A Human Pluripotent Stem Cell Model for Elucidating Cell Fate Defects in McCune-Albright Syndrome

用于阐明 McCune-Albright 综合征细胞命运缺陷的人类多能干细胞模型

• 批准号: 9258289
• 负责人: Kelly Lee Wentworth
• 金额: $ 6.74万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258289
• 关键词: Acromegaly; Address; Adrenal Glands; Affect; Animal Model; Award; Binding Proteins; Biological; CRISPR/Cas technology; Cell Lineage; Cell model; Cells; Complex; Coupled; Critical Pathways; Cyclic AMP; Data; Defect; Development; Disease; Disease model; Ductal Epithelial Cell; Embryo; Embryonic Development; Endocrinology; Engineering; Forskolin; Future; G Protein-Coupled Receptor Signaling; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; G-substrate; GNAS gene; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genes; Germ Layers; Germ-Line Mutation; Goals; Human; Human body; Hyperpigmentation; Hyperthyroidism; Induced Mutation; Inherited; Integral Membrane Protein; Knowledge; Lead; Ligands; Light; Literature; Maintenance; Malignant Neoplasms; McCune-Albright Syndrome; Mediating; Missense Mutation; Modeling; Mosaicism; Mutation; Organ; Osteoblasts; Pancreas; Pancreatic duct; Pathway interactions; Pharmacology; Physicians; Pituitary Gland; Pituitary-dependent Cushing' s disease; Play; Polyostotic fibrous dysplasia; Positioning Attribute; Preclinical Drug Evaluation; Precocious Puberty; Production; Proteins; Receptor Signaling; Role; Scientist; Signal Pathway; Signal Transduction; Signaling Protein; Skin; Solid; Techniques; Technology; Testing; Thyroid Gland; Time; Tissues; Training; Training Programs; Transgenic Organisms; Triad Acrylic Resin; United States National Institutes of Health; Vertical Disease Transmission; adult stem cell; cell type; extracellular; guanine nucleotide binding protein; human disease; human pluripotent stem cell; induced pluripotent stem cell; insight; interest; novel; osteogenic; pluripotency; post-doctoral training; receptor; screening; self-renewal; skills; skin lesion; stem cell fate; tool

Project Summary: G-protein coupled receptor (GPCR) signaling pathways mediate a wide spectrum of biological activities in humans. McCune-Albright Syndrome (MAS) is a mosaic disease caused by a somatic activating mutation in the GNAS gene (c.602G>A, p.R201H). The GNAS complex locus encodes the stimulatory alpha subunit of the guanine nucleotide binding protein (Gsα) and regulates production of cAMP. MAS is characterized by the classic triad of polyostotic fibrous dysplasia, café-au-lait skin lesions, and precocious puberty. It can also cause hyperthyroidism, Cushing’s disease, acromegaly, and malignancies of the thyroid, pituitary and pancreas. The R201H mutation is thought to occur post-zygotically since tissues from all 3 germ layers can be affected. There is no known vertical transmission of MAS in humans; therefore, germline mutations are thought to be embryonically lethal; however, the precise mechanism leading to lethality is not fully understood. There is emerging literature suggesting that GPCR signaling pathways play a role in early development and stem cell fate, and we hypothesize that the early lethality seen in MAS may be a consequence of over-activation of the Gs-signaling pathway which may create a critical block in the development of certain cell lineages. Unfortunately, our ability to study this mechanism further is hampered by our lack of animal models carrying the GNAS R201H mutation in the endogenous locus. We propose developing a novel, robust human model of MAS using induced pluripotent stem cells (iPSCs) to explore how activated GNAS and elevated cAMP levels affect stem cell fate and lineage commitment in MAS. First, we will test how increased cAMP levels, pharmacologically induced by forskolin, affect pluripotency and lineage commitment in control human iPSCs. Next, we will create a human model of MAS by introducing the R201H mutation into control iPSCs at the endogenous locus using CRISPR/Cas9 gene-editing techniques. We will then compare these two models and examine whether pharmacologically-induced and R201H mutation-induced cAMP activation have similar effects on iPSC pluripotency and lineage commitment. We will then differentiate our engineered R201H iPSCs into osteogenic precursors and examine the effect of the mutation on osteoblast commitment and maturation. Our results will provide insight into the potential mechanisms contributing to early cell fate changes and embryonic lethality in MAS. This new knowledge will guide future studies in mature cell types that can be generated from iPSCs, including adrenal cortical cells, pituitary cells, and pancreatic ductal cells. As GPCR signaling pathways mediate many critical biological activities in the human body, this model will support the study of Gs-signaling in other tissues and diseases. The results of our current study will also be critical for developing screening tools and identifying key endpoints for GNAS-specific high-throughput drug screens. Finally, this training plan will help the candidate gain the necessary skills to apply for a NIH K08 award and develop into an independent translational clinician-scientist in the field of endocrinology.

项目概要： G蛋白偶联受体（GPCR）信号通路介导了广泛的生物活性， 人类McCune-Albright综合征（MAS）是一种由体细胞激活突变引起的镶嵌病， GNAS基因（c.602G>A，p.R201H）。GNAS复合物基因座编码GNAS的刺激性α亚基。 鸟嘌呤核苷酸结合蛋白（Gsα），调节cAMP的产生。MAS的特点是 典型的多发性骨纤维异常增殖症、咖啡牛奶样皮肤病变和性早熟三联征。它也可能导致 甲状腺功能亢进症、库欣病、肢端肥大症和甲状腺、垂体和胰腺的恶性肿瘤。的 R201 H突变被认为发生在合子后，因为所有3个胚层的组织都可能受到影响。 没有已知的垂直传播的MAS在人类;因此，生殖系突变被认为是 胚胎致死;然而，导致致死的确切机制尚未完全了解。有 新出现的文献表明，GPCR信号通路在早期发育和干细胞中发挥作用， 命运，我们假设在MAS中看到的早期致死性可能是过度激活的结果。 可能在某些细胞谱系的发育中产生关键阻滞的信号通路。 不幸的是，我们进一步研究这一机制的能力受到了缺乏携带 内源性基因座中的GNAS R201 H突变。我们建议开发一种新的，健壮的人类 使用诱导多能干细胞（iPSC）的MAS模型，以探索如何激活GNAS和 升高的cAMP水平影响MAS中的干细胞命运和谱系定型。首先，我们将测试如何 毛喉素诱导的cAMP水平升高影响多能性和谱系定型， 对照人iPSC。接下来，我们将通过将R201 H突变引入到 使用CRISPR/Cas9基因编辑技术在内源基因座控制iPSC。我们将比较 这两个模型，并检查是否药理学诱导和R201 H突变诱导的cAMP 在一些实施方案中，iPSC活化对iPSC多能性和谱系定型具有类似的作用。然后我们将区分我们的 将R201 H iPSCs工程化为成骨前体，并检查突变对成骨细胞的影响。 承诺和成熟。我们的研究结果将提供深入了解的潜在机制，有助于早期 细胞命运的变化和胚胎致死性。这一新知识将指导未来的成熟细胞研究。 可以从iPSC产生的类型，包括肾上腺皮质细胞，垂体细胞和胰腺导管细胞。 细胞由于GPCR信号通路介导人体中许多关键的生物活性， 将支持在其他组织和疾病中的GS信号传导的研究。我们目前的研究结果也将 对于开发筛选工具和确定GNAS特异性高通量药物的关键终点至关重要 卡位最后，本培训计划将帮助候选人获得申请NIH K 08的必要技能 奖励并发展成为内分泌学领域的独立翻译临床医生-科学家。