Identify mechanisms of dedifferentiation during limbal stem cell niche reconstruction.

项目摘要 角膜缘干细胞（LSC）在体内环境稳定和伤口愈合中起着重要作用。 对LSC的损伤和与LSC缺陷（LSCD）相关的其他病理导致角膜损伤。 新生血管形成、角膜混浊和视力丧失。目前的治疗模式是基于 将LSC移植到患者体内以恢复LSC生态位。在这方面作出了巨大努力， 在过去的几年里，我们的目标是确定不同程度的LSC移植来源， 成功然而，我们最近的工作表明，LSC的移植可能不是最好的治疗方法。 LSCD的唯一有效治疗范例。我们发现， 失去干细胞标记物K15的人能够去分化并重新填充干细胞龛。如果 该过程可以被控制和操纵，它可以提供替代治疗， 避免复杂的干细胞移植。我们已经证明了利基市场的重要性 对于去分化的过程，证明了细胞的去分化和再增殖， 小生境需要保持在小生境中的细胞与角膜上皮细胞之间的通信。 在这里，我们将使用角膜器官培养的尖端长期多日成像来研究 在龛和上皮细胞之间的通信的两侧。在目标#1中，我们将采取 药理学方法，筛选99种不同的化合物，并获得长时程 在不同药物存在下LSC生态位恢复的电影。含量高 分析将提供关于生态位所使用的不同信号通路的关键信息， 募集并诱导上皮细胞的去分化。在目标#2中，我们将专注于接收器 通信的一面，并分析用于重新填充利基的细胞的来源。使用 先进的光片显微镜方法，我们将跟踪单个细胞，并发现 能够去分化的细胞的身份。单细胞追踪将显示 去分化的能力是普遍存在的，或者是否只有一小部分细胞维持去分化的能力。 去分化的能力。这些目标的成功实现将为以下方面提供重要见解： LSC生态位恢复的生理过程，并将铺平道路， LSCD的新疗法

Project Summary The corneal limbal stem cells (LSC) play a vital function in homeostasis and wound healing. Damage to the LSC and other pathologies associated LSC deficiency (LSCD) cause cornea neovascularization, corneal opacification, and vision loss. Current treatment paradigm is based on the transplantation of LSC into patients to restore the LSC niche. Substantial effort over the last few years aims at identifying sources of LSC for transplantation with varying degree of success. However, our recent work indicates that transplantation of LSC might not be the the only useful treatment paradigm for LSCD. We showed that committed epithelial cells that have lost the stem cell marker K15 are able to dedifferentiate and repopulate the stem cell niche. If this process can be controlled and manipulated it could provide alternative treatment that can avoid the need for complex stem cell transplantation. We have shown that the niche is important for the process of dedifferentiation demonstrating that dedifferentiation and repopulation of the niche require communication between cells that remain in the niche and corneal epithelial cells. Here we will use cutting edge long term multi-day imaging of corneal organ culture to investigate both sides of the communication between the niche and epithelial cells. In Aim #1 we will take a pharmacological approach and screen 99 different compounds and acquire long timelapse movies of the restoration of the LSC niche in the presence of different drugs. The high content analysis will provide key information on the different signaling pathways used by the niche to recruit and induce the dedifferentiation of epithelial cells. In Aim #2 we will focus on the receiver side of the communication and analyze the source of cells used to repopulate the niche. Using advanced light sheet microscopy approaches we will track individual cells and discover the identity of the cells that are capable of dedifferentiation. Single cell tracking will show whether the ability to dedifferentiate is ubiquitous or whether only a small subset of cells maintain the ability to dedifferentiate. The successful completion of these aims will provide key insights into the physiological process of LSC niche recovery and will pave the way to the development of new treatments to LSCD.