TSHR and thyrocyte development

简介（由申请人提供）：甲状腺疾病通常是由甲状腺细胞异常增殖和分化引起的，可导致各种可能致命的并发症。这些疾病发生的机制在很大程度上是未知的，尚未被发现。目前，本研究的长期目标是阐明胚胎干细胞分化为最终甲状腺细胞的分子机制。多能胚胎干细胞来源于早期胚胎的内部细胞群，可以自我更新并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。操纵小鼠胚胎干细胞的多能性潜能，使我们能够在适当的条件下直接向甲状腺谱系分化。为了扩展这个小鼠模型，我们也在利用nih注册的人类胚胎干细胞开发一个类似的模型。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）转染，可以将成人皮肤成纤维细胞重编程为胚胎干细胞样状态。我们的iPS细胞在许多方面与胚胎干细胞相似，包括主要干细胞基因和蛋白质的表达、胚状体的形成、畸胎瘤的形成、效力和分化。在本应用中，我们将扩展人类胚胎干细胞的研究，重点关注胚胎干细胞分化过程中内胚层诱导的调节机制及其对甲状腺谱系的规范。此外，我们提出建立和繁殖突变TSHR甲状腺功能减退小鼠模型的小鼠iPS细胞。甲状腺功能减退小鼠表现出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。Specific Aim 1的目标是从人类胚胎干细胞中生成甲状腺滤泡细胞。Specific Aim 2的目标是从正常和突变的TSHR成纤维细胞中产生和表征小鼠iPS细胞。我们将利用已经建立的方法，通过四种转录因子的异位表达，将正常和突变TSHR小鼠的皮肤成纤维细胞重编程为iPS细胞。我们将测试疾病特异性iPS细胞可以被诱导进入甲状腺滤泡细胞的假设，以探索导致疾病表型的生物学过程。最后，我们将通过体外和体内实验，将这些小鼠iPS细胞系与我们已经建立的小鼠TSHR-/- ES细胞系进行比较，来表征这些小鼠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化物运输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细胞系作为模拟人类疾病的研究工具的能力，以及2)使用患者来源的iPS细胞系进行候选疗法和筛选分析的复杂测试的机会。

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.