Transplantable limbal cells from induced pluripotent stem cells

来自诱导多能干细胞的可移植角膜缘细胞

• 批准号: 8503490
• 负责人: Alexander V Ljubimov
• 金额: $ 41.71万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8503490
• 关键词: Address; Affect; Allografting; Aniridia; Autologous Transplantation; Back; Basement membrane; Bilateral; Blindness; Burn injury; Cell Culture Techniques; Cell Transplantation; Cell Transplants; Cells; Chemicals; Chronic; Cicatrix; Clinical; Cornea; Country; Data; Derivation procedure; Differentiation Antigens; Engineering; Ensure; Epigenetic Process; Epithelial; Epithelial Cells; Eye diseases; Fibroblasts; Future; Generations; Goals; Graft Survival; Growth; Growth Factor; Health; Hereditary Disease; Homologous Transplantation; Human; In Vitro; Infection; Inflammation; Karyotype; Keratoplasty; Lead; Membrane; Memory; Methods; Monitor; Mus; Nude Mice; Ocular cicatricial pemphigoid; Organ Culture Techniques; Oryctolagus cuniculus; Outcome; Parents; Patients; Plasmids; Procedures; Process; Production; Protocols documentation; Rattus; Research; Skin; Source; Standardization; Stem cell transplant; Stem cells; Stevens-Johnson Syndrome; Structure; Telomerase; Time; Tissues; Transplantation; Tumorigenicity; Vascularization; Vision; Visual; Work; Wound Healing; Xenobiotics; base; corneal epithelial stem cells; corneal epithelium; corneal scar; extracellular; improved; in vivo; induced pluripotent stem cell; inhibitor/antagonist; limbal; microbial; novel; novel strategies; public health relevance; stem cell differentiation; stem cell niche; stemness; success

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.

描述（由申请人提供）：全世界约有 6-800 万人患有角膜失明。其临床重要原因之一是角膜缘上皮干细胞（LESC）缺乏（LSCD）。它可能是由于遗传性疾病、烧伤、感染和慢性炎症导致的，导致角膜疤痕、血管化和结膜化，从而导致视力丧失。普通的 LESC移植可以改善视力。美国批准用于治疗 LSCD 的自体角膜缘或同种异体角膜缘移植物的 3-5 年移植物存活率仅为 30-45%。在国外，培养的LESC进行移植，1-3年的成功率为76%。然而，该程序也有缺点，包括缺乏标准化和同种异体移植存活率低、培养中 LESC 传代数量有限以及小鼠 3T3 饲养层的常规使用。迄今为止，显然需要一种可再生和标准化的 LESC 来源来治疗 LSCD。在这项提案中，我们采用诱导多能干细胞（iPSC）来解决这一重大临床问题的新方法。与 LESC 不同，iPSC 是不朽的，可以持续繁殖和储存。标准 iPSC 衍生和生长方案符合 GLP 和 GMP。我们的目标是通过生成 iPSC 并将其分化回 LESC，获得用于治疗 LSCD 的可再生 LESC 来源。我们首次提出使用LESC培养物来获得iPSC以再分化回角膜缘细胞。我们的策略基于以下假设：iPSC 保留亲代细胞表观遗传特征的机制可能会促进 iPSC 分化回 LESC。我们还建议使用 iPSC 分化为 LESC 的自然生态位，例如裸露的人体器官培养角膜或羊膜 (HAM)。 HAM 被用作可移植 LESC 的基质，但在 LESC 培养之前处理和去除羊膜细胞非常繁琐。我们开发了一种剥除 HAM 的新方法，支持 LESC 和 iPSC 的可靠生长。这些由我们的初步数据支持的新方法可能会迅速推进 LESC 移植领域的发展。我们假设从角膜缘细胞制造 iPSC 将通过表观遗传记忆机制促进其再分化为 LESC。这种再分化可能通过 LESC 细胞外生态位（例如角膜缘基底膜或 HAM）与生长因子相结合来增强。我们的目标是获得可靠且可再生的移植级 LESC 来源。具体目标 1. 从培养的人类供体角膜缘干细胞中产生 iPSC 并对其进行表征。具体目标 2. 使用可溶性因子和细胞外生态位（HAM 和裸露的人角膜或角膜缘边缘）的组合，将角膜缘来源的 iPSC 定向分化回 LESC。具体目标3.实现将iPSC来源的角膜缘细胞移植到器官培养人角膜的裸露角膜缘区，恢复正常的干细胞区室结构和功能。健康相关性：我们获得 LESC 衍生的 iPSC 并利用天然细胞外生态位将其分化回 LESC 的新策略将为 LSCD 产生可靠且可再生的可移植 LESC 来源。