Ex Vivo Expansion of Human Limbal Stem Cells for Transplantation

人角膜缘干细胞的体外扩增用于移植

• 批准号: 8373855
• 负责人: Sophie Deng
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8373855
• 关键词: 3T3 Cells; Adipose tissue; Adverse effects; Animals; Applications Grants; Autologous Transplantation; Biomedical Engineering; Blindness; Bone Marrow; Cattle; Cell Proliferation; Cell Therapy; Cell Transplantation; Cells; Clinical; Cornea; Corneal Diseases; Data; Development; Epithelial; Epithelial Cells; Genetic; Goals; Graft Survival; Growth; Health Hazards; Human; In Vitro; Individual; Keratoplasty; Laboratories; Ligands; Lithium Chloride; Membrane; Mesenchymal; Mesenchymal Stem Cells; Methods; Mus; Natural regeneration; Notch Signaling Pathway; Notch and Wnt Signaling Pathway; Outcome; Patients; Phenotype; Play; Population; Protocols documentation; Publishing; RNA; Regulation; Reporting; Research; Role; Serum; Signal Pathway; Signal Transduction; Source; Stem cell transplant; Stem cells; Stromal Cells; System; Testing; Toxic effect; Transcript; Translational Research; Transplantation; Transplantation Conditioning; Undifferentiated; United States Food and Drug Administration; Vision; Wound Healing; Xenobiotics; base; blind; cell growth; cell type; cellular engineering; conjunctiva; corneal epithelium; human FZD7 protein; improved; inhibitor/antagonist; innovation; knock-down; limbal; mouse model; notch protein; novel; ocular surface; progenitor; receptor; self-renewal; small hairpin RNA; small molecule; stem; stem cell population; tissue culture; transplantation typing

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.

描述（由申请人提供）：在组织培养中扩增的自体角膜上皮干/祖细胞（CESC）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，LSCD是许多常见角膜疾病中显著视力丧失和失明的主要原因，无论是原发性还是继发性。培养物中干/祖细胞群的较高扩增效率对应于较好的长期移植物存活。最有效的扩增方法需要小鼠3 T3饲养细胞，其使用小牛血清生长，并且是污染扩增细胞的小鼠RNA的来源。这种动物产品的交叉污染造成了相当大的潜在健康危害，因此使得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。需要新的细胞工程方法，实现相同或更好的效率，扩大CESC在无异生物质的条件下，以实现可接受的临床结果。本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。本具体申请的目的是鉴定能够特异性和有效地扩增用于移植的人角膜上皮细胞的干/祖细胞群的最佳细胞工程系统。核心假设是合适的人类饲养细胞 可以替代小鼠3 T3细胞以提供适当的微环境来支持CESC的生长，并且在接收额外的适当外部信号时，CESC的扩增可以在培养中进一步优化。这个假设是根据我实验室的数据提出的。为了实现该转化研究应用的目的，提出了两个具体目标：1）使用可以有效扩增CESC的人饲养层建立无异生物质的培养系统;以及2）通过使用小分子或生物工程化的人饲养细胞调节Wnt和/或Notch信号传导途径来鉴定CESC的最佳扩增条件。根据第一个目标，将测试五名人类饲养候选人培养CESC的能力。这些生物工程化CESC的功能方面将在完善的LSCD小鼠模型中进行测试。在第二个目标下，将使用人饲养细胞进一步优化CESC的增殖，所述人饲养细胞被工程化以过表达角膜缘特异性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化CESCs，而不会对靶细胞进行永久的遗传改变。这种方法有三个主要优点：可逆性，特异性和翻译。这将消除任何潜在的永久性副作用或毒性。拟议的转化研究是重要的，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。 公共卫生关系：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（CESC）的体外扩增方案，在无异生物质的条件下移植到人体内，以及一种新的生物工程方法，以提高功能性人类CESC的扩增效率。因此，拟议的研究将能够启动一个安全有效的患者特异性干细胞为基础的治疗角膜缘干细胞缺乏症。