为基质的中间部分，以及保持角膜水合的内皮。我们不知道这些层中的干细胞是否彼此行为相同，或者是否有几种类型以不同的方式对角膜损伤做出反应。允许单个细胞单独研究的技术的巨大进步使得人类细胞图谱的开发成为可能，该图谱目前专注于皮肤和血液等组织。在这个项目中，我们建议集中在三个不同的区域的成人尸体人角膜（中央，周边和角膜缘）已被证明是不同的细胞组成和密度。单细胞分析将使我们能够知道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个层中有多少细胞类型。由于我们的眼睛主要在出生前发育，因此我们还建议查看经母亲同意捐赠用于研究的流产标本样本。通过了解从发育到成年的细胞类型的复杂性，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我们如何提高临床移植的成功率。该项目的最终影响将是促进对世界上相当大一部分由角膜损伤和疾病引起的失明进行更安全和更有效的治疗。

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.