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）：角膜失明影响全球约600 - 800万人。其临床上重要的原因之一是角膜缘上皮干细胞（LESC）缺乏症（LSCD）。它可能是由于遗传性疾病、烧伤、感染和慢性炎症，导致角膜瘢痕形成、血管形成和结膜形成，从而导致视力丧失。正常 LESC移植可改善视力。在美国批准用于LSCD的角膜缘自体或同种异体移植物的3-5年移植物存活率仅为30-45%。在其他国家，移植培养的LESC，1-3年成功率为76%。然而，这种方法也有缺点，包括缺乏标准化和低同种异体移植物存活率，培养中LESC传代次数有限，以及常规使用小鼠3 T3饲养层。迄今为止，显然需要用于治疗LSCD的可再生和标准化的LESC来源。在这个提议中，我们采用了一种新的方法来解决这个重要的临床问题，使用诱导多能干细胞（iPSC）。与LESC不同，iPSC是不朽的，允许持续的传播和存储。标准iPSC衍生和生长方案符合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培养物获得iPSC以再分化回角膜缘细胞。我们的策略是基于这样的假设，即iPSC分化回LESC可以通过iPSC保留亲本细胞表观遗传特征的机制来促进。我们还建议使用iPSC分化为LESC的自然生态位，例如裸露的人类器官培养的角膜或羊膜（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是繁琐的。我们开发了一种剥脱HAM的新方法，支持LESC和iPSC的可靠生长。这些新的方法得到了我们初步数据的支持，可能会迅速推进LESC移植领域。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它们再分化为LESC。这种再分化可以通过LESC细胞外小生境如角膜缘基底膜或HAM与生长因子组合来增强。我们的目标是获得可靠和可再生的移植级LESC来源。具体目标1。从培养的人供体角膜缘干细胞产生并表征iPSC。具体目标2。使用可溶性因子和细胞外小生境（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。目的：实现iPSC衍生的角膜缘细胞移植到器官培养的人角膜缘裸露区，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并使用天然细胞外小生境将它们分化回LESC的新策略将产生用于LSCD的可移植LESC的可靠和可再生来源。