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6-80万人。其临床上重要的原因之一是缘长上皮干细胞（LESC）缺陷（LSCD）。这可能是由于遗传疾病，烧伤，感染和慢性炎症，导致角膜疤痕，血管形成和结膜化导致视力丧失。普通的 LESC移植可以改善视力。在美国批准的LSCD批准的角膜膜自动或同种异体移植物只有30 - 45％3 - 5年的移植物存活率。在其他国家，培养的LESC被移植，成功率为76％。但是，此过程还具有缺点，包括缺乏标准化和低不同的同种异体存活率，培养物中的LESC段落数量有限以及常规使用小鼠3T3馈线层。迄今为止，显然需要可再生和标准化的LESC来处理LSCD。在此提案中，我们使用诱导的多能干细胞（IPSC）采取了一种新的方法来解决这一重大临床问题。与LESC不同，IPSC是不朽的，可以持续传播和银行业务。标准的IPSC推导和增长方案适合GLP和GMP。我们旨在通过生成IPSC并将其区分回LESC来获得可再生的LESC来源来治疗LSCD。我们首次建议使用LESC培养物获得IPSC，以将其重新分化回边缘细胞。我们的策略是基于以下假设：IPSC分化可以通过母细胞表观遗传学特征的IPSC保留机理来促进与LESC的分化。我们还建议将天然利基用于IPSC与LESC的分化，例如裸露的人体培养角膜或羊膜膜（HAM）。 HAM用作可移植LESC的矩阵，但在LESC培养之前使用并去除羊膜细胞是很乏味的。我们开发了一种新颖的方法来剥去火腿，支持LESC和IPSC的可靠增长。这些以我们的初步数据为支持的新型方法可能会迅速推动LESC移植的领域。我们假设从边缘细胞中制造IPSC将通过表观遗传记忆机制促进其将其重新分化为LESC。 LESC细胞外壁细分市场可以增强这种重新分化，例如缘地下膜或HAM，结合生长因子。我们旨在获得可靠的可再生移植级LESC来源。特定目的1。从培养的人类管状干细胞中产生和表征IPSC。特定的目标2。使用可溶性因子和细胞外壁细胞生态位（HAM和HAM和裸露的人角膜或边缘边缘）的组合将缘源性的IPSC区分为LESC。具体目的3。要实现IPSC衍生的边缘细胞移植到器官培养的人角膜的裸缘区域，以恢复正常的干细胞室结构和功能。健康相关性：我们获得LESC衍生的IPSC并使用天然细胞外壁基将其返回LESC的新型策略将产生LSCD的可靠且可再生的可移植LESC来源。