Transplantable limbal cells from induced pluripotent stem cells

描述(申请人提供)：全球约有600-800万人患有角膜失明。角膜缘上皮干细胞缺乏症(LSCD)是其重要的临床原因之一。它可能是由于遗传性疾病、烧伤、感染和慢性炎症造成的，导致角膜瘢痕形成、血管形成和结膜形成，导致视力丧失。正常 LESC移植可提高视力。美国批准用于LSCD的自体或异体角膜缘移植仅有30-45%的移植物3-5年存活率。在其他国家，培养的LESC被移植，1-3年的成功率为76%。然而，这种方法也有缺陷，包括缺乏标准化和同种异体移植物存活率低，培养的LESC传代数量有限，以及常规使用小鼠3T3饲养层。到目前为止，治疗LSCD显然需要一种可再生和标准化的LESC来源。在这项建议中，我们采用了一种新的方法来解决这一重大的临床问题，使用诱导多能干细胞(IPSC)。与LESC不同，IPSC是不朽的，允许持续传播和银行业务。标准的IPSC派生和生长协议适用于GLP和GMP。我们的目标是通过产生IPSC并将其分化回LESC来获得用于治疗LSCD的可再生LESC源。我们首次提出使用LESC培养来获得IPSC，以便重新分化回角膜缘细胞。我们的策略是基于这样的假设，即IPSC通过保留亲本细胞表观遗传特征的机制可能促进了IPSC向LESC的分化。我们还建议使用自然生态位将IPSC分化为LESC，例如裸露的人器官培养的角膜或羊膜(HAM)。HAM被用作可移植的LESC的基质，但在LESC培养前处理和去除羊水细胞是一件繁琐的事情。我们开发了一种新的火腿去皮方法，支持了LESC和IPSC的可靠增长。在我们初步数据的支持下，这些新的方法可能会迅速推进LESC移植领域。我们推测，从角膜缘细胞分化为IPSC将通过表观记忆机制促进其向LESC的再分化。这种再分化可能被LESC胞外生态位如角膜缘基底膜或HAM结合生长因子所增强。我们的目标是获得可靠的、可再生的移植级LESC源。具体目的1.从体外培养的人角膜缘干细胞中分离培养出IPSC，并对其进行鉴定。具体目标2.利用可溶性因子和细胞外生态位(火腿和裸露的人角膜或角膜缘)相结合的方法，定向将角膜缘来源的IPSC重新分化为LESC。具体目的3.实现IPSC来源的角膜缘细胞移植到器官培养的人角膜缘剥离区，恢复正常干细胞室的结构和功能。与健康相关：我们的新策略是获得LESC来源的IPSC，并使用天然的胞外利基将它们分化回LESC，这将为LSCD提供可靠和可再生的LESC来源。

DESCRIPTION (provided by applicant): Corneal blindness affects about 6-8 million people worldwide. One of its clinically important causes is limbal epithelial stem cell (LESC) deficiency (LSCD). It may be due to genetic diseases, burns, infections, and chronic inflammation and results in corneal scarring, vascularization and conjunctivalization leading to vision loss. Normal LESC transplantation can improve vision. Keratolimbal auto- or allografts approved in U.S. for LSCD have only 30-45% 3-5-year graft survival. In other countries, cultured LESC are transplanted, with a 1-3 years success rate of 76%. However, this procedure also has drawbacks including lack of standardization and low allograft survival, limited number of LESC passages in culture, and routine use of mouse 3T3 feeder layer. To date, there is a clear need for a renewable and standardized source of LESC for treating LSCD. In this proposal, we take a novel approach to this significant clinical problem using induced pluripotent stem cells (iPSC). Unlike LESC, iPSC are immortal, allowing continual propagation and banking. Standard iPSC derivation and growth protocols are amenable to GLP and GMP. We aim at obtaining a renewable LESC source for treating LSCD by generating iPSC and differentiating them back to LESC. We propose for the first time to use LESC cultures to obtain iPSC for redifferentiation back to limbal cells. Our strategy is based on the hypothesis that iPSC differentiation back to LESC may be facilitated by the mechanism of retention by iPSC of parent cell epigenetic signatures. We also propose to use a natural niche for iPSC differentiation to LESC, such as denuded human organ-cultured corneas or amniotic membrane (HAM). HAM is used as matrix for transplantable LESC, but it is tedious to work with and to remove amniotic cells before LESC culture. We developed a novel method for denuding HAM, supporting a reliable growth of LESC and iPSC. These novel approaches backed by our preliminary data may quickly advance the field of LESC transplantation. We hypothesize that making iPSC from limbal cells will facilitate their redifferentiation to LESC via epigenetic memory mechanism. This redifferentiation may be enhanced by LESC extracellular niche such as limbal basement membrane or HAM, combined with growth factors. We aim at obtaining reliable and renewable transplant-grade LESC source. Specific Aim 1. To generate and characterize iPSC from cultured human donor limbal stem cells. Specific Aim 2. To directionally differentiate limbal-derived iPSC back into LESC using a combination of soluble factors and extracellular niche (HAM and denuded human corneas or limbal rims). Specific Aim 3. To achieve transplantation of iPSC-derived limbal cells to the denuded limbal zone of organ-cultured human corneas, to restore normal stem cell compartment structure and function. Health relevance: our novel strategy of obtaining LESC-derived iPSC and differentiating them back to LESC using natural extracellular niche will yield a reliable and renewable source of transplantable LESC for LSCD.