Human pluripotent stem cells

人类多能干细胞

• 批准号: 10691970
• 负责人: Pamela Robey
• 金额: $ 160.13万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10691970
• 关键词: 3-Dimensional; 8 year old; Address; Affect; Age; Area; Atrophic; Autophagocytosis; Back; Binding; Biological Assay; Biological Models; Blindness; Brain; CLN3 gene; CRISPR/Cas technology; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell model; Cells; Cephalic; Code; Collaborations; Complex; Cranial nerve palsies; Data; Derivation procedure; Development; Disease; Disease model; Educational process of instructing; Epithelial Cells; Exhibits; Fibroblasts; Functional disorder; GABA transporter; GTP-Binding Proteins; Gene Targeting; Generations; Genes; Genetic; Genetic Diseases; Genetic Engineering; Genome; Genomic Instability; Glial Fibrillary Acidic Protein; Goals; Golgi Apparatus; Hormones; Human; Hypoxia; Immune System Diseases; Immunocompromised Host; Immunofluorescence Immunologic; In Vitro; Instruction; Integral Membrane Protein; Knock-out; Lead; Ligands; Limb structure; Maintenance; Mentors; Meta-Analysis; Metabolic; Methods; Microtubule-Associated Protein 2; Mission; Mitochondria; Modeling; Monitor; Motor Neuron Disease; Mus; Mutation; Myocardial dysfunction; National Human Genome Research Institute; National Institute of Child Health and Human Development; National Institute of Dental and Craniofacial Research; National Institute of Neurological Disorders and Stroke; National Institute on Alcohol Abuse and Alcoholism; Neocortex; Neurites; Neurodegenerative Disorders; Neuroglia; Neurons; Nursery Schools; Organoids; PINK1 gene; Parkin; Parkinson Disease; Pathway interactions; Patients; Phenotype; Pituitary Diseases; Pituitary Gland; Pituitary Gland Adenoma; Pituitary-dependent Cushing' s disease; Pluripotent Stem Cells; Population; Preclinical Drug Development; Prevalence; Process; Property; Protein-Serine-Threonine Kinases; Proteins; Protocols documentation; Quality Control; RNA interference screen; Rattus; Reporter; Rhodopsin; Salivary; Salivary Glands; Sampling; Sendai virus; Sensorineural Hearing Loss; Side; Sjogren' s Syndrome; Skin; Spielmeyer-Vogt Disease; Structure; Symptoms; Syndrome; System; Technology; Teenagers; Teratoma; Testing; United States National Institutes of Health; Work; Xerostomia; acetylcholine transporter; adenoma; aquaporin 5; behavioral study; cell community; cell type; clinically relevant; comparative; differential expression; differentiation protocol; disease-causing mutation; dopamine transporter; drug discovery; established cell line; eye dryness; human embryonic stem cell; human pluripotent stem cell; in vitro Model; induced pluripotent stem cell; induced pluripotent stem cell technology; inhibitor; insight; interest; metabolome; motor disorder; mouse model; mutant; neural; neurogenesis; pandemic disease; pluripotency; programs; protein folding; rho; serotonin transporter; skeletal abnormality; stem cell growth; stem cells; synaptogenesis; tool; tumorigenesis

During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below. 1. Plasticity of metabolic remodeling in naive-like pluripotent stem cells One of the most important properties of human pluripotent stem cells (hPSCs) is related to their ground or nave pluripotent state, which may have major impacts on hPSC growth, genetic engineering, disease modeling, and drug discovery. We have derived and comprehensively characterized naive-like hPSCs (NLPs) under various normoxic and hypoxic conditions. Our comparative meta-analysis indicates the existence of heterogeneous pluripotent states in diverse NLPs generated from different nave protocols. Interestingly, some NLPs exhibit much lower single cell plating efficiency, and commonly lack unique mouse and human NLP marker expression. Evidently, these cells represent an unrecognized minimal nave-like state downstream of formative pluripotency. Moreover, we revealed a unique metabolome associated with a limited metabolic reprograming capacity in these cells. Our current data provide significant insights into pluripotent state transitions and their associated downstream lineage priming. 2. Disease model generation Sjogrens syndrome Sjogrens syndrome is a disorder of the immune system characterized by two main symptoms which are dry eyes and dry mouth. Previously, we generated human iPSC lines from the salivary epithelial cells of Sjogrens syndrome patients in collaboration with Dr. Youmgmi Ji at NIDCR. Teratoma formation in immunocompromised rats is being carried out to determine the completeness of the reprogramming of the epithelial cells into pluripotent stem cells. To study the behavior of the diseased cells, they will be differentiated to form salivary gland organoids in vitro in comparison with those derived from normal iPSCs. To determine the optimal differentiation process for generating salivary gland organoids, marker cell lines are being made in which endogenous AQP5 protein, one of the salivary gland-specific markers, is tagged with GFP. Pituitary gland organoid development: In collaboration with Dr. Prashant Chittiboina at the NINDS, we study pituitary adenomas including releasing adenomas that causes Cushing's disease. Although very common (10% of human population), most adenomas are mutationally bland and have a varied phenotypic presentation. Currently, there are no established cell lines or mouse models that capture pituitary tumorigenesis. We are starting an hiPSC differentiation and pituitary organoid program to study this disease. The ongoing work includes culture and maintenance of hiPSCs, formulating various differentiation protocols to increase the efficiency of generating hormone-producing cells, and characterizing cellular properties of differentiated cells with immunofluorescence. This study will likely establish clinically relevant models in vitro to address primary/secondary pituitary disorders in human patients. As part of the study, they are developing the pituitary organoid from human iPSC lines. Pituitary gland is composed of several highly specialized cells, making it hard to track the differentiation process of each type of cells. To help achieve this, four key pituitary markers - SIX1, GATA2, POU1F1, TBX19 -are being tagged with GFP separately to generate marker hiPSC lines for cranial placodes, gonadotroph, somatotroph, and corticotroph, respectively. Batten disease In collaboration with Dr. Hee-Yong Kim at the NIAAA, we are using hiPSCs to investigate the effect of G-protein ligands on human brain development, including neurogenesis, neurite outgrowth, and synaptogenesis. In collaboration with Dr. An Dang Do and Dr. Forbes Porter at the NICHD, we study Batten disease, caused by mutations in the CLN3 gene (lysosomal/endosomal transmembrane proten), which codes for a transmembrane protein of unknown function. Batten disease is a fatal, neurodegenerative disease, characterized by lysosomal storage of proteins and other components, which has an estimated prevalence of 1:100,000. Classic CLN3 symptoms present asynchronously with vision loss occurring around pre-kindergarten age, neurodevelopmental plateauing and decline around 7-8 years of age, and motor and cardiac dysfunctions around mid-late teens. Currently, no reliable models are available for studying CLN3. We used reprogrammed hiPSC lines to test the hypothesis that CLN3 expression differentially affects cellular pathways at different stages of development. We are performing RNAi screens in these hiPSC differentiated models to identify potential targets and inhibitors associated with CLN3. Madras motor neuron disease Madras motor neuron disease (MMND) is characterized by weakness and atrophy of limbs, multiple lower cranial nerve palsies and sensorineural hearing loss. To study the pathophysiology of MMND, skin fibroblasts from two patients suffering from MMND were reprogrammed to hiPSC lines using Sendai virus. in collaboration with Dr. Christopher Grunseich at NINDS. Saul-Wilson syndrome Saul-Wilson syndrome (SWS) is a genetic disease characterized by short stature and other skeletal abnormalities. SWS is caused by mutations in the COG4 gene. This gene provides instructions for making one piece of a group of proteins known as the conserved oligomeric Golgi (COG) complex. Dr. Carlos Ferreira at NHGRI generated human iPSC lines from patients suffering from SWS. To analyze the cellular phenotype of the mutant cells, it is essential to have a control cell line without the mutation in the same genetic background (isogenic control). Using CRISPR/cas9 gene targeting technology, isogenic control hiPSC lines have been generated from SWS patient hiPSCs by converting the mutant sequence back to normal. PINK1 deficient mice PTEN-induced kinase 1 is a mitochondrial serine/threonine-protein kinase encoded by the PINK1 gene. PINK1 activity causes the Parkin protein to bind to depolarized mitochondria to induce autophagy of those mitochondria. Mutations in the PINK1 protein have been found to lead to a build-up of improperly folded proteins in the mitochondria in a number of Parkinsons patients. To generate a cellular model system for studying PINK1-induced Parkinsons disease, the PINK1 gene was knocked out using CRISPR/cas9 genome manipulation technology. 3. Reporter cell line generation GLIA markers We have been developing reliable protocols to differentiate hiPSCs into glial cells. As part of this, three endogenous glia-specific proteins GFAP, OLIG2, and CX3CR1 - have been tagged with GFP. The expression of GFP is being assessed after proper differentiation. Neuron subtypes The mammalian neocortex is a complex, highly organized structure that contains hundreds of different neuronal cell types. The generation of functionally specialized neural subtypes from hPSCs can be achieved by manipulating fundamental developmental principles. However, fine-tuning of these developmental principles to generate each neuronal subtypes is hampered by the lack of the tools to track the differentiation process. To generate such tools, selected endogenous neuron-specific proteins are being tagged with GFP. The following markers have been tagged so to date: MAP2 (neuron-specific microtubule associated protein 2), RHO (rhodopsin), SLC6A3 (dopamine transporter), SLC6A4 (serotonin transporter), SLC18A3 (acetylcholine transporter), and SLC32A1 (GABA transporter).

在上一财年，NIH SCCF 在许多领域取得了进展，如下所示。 1. 幼稚多能干细胞代谢重塑的可塑性 人类多能干细胞 (hPSC) 最重要的特性之一与其基态或原始多能状态有关，这可能对 hPSC 生长、基因工程、疾病建模和药物发现产生重大影响。我们在各种常氧和低氧条件下衍生并全面表征了幼稚型 hPSC (NLP)。我们的比较荟萃分析表明，不同 nave 协议生成的不同 NLP 中存在异质多能状态。有趣的是，一些 NLP 表现出低得多的单细胞铺板效率，并且通常缺乏独特的小鼠和人类 NLP 标记表达。显然，这些细胞代表了形成多能性下游的一种未被识别的最小中枢样状态。 此外，我们揭示了与这些细胞中有限的代谢重编程能力相关的独特代谢组。我们当前的数据为多能状态转变及其相关的下游谱系启动提供了重要的见解。 2. 疾病模型生成 干燥综合征 干燥综合征是一种免疫系统疾病，其特征是两个主要症状：眼干和口干。此前，我们与 NIDCR 的 Youmgmi Ji 博士合作，从干燥综合征患者的唾液上皮细胞中生成了人类 iPSC 系。 正在免疫功能低下的大鼠中进行畸胎瘤形成，以确定上皮细胞重编程为多能干细胞的完整性。 为了研究患病细胞的行为，他们将在体外分化形成唾液腺类器官，与正常 iPSC 衍生的细胞进行比较。为了确定生成唾液腺类器官的最佳分化过程，正在制作标记细胞系，其中内源性 AQP5 蛋白（唾液腺特异性标记物之一）被 GFP 标记。 垂体类器官发育： 我们与 NINDS 的 Prashant Chittiboina 博士合作，研究垂体腺瘤，包括导致库欣病的释放腺瘤。尽管很常见（占人类人口的 10%），但大多数腺瘤突变平淡，并且具有不同的表型表现。目前，还没有建立捕获垂体肿瘤发生的细胞系或小鼠模型。我们正在启动 hiPSC 分化和垂体类器官项目来研究这种疾病。正在进行的工作包括 hiPSC 的培养和维护、制定各种分化方案以提高产生激素的细胞的效率，以及通过免疫荧光表征分化细胞的细胞特性。这项研究可能会建立临床相关的体外模型来解决人类患者的原发性/继发性垂体疾病。作为研究的一部分，他们正在利用人类 iPSC 系开发垂体类器官。垂体由几种高度特化的细胞组成，因此很难追踪每种细胞的分化过程。为了帮助实现这一目标，四个关键垂体标记 - SIX1、GATA2、POU1F1、TBX19 - 分别用 GFP 进行标记，以分别生成颅板、促性腺激素细胞、生长激素细胞和促肾上腺皮质激素细胞的标记 hiPSC 系。 巴顿病 我们与 NIAAA 的 Hee-Yong Kim 博士合作，利用 hiPSC 来研究 G 蛋白配体对人脑发育的影响，包括神经发生、神经突生长和突触发生。我们与 NICHD 的 An Dang Do 博士和 Forbes Porter 博士合作，研究了由 CLN3 基因（溶酶体/内体跨膜蛋白）突变引起的 Batten 病，该基因编码一种功能未知的跨膜蛋白。 Batten 病是一种致命的神经退行性疾病，其特征是溶酶体储存蛋白质和其他成分，估计患病率为 1:100,000。典型的 CLN3 症状与学龄前左右的视力丧失、7-8 岁左右的神经发育平台期和衰退以及青少年中晚期左右的运动和心脏功能障碍同时出现。目前，没有可靠的模型可用于研究 CLN3。我们使用重编程的 hiPSC 系来检验 CLN3 表达对不同发育阶段的细胞通路产生不同影响的假设。我们正在这些 hiPSC 分化模型中进行 RNAi 筛选，以确定与 CLN3 相关的潜在靶点和抑制剂。 马德拉斯运动神经元病 马德拉斯运动神经元病（MMND）的特点是四肢无力和萎缩、多发性下颅神经麻痹和感音神经性听力损失。为了研究 MMND 的病理生理学，使用仙台病毒将两名 MMND 患者的皮肤成纤维细胞重新编程为 hiPSC 系。与 NINDS 的 Christopher Grunseich 博士合作。 索尔-威尔逊综合征 索尔-威尔逊综合征 (SWS) 是一种以身材矮小和其他骨骼异常为特征的遗传性疾病。 SWS 是由 COG4 基因突变引起的。该基因提供了制造一组称为保守寡聚高尔基体 (COG) 复合体的蛋白质中的一个片段的指令。 NHGRI 的 Carlos Ferreira 博士从 SWS 患者身上培育出人类 iPSC 系。为了分析突变细胞的细胞表型，必须有一个在相同遗传背景下没有突变的对照细胞系（等基因对照）。使用 CRISPR/cas9 基因打靶技术，通过将突变序列恢复正常，从 SWS 患者 hiPSC 中产生了等基因对照 hiPSC 系。 PINK1缺陷小鼠 PTEN 诱导激酶 1 是一种由 PINK1 基因编码的线粒体丝氨酸/苏氨酸蛋白激酶。 PINK1 活性导致 Parkin 蛋白与去极化线粒体结合，诱导这些线粒体的自噬。研究发现，PINK1 蛋白的突变会导致许多帕金森病患者线粒体中不正确折叠的蛋白质堆积。为了生成用于研究 PINK1 诱导的帕金森病的细胞模型系统，使用 CRISPR/cas9 基因组操作技术敲除 PINK1 基因。 3. 报告细胞系的生成 GLIA标记 我们一直在开发可靠的方案来将 hiPSC 分化为神经胶质细胞。作为其中的一部分，三种内源性神经胶质细胞特异性蛋白 GFAP、OLIG2 和 CX3CR1 已被 GFP 标记。适当分化后评估 GFP 的表达。 神经元亚型 哺乳动物新皮质是一个复杂的、高度组织化的结构，包含数百种不同的神经元细胞类型。从 hPSC 中生成功能特化的神经亚型可以通过操纵基本的发育原理来实现。然而，由于缺乏跟踪分化过程的工具，对这些发育原理进行微调以生成每种神经元亚型受到阻碍。为了生成这样的工具，选择的内源性神经元特异性蛋白被 GFP 标记。迄今为止，已标记以下标记：MAP2（神经元特异性微管相关蛋白 2）、RHO（视紫红质）、SLC6A3（多巴胺转运蛋白）、SLC6A4（血清素转运蛋白）、SLC18A3（乙酰胆碱转运蛋白）和 SLC32A1（GABA 转运蛋白）。