Ex Vivo Expansion of Human Limbal Stem Cells for Transplantation

描述（由申请人提供）：自体角膜上皮干细胞/祖细胞（CESCs）在组织培养中扩增的移植成功地恢复了视力，并彻底改变了角膜缘干细胞缺乏症（LSCD）的治疗方法。LSCD是许多常见角膜疾病中严重视力丧失和失明的主要原因，无论是原发性还是继发性。培养过程中，干细胞/祖细胞群体的扩增效率越高，移植物的长期存活率越高。最有效的扩增方法需要小鼠3T3饲养细胞，这些细胞是用小牛血清培养的，是污染扩增细胞的小鼠RNA的来源。这种由动物产品造成的交叉污染构成了相当大的潜在健康危害，因此这种培养方法不太可能被美国食品和药物管理局批准用于人类。为了获得可接受的临床结果，需要新的细胞工程方法，在无外源药物的条件下实现相同或更好的扩展CESCs的效率。我的实验室的长期目标是阐明控制CESC自我更新和分化的调节因素，并开发针对LSCD的患者特异性干细胞疗法。这一特殊应用的目的是确定最佳的细胞工程系统，可以特异性和有效地扩增用于移植的人角膜上皮细胞的干细胞/祖细胞群。中心假设是适当的人类饲养细胞

DESCRIPTION (provided by applicant): Transplantation of autologous corneal epithelial stem/progenitor cells (CESCs) expanded in tissue culture has successfully restored vision and revolutionized the treatment for limbal stem cell deficiency (LSCD) which is a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders. A higher expansion efficiency of the stem/progenitor cell population in culture corresponds to a better long-term graft survival. The most efficient expansion method requires mouse 3T3 feeder cells which are grown using calf serum and are the source of mouse RNA that contaminates the expanded cells. This cross-contamination by animal products poses considerable potential health hazards and therefore makes this culture method unlikely to be approved by the US Food and Drug Administration to be used in humans. New cell engineering methods that achieve the same or better efficiency of expanding CESCs under xenobiotic-free conditions are needed to achieve acceptable clinical outcomes. The long-term goal of my laboratory is to elucidate the regulatory factors that govern CESC self-renewal and differentiation, and to develop patient- specific stem cell-based therapies for LSCD. The objective of this particular application is to identify optimal cell engineering systems that can specifically and efficiently expand the stem/progenitor cell population of human corneal epithelial cells for transplantation. The central hypothesis is that appropriate human feeder cells can replace mouse 3T3 cells to provide a proper microenvironment to support the growth of CESCs, and upon receiving additional appropriate external signals the expansion of CESCs could be further optimized in culture. The hypothesis has been formulated based on the data produced in my laboratory. To achieve the objective of this translational research application, two specific aims are proposed: 1) Establish a xenobiotic-free culturing system using a human feeder layer that can efficiently expand CESCs; and 2) Identify an optimal expansion condition of CESCs by modulating the Wnt and/or Notch signaling pathway using small molecules or bioengineered human feeder cells.) Under the first aim, five human feeder candidates will be tested for their ability to grow CESCs. The functional aspect of these bioengineered CESCs will be tested in a well-established mouse model of LSCD. Under the second aim, proliferation of CESCs will be further optimized using human feeder cells that are engineered to over express limbal-specific Wnt molecules and Notch ligands, and small molecules of Wnt activators and Notch inhibitors. The approach is innovative, because it utilizes a novel method to bioengineer CESCs without permanent genetic alternation of the target cells. This approach has three major advantages: reversible, specific and translational. This will eliminate any potential permanent side effects or toxicity. The proposed translational research is significant, because the results from any of the two aims can be readily adapted for clinical development. PUBLIC HEALTH RELEVANCE: The goal of this proposed research is to establish an ex vivo expansion protocol for limbal stem cells, or corneal epithelial stem/progenitor cells (CESCs), in xenobiotic-free conditions for transplantation into humans and a novel bioengineering approach to increase the efficiency of expansion of functional human CESCs. Thus, the proposed research will enable the initiation of a safe and effective patient specific stem cell-based therap for limbal stem cell deficiency.