Regenerative Airway Epithelium by Embryonic Stem Cells

描述（由申请人提供）：再生医学的一个主要研究领域是细胞治疗各种疾病的潜在应用。胚胎干细胞（ES）是一种可自我再生的多能细胞，来源于囊胚期胚胎的内部细胞群。胚胎干细胞有潜力为各种退行性和非退行性人类疾病提供创新的治疗选择，包括替换功能失调的气道上皮。为了实现这一目标，需要制定明确和有效的方案来指导干细胞向气道上皮细胞谱系的承诺和分化，以及它们在体外的选择性纯化和增殖。这种方案的发展将减少胚胎干细胞在移植时自发分化为不同谱系的可能性，并降低畸胎瘤形成的风险。此外，这些方案可以为研究呼吸道上皮细胞的发育生物学提供有用的体外模型，并促进干细胞的遗传操作用于治疗应用。该应用的假设是，胚胎干细胞可以在体外培养分化为气道上皮细胞系，在体内具有再生气道上皮的全部潜力。为了验证这一假设，采用了三种方法。1)定义“最低优化的无血清培养条件”，该条件将指导小鼠（D3和Bruce4）和人（H9） ES细胞在不同培养时间点根据特异性气道上皮细胞、细胞生长和细胞形态的分化标记表达向气道上皮细胞系分化，使用负/正一因子方法。2)在“最低优化无血清培养条件”下，对小鼠（D3和Bruce4）和人（H9） ES细胞衍生的气道上皮细胞系进行形态学和免疫组织化学评估，通过将剥去的大鼠气管移植物移植到SCID/裸鼠中进行气道上皮再生。3)表征gfp标记的Bruce4 ES细胞在“最低优化无血清培养条件”下，在C57BL/6小鼠气道上皮修复过程中定位/分化的模式，C57BL/6小鼠气道上皮特异性毒物萘处理。

DESCRIPTION (provided by applicant): A major area of research in regenerative medicine is the potential application of cell therapy for various disorders. Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cells have the potential to provide innovative therapeutic options for a wide variety of degenerative and non-degenerative human disorders, including the replacement of dysfunctional airway epithelia. To achieve this goal, it would require the development of well defined and efficient protocols for directing the commitment and differentiation of stem cells into the airway epithelial cell lineage, together with their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of ES cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation. Additional, such protocols could provide useful in vitro models for studying the developmental biology, of respiratory tract epithelial cells, as well as facilitate the genetic manipulation of stem cells for therapeutic application. The hypothesis of this application is that ES cells can be in vitro trained to differentiate to the airway epithelial cell lineages that have the full potential to regenerate airway epithelium in vivo. To test this hypothesis, three approaches are undertaken. 1) Define "minimally optimized serum-free culture conditions" that will direct both mouse (D3 and Bruce4) and human (H9) ES cells to differentiate to the airway epithelial cell lineages based on differentiation marker expression that are specific for airway epithelial cells, cell growth, and cell morphology using minus/plus one factor approach at various culture time points. 2) Assess morphologically and immunohistochemically the potential of the airway epithelial cell lineages derived from mouse (D3 and Bruce4) and human (H9) ES cells under the "minimally optimized serum-free culture conditions" to regenerate airway epithelium through the repopulation of denuded rat tracheal graft implanted in SCID/nude mice. 3) Characterize the patterns of localization/differentiation of in vivo transplanted cell lineages derived from GFP-tagged Bruce4 ES cells under the "minimally optimized serum-free culture conditions" during the repair process of the airway epithelium in C57BL/6 mice that have been previously treated with an airway epithelium-specific toxicant, naphthalene.