TSHR and Thyrocyte Development

描述(申请人提供)：甲状腺疾病通常是由异常的甲状腺细胞增殖和分化引起的，这可能会导致各种可能致命的并发症。这些疾病发展的机制在很大程度上是未知的，还有待发现。目前，我们父母资助的长期目标是阐明胚胎干细胞分化为确定的甲状腺细胞的分子机制。来自早期胚胎内细胞团的多能ES细胞既可以自我更新，也可以分化为体内所有类型的细胞，因此提供了一个关键的甲状腺发育模型。在体外对小鼠胚胎干细胞多潜能的操纵使我们能够在适当的条件下直接向甲状腺分化。为了扩展这个小鼠模型，我们还在努力开发一个类似的模型，使用NIH注册的人类ES细胞。我们最近开发了一种诱导多能干细胞(IPS)技术，通过逆转录病毒介导的四种转录因子(Oct4、Sox2、c-Myc和Klf4)的转染，可以将成人皮肤成纤维细胞重新编程为ES细胞样状态。我们操纵的iPS细胞在许多方面与ES细胞相似，包括主要干细胞基因和蛋白的表达、类胚体形成、畸胎瘤形成、潜能和分化。在这一应用中，我们将扩大对人类ES细胞的研究，重点是在ES细胞分化过程中调节内胚层诱导的机制及其对甲状腺谱系的指定。此外，我们建议从突变型TSHR甲状腺功能低下小鼠模型中建立和繁殖小鼠iPS细胞。甲状腺功能低下小鼠表现为严重的先天性甲状腺功能减退和甲状腺增生，为探讨该病的病理生理表现提供了机会。特定目标1的目标是从人ES细胞中产生甲状腺滤泡细胞。特异性目标2的目标是从正常和突变的TSHR成纤维细胞中培养和鉴定小鼠iPS细胞。我们将使用我们已建立的方法，通过四种转录因子的异位表达，将正常和突变的TSHR小鼠的皮肤成纤维细胞重新编程为iPS细胞。我们将测试疾病特异性iPS细胞可以被诱骗成甲状腺滤泡细胞的假设，以探索导致疾病表型的生物过程。最后，我们将通过体外和体内实验，与我们已建立的小鼠TSHR-/-ES细胞系相比较，表征这些小鼠iPS细胞系的甲状腺细胞分化潜力。更具体地说，为了启动新的治疗方法，我们将使用这些疾病-iPS细胞作为检测工具来验证碘转运和甲状腺激素合成和分泌的缺陷。这些目标的成功将提供许多治疗前景，包括：1)建立患者来源的iPS细胞系作为人类疾病模型的研究工具的能力，以及2)使用患者来源的iPS细胞系对候选疗法和筛选分析进行复杂测试的机会。 公共卫生相关性：甲状腺疾病通常是由甲状腺细胞增殖和分化异常引起的，这可能会导致各种可能致命的并发症。这些疾病发展的机制在很大程度上是未知的，还有待发现。该项目的目标是将胚胎干细胞和诱导多能干细胞的优势与小鼠模型中可用的强大遗传工具结合起来，以采取一种独特的方法来治疗甲状腺发育和疾病。

DESCRIPTION (provided by applicant): Thyroid diseases are commonly caused by abnormal thyroid cell proliferation and differentiation, which can lead to various, possibly fatal complications. The mechanisms by which these diseases develop by are largely unknown and have yet to be discovered. Presently, the long-term goal of our parent grant is to elucidate the molecular mechanisms by which embryonic stem (ES) cells differentiate into definitive thyrocytes. Pluripotent ES cells, derived from the inner cell mass of early embryos, can both self-renew and differentiate into all cell types in the body and as such provide a key thyroid developmental model. The manipulation of the pluripotent potential of murine ES cells in vitro has allowed us to direct differentiation towards the thyroid lineage under the appropriate conditions. To expand upon this mouse model, we are also working to develop a similar model using NIH-registry human ES cells. We have recently developed a form of induced pluripotent stem (iPS) cell technology, which can reprogram adult skin fibroblasts into an ES cell-like state using the retrovirus-mediated transfection of four transcription factors (Oct4, Sox2, c-Myc, and Klf4). Our manipulated iPS cells are similar to ES cells in many respects, including the expression of main stem cell genes and proteins, embryoid body formation, teratomas formation, potency and differentiation. In this application, we will expand on human ES cell studies focused on the mechanisms regulating endoderm induction and its specification to thyroid lineage during ES cell differentiation. Furthermore, we propose to establish and propagate murine iPS cells from a mutant TSHR hypothyroid mouse model. The hypothyroid mice display severe congenital hypothyroidism and thyroid hyperplasia and provide an opportunity to explore the pathophysiological manifestations of this disease. The goal of Specific Aim 1 is to generate thyroid follicular cells from human ES cells. The goal of Specific Aim 2 is to produce and characterize murine iPS cells from normal and mutant TSHR fibroblasts. We will use our established methods to reprogram skin fibroblasts of normal and mutant TSHR mice into iPS cells by the ectopic expression of four transcription factors. We will test the hypothesis that the disease-specific iPS cells can be coaxed into thyroid follicular cells to explore the biological processes that lead to disease phenotypes. Finally, we will characterize the thyrocyte differentiation potential of these murine iPS cell lines in comparison to our well-established murine TSHR-/- ES cell lines by using in vitro and in vivo assays. More specifically, to initiate effort to new therapeutic approaches, we will use these disease-iPS cells as assay tools to validate the defects of iodide transport and thyroid hormone synthesis and secretion. The success of these aims will provide a number of therapeutic promises including: 1) the ability to establish patient-derived iPS cell lines as a research tool to model human disease, and 2) the opportunity to use patient-derived iPS cell lines to perform sophisticated testing of candidate therapeutics and screening assays. PUBLIC HEALTH RELEVANCE: Thyroid diseases are commonly caused by abnormal thyroid cell proliferation and differentiation, which can lead to various, possibly fatal complications. The mechanisms by which these diseases develop by are largely unknown and have yet to be discovered. The goal of this project is to combine the advantages of embryonic stem cells and induced pluripotent stem cells with the powerful genetic tools available in mouse models to take a unique approach to thyroid development and disease.