Ex Vivo Expansion of Human Limbal Stem Cells for Transplantation

描述(申请人提供)：在组织培养中扩增的自体角膜上皮干细胞(CESCs)移植成功地恢复了视力，并使角膜缘干细胞缺乏症(LSCD)的治疗发生了革命性变化，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，无论是原发的还是继发性的。干细胞/祖细胞在培养中的扩增效率越高，移植物的长期存活率就越好。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，是污染扩大细胞的小鼠RNA的来源。这种动物产品的交叉污染构成了相当大的潜在健康危害，因此使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来达到在无异物条件下扩增CESCs的相同或更高的效率。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，并开发针对患者的基于干细胞的LSCD治疗方法。这一特殊应用的目的是确定最佳的细胞工程系统，该系统可以特异性和有效地扩大用于移植的人角膜上皮细胞的干/祖细胞群。中心假设是合适的人类饲养层细胞 可以替代小鼠3T3细胞，为CESCs的生长提供合适的微环境，在接收到额外的合适的外部信号后，CESCs的扩增可以在培养中进一步优化。这一假设是基于我的实验室产生的数据而提出的。为了实现这一翻译研究应用的目标，提出了两个具体的目标：1)建立一个使用人饲养层的无异物培养系统，可以有效地扩增CESCs；2)通过使用小分子或生物工程人饲养细胞来调节Wnt和/或Notch信号通路，确定CESCs的最佳扩增条件。)在第一个目标下，五名人工饲养员候选人将接受培养CESCs的能力测试。这些生物工程CESCs的功能方面将在建立良好的LSCD小鼠模型中进行测试。在第二个目标下，CESCs的增殖将进一步优化，使用人类饲养细胞，这些细胞被设计为过度表达特定于角膜缘的Wnt分子和Notch配体，以及Wnt激活剂和Notch抑制剂的小分子。这种方法是创新的，因为它利用了一种新的方法来生物工程CESCs，而不需要对目标细胞进行永久性的遗传改变。这种方法有三大优势：可逆性、特效性和转化性。这将消除任何潜在的永久性副作用或毒性。这项拟议的转化性研究意义重大，因为这两个目标中的任何一个的结果都可以很容易地适应临床开发。 公共卫生相关性：这项拟议研究的目标是建立角膜缘干细胞或角膜上皮干/祖细胞(CESCs)在无异种生物条件下移植到人类体内的体外扩增方案，并建立一种新的生物工程方法来提高功能性人类CESCs的扩增效率。因此，拟议的研究将使安全和有效的患者特异性干细胞治疗角膜缘干细胞缺乏症成为可能。

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.