A Pluripotent Stem Cell Reagent for Corneal Regeneration

描述（由申请人提供）：在美国，每年约有40，000人因角膜混浊需要角膜移植术，全世界多达1000万人患有双侧角膜失明。 大多数受影响的人没有接受治疗，因为组织无法移植。 获得供体角膜的困难刺激了对角膜再生的替代方法的研究，包括组织的生物工程和直接基于细胞的治疗。 基于细胞的治疗的潜力是由同种异体组织耐受良好且移植后很少排斥的事实促进的。 最近的进展，在设备和细胞为基础的治疗方法，使用动物模型显示出很大的前景，为生物工程解决方案的病理角膜基质和内皮。 然而，细胞技术发展的一个主要障碍是细胞来源。 在角膜基质的角膜细胞和角膜内皮细胞以及成体干细胞的情况下，在培养中扩增的潜力是有限的，并且细胞的性质根据个体供体和制备方法而变化，在一致性和质量控制方面提出了挑战。 在这个项目中，我们将开发一个干细胞为基础的试剂，角膜工程使用人类胚胎干细胞（hESC），利用两个最近的发展hESC研究。 我们的实验室已经证明了人胚胎干细胞转化为角膜祖细胞。 我们发现角膜祖细胞很容易分化为人角膜细胞。 与此同时，其他研究人员描述了一种新的方法，从hESC中产生基本上无限数量的同质神经嵴细胞。 我们建议联合收割机这些技术，以产生一个标准化的人胚胎干细胞衍生的干细胞试剂的潜力，直接分化为角膜细胞和角膜内皮细胞。 该项目将：1）定义一个标准的操作程序，从hESC产生角膜祖细胞。 (2)证明hESC衍生的角膜祖细胞的均一性、稳定性、安全性和冷冻储存。 (3)开发两种基于hESC的荧光报告细胞系，用于评估来源于hESC的角膜基质细胞和内皮细胞的质量和均一性。 (4)使用体外功能测定证明hESC衍生的角膜细胞的功能。 该项目应用新的技术发展，以解决一个重要的障碍，角膜细胞为基础的再生医学。 该项目的成功完成将提供一种定义明确、安全、无限量的细胞基试剂，用于改善角膜失明的生物工程和细胞治疗方法。

DESCRIPTION (provided by applicant): About 40,000 individuals annually require keratoplasty for corneal opacity in the USA and up to 10 million people world-wide suffer bilateral corneal blindness. Most affected individuals do not receive treatment because tissue is not available to transplant. Difficulty in obtaining donor corneas has spurred research into alternative approaches to corneal regeneration including bioengineering of tissues and direct cell-based therapy. The potential for cellular based therapy is facilitated by the fact that allogenic tissues are well tolerated and seldom rejected after transplantation. Recent advances in devices and in cell-based therapeutic approaches using animal models show great promise for bioengineering solutions to pathologies of the corneal stroma and endothelium. A major barrier to the advancement of cell-based technologies, however, is a source for cells. In the case of keratocytes of corneal stroma and corneal endothelial cells, as well as of adult stem cells, the potential for expansion in culture is limited, and the properties of the cells vary according to individual donors and preparation methods, presenting a challenge in terms of consistency and quality control. In this project we will develop a stem cell-based reagent for corneal engineering using human embryonic stem cells (hESC), taking advantage of two recent developments in hESC research. Our lab has demonstrated conversion of hESC into corneal progenitor cells. We showed that the corneal progenitor cells readily differentiate to human keratocytes. Simultaneously, other researchers described a new method to produce essentially limitless numbers of homogeneous neural crest cells from hESC. We propose to combine these techniques to generate a standardized hESC-derived stem cell reagent with the potential to differentiate directly into corneal keratocytes and corneal endothelial cells. The project will: 1) Define a standard operating procedure to generate corneal progenitor cells from hESC. (2) Demonstrate homogeneity, stability, safety, and cryo-storage of the hESC-derived corneal progenitor cells. (3) Develop two hESC-based fluorescent reporter cell lines used to assess the quality and homogeneity of the corneal keratocytes and endothelial cells derived from the hESC. (4) Demonstrate functionality of the hESC-derived corneal cells using in vitro functional assays. This project applies novel technical developments to address an important barrier to corneal cell- based regenerative medicine. Successful completion of this project will provide a well-defined, safe, cell-based reagent available in unlimited quantities for use in bioengineering and cytotherapeutic approaches for the amelioration of corneal blindness.