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疗法在各种疾病中的潜在应用。胚胎干细胞是一种来源于囊胚期胚胎内细胞团的自我更新的多能细胞。ES细胞有可能为多种退行性和非退行性人类疾病提供创新的治疗选择，包括替换功能障碍的呼吸道上皮。为了实现这一目标，需要制定明确和有效的方案，指导干细胞定向和分化为呼吸道上皮细胞系，并在体外选择性纯化和增殖。此类方案的开发将降低ES细胞在移植后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，以及促进用于治疗应用的干细胞的遗传操作。这一应用的假设是，ES细胞可以在体外培养分化为具有在体内再生呼吸道上皮细胞的全部潜力的呼吸道上皮细胞系。为了检验这一假设，我们采取了三种方法。1)定义“最低限度优化的无血清培养条件”，该条件将引导小鼠(D3和Bruce4)和人(H9)ES细胞根据不同培养时间点的分化标志物表达(针对呼吸道上皮细胞、细胞生长和细胞形态)分化为呼吸道上皮细胞系。2)在“最低限度优化的无血清培养条件”下，通过裸鼠气管移植于SCID/裸鼠体内，从形态学和免疫组织化学的角度评价来源于小鼠(D3和Bruce4)和人(H9)ES细胞的呼吸道上皮细胞系再生呼吸道上皮的能力。3)研究GFP标记的Bruce4 ES细胞来源的体内移植细胞系在“最低优化的无血清培养条件”下的定位/分化模式，在C57BL/6小鼠的呼吸道上皮修复过程中，这些小鼠以前曾接受过呼吸道上皮特异性毒物--萘的处理。

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.