Establishment of xenopus stem cell lines

非洲爪蟾干细胞系的建立

• 批准号: 10667834
• 负责人: Nadege Gouignard
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF WISCONSIN MILWAUKEE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667834
• 关键词: Adult; Amphibia; Animal Cap; Animals; Award; Biochemical; Biological Models; Biology; Brain; Cell Line; Cell Separation; Cells; Cellular Morphology; Characteristics; Chick; Communities; Defect; Development; Developmental Process; Disease; ES Cell Line; Embryo; Endocrine System Diseases; Eye; Fertilization in Vitro; Gastrointestinal Diseases; Genome; Heart; Homologous Gene; Hour; Human; In Vitro; Inner Cell Mass; Kidney Diseases; Limb structure; Malignant Neoplasms; Mammalian Cell; Methods; Modeling; Molecular; Molecular Analysis; Morphology; Mouse Cell Line; Mus; Natural regeneration; Neural Crest; Neurodevelopmental Disorder; Nobel Prize; Optic Nerve; Organ; Organoids; Pancreatic Diseases; Physiological; Population; Primary Cell Cultures; Process; Research; Research Personnel; Research Project Grants; Species Specificity; Spinal Cord; Syndrome; System; Tadpoles; Time; Tissues; Transfection; Xenopus; Xenopus laevis; animal tissue; blastocyst; brain tissue; cell behavior; cell type; ciliopathy; congenital heart disorder; embryo tissue; embryonic stem cell; genome editing; human disease; in vivo; in vivo Model; induced pluripotent stem cell; lens; mutant; organ growth; orofacial; pluripotency; programs; somatic cell nuclear transfer; species difference; stem cell fate; stem cells; stem-like cell; tissue culture; tool; transcription factor; xenopus development

PROJECT SUMMARY Xenopus laevis is a remarkable model system used for decades to answer fundamental questions in cell, developmental, and evolutionary biology. Several Nobel prizes were awarded for groundbreaking research in Xenopus. Xenopus embryos have comparable organ development and morphology to mammalian systems, but with the added benefit of being able to regenerate adult tissues, such as the optic nerve, lens, spinal cord, and limb tissue. Over the years, Xenopus has been used to model several human diseases and syndromes, including congenital heart disorders, heterotaxia, gastrointestinal and pancreatic diseases, endocrine disorders, kidney disease, cancer, ciliopathies, orofacial defects, and neurodevelopmental disorders. Xenopus is a powerful in vivo model system, but robust complementary in vitro tools are still limited. While animal caps and tissue explants can be easily isolated from Xenopus embryos, and cultured in vitro, these cells do not survive for extended time periods. Furthermore, cellular and intracellular processes are often difficult to document and analyze in vivo. These limitations prompted the establishment of several cell lines in the 1990's, but they fell out of favor for the more amenable mammalian in vitro systems. Since then, the Xenopus community has relied on the use of human or mouse cell lines, including embryonic and induced stem cells, which introduce inherent variability due to species differences. Even though the blueprint of vertebrate development across species is largely conserved, several aspects of cellular, tissue, and organ biology have species specific characteristics. An example among many, during development Xenopus neural crest expresses both the transcription factors Snai1 and Snai2, while mouse neural crest only expresses Snai1 and the chick neural crest only Snai2. These differences constrain the use of cross species systems experimentally. As a result, we and others are engaged in efforts to expand the in vitro toolbox of the Xenopus community. We propose to generate new Xenopus stem cell lines to enhance current and future research projects for the Xenopus community. Stem cells represent a normal physiological state, their genomes lack abnormalities typically found in most tissue culture lines, and they can be differentiated into many different cell types and organoids. Furthermore, a strong advantage offered by Xenopus stem cells would be the opportunity to perform genome editing efficiently, as well as somatic cell nuclear transfer (SCNT) to generate F0 homozygous null animals to create new mutants. Currently, stem cell lines are obtained via two methods: embryonic stem cells isolated from the inner cell mass of mammalian blastocyst-stage embryos, and induced pluripotent stem cells obtained via reprogramming of mature cells to re-initiate endogenous pluripotency programs. In this application we propose to generate embryonic stem cell lines from animal pole cells isolated at the blastula stage and reprogram newly generated primary cell lines derived from tadpole tissues to produce induced pluripotent stem cells.

项目总结 非洲爪哇是一个了不起的模型系统，几十年来一直用来回答细胞中的基本问题， 发育生物学和进化生物学。几个诺贝尔奖被授予开创性的研究 非洲爪哇。非洲爪哇胚胎的器官发育和形态与哺乳动物系统相似，但 另一个好处是能够再生成人组织，如视神经、晶状体、脊髓和 四肢组织。多年来，非洲爪哇已被用于模拟几种人类疾病和综合征，包括 先天性心脏病、异位症、胃肠和胰腺疾病、内分泌紊乱、肾脏 疾病、癌症、纤毛疾病、口腔面部缺陷和神经发育障碍。 非洲爪哇是一个强大的体内模型系统，但强大的补充体外工具仍然有限。而当 可以很容易地从非洲爪哇胚胎中分离出动物帽和组织外植体，并在体外培养这些细胞 不能在长时间内存活。此外，细胞和细胞内的过程通常很难 在活体内记录和分析。这些限制促使在20世纪90年代的S建立了几个细胞系， 但它们失去了对更具顺应性的哺乳动物体外系统的青睐。从那时起，非洲爪哇社区 依赖于人类或小鼠细胞系的使用，包括胚胎和诱导干细胞，它们引入了 由于物种差异而产生的固有的可变性。即使整个脊椎动物的发展蓝图 物种在很大程度上是保守的，细胞、组织和器官生物学的几个方面都有物种特有的 特点。其中一个例子是，在发育过程中，非洲爪哇神经冠既表达了 转录因子Snai1和SNAI2，而小鼠神经峰只表达Snai1和鸡神经峰 只有SNAI2。这些差异在实验上限制了跨物种系统的使用。因此，我们和 其他人则致力于扩大非洲爪哇群体的体外工具箱。我们建议生成 新的非洲爪哇干细胞系，以加强非洲爪哇社区当前和未来的研究项目。 干细胞代表一种正常的生理状态，它们的基因组缺乏大多数 组织培养系，它们可以分化成许多不同的细胞类型和细胞器。此外，a 非洲爪哇干细胞提供的强大优势将是有效地进行基因组编辑的机会， 以及体细胞核移植(SCNT)，以产生F0纯合子空动物，以创造新的突变体。 目前，获得干细胞系的方法有两种：从细胞内分离胚胎干细胞 哺乳动物囊胚期胚胎的质量和通过重编程获得的诱导多能干细胞 以重新启动内源性多能性程序。在此应用程序中，我们建议生成 从囊胚期分离的动物极细胞中分离出胚胎干细胞并对新生成的细胞进行重组 源自蝌蚪组织的原代细胞系可产生诱导的多能干细胞。