Human pluripotent stem cells

人类多能干细胞

基本信息

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
项目类别：
财政年份：
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&apos 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&apos 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

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&apos 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&apos 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

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项目摘要

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).

在上一个财政年度，NIH SCCF在以下强调的许多领域取得了进展。 1。在天真的多能干细胞中代谢重塑的可塑性人多能干细胞（HPSC）最重要的特性之一与其地面或中殿多能状态有关，这可能对HPSC生长，基因工程，疾病建模和药物发现有重大影响。在各种常氧和低氧条件下，我们已经得出并全面地表征了幼稚的HPSC（NLP）。我们的比较荟萃分析表明，在不同的中殿方案产生的不同NLP中存在异质多能状态。有趣的是，某些NLP表现出较低的单细胞电镀效率，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性多能的下游未识别的最小中殿样状态。此外，我们揭示了与这些细胞中代谢能力有限的代谢能力有限的独特代谢组。我们当前的数据为多能状态过渡及其相关的下游谱系启动提供了重要的见解。 2。疾病模型产生 Sjogrens综合征 Sjogrens综合征是一种免疫系统的疾病，其特征是两种主要症状是干眼和口干。以前，我们与NIDCR的Youmgmi JI博士合作，从Sjogrens综合征患者的唾液上皮细胞中产生了人IPSC系。正在进行免疫功能低下的大鼠中的畸胎瘤形成，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，与正常IPSC相比，它们将在体外区分以形成唾液腺类器官。为了确定产生唾液腺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）用GFP标记。垂体腺体器官发育：在与Ninds的Prashant Chittiboina博士合作的情况下，我们研究了垂体腺瘤，包括释放引起库欣病的腺瘤。尽管非常普遍（人口占10％），但大多数腺瘤在突变上是平淡的，并且表型表现不同。当前，尚无已建立的细胞系或小鼠模型来捕获垂体肿瘤发生。我们正在启动HIPSC分化和垂体器官程序来研究这种疾病。正在进行的工作包括培养和维持HIPSC，制定各种分化方案，以提高产生激素的细胞的效率，并表征具有免疫荧光的分化细胞的细胞性质。这项研究可能会在体外建立与临床相关的模型，以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，他们正在从人IPSC系中开发垂体器官。垂体由几个高度专业的细胞组成，因此很难跟踪每种细胞的分化过程。为了帮助实现这一目标，四个关键的垂体标记-SIX1，GATA2，POU1F1，TBX19-分别用GFP标记，以生成用于颅骨位置的标记hipsc系，分别为颅骨斑块，促性腺激素，somatotroph和皮质营养素。板条疾病与NIAAA的Hee-Yong Kim博士合作，我们正在使用HIPSC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。通过与NICHD的Ang Do和Forbes Porter博士合作，我们研究了由CLN3基因突变（溶酶体/内体跨膜蛋白）引起的Batten疾病，该疾病代码编码未知功能的跨膜蛋白。巴顿疾病是一种致命的神经退行性疾病，其特征是蛋白质和其他成分的溶酶体储存，估计患病率为1：100,000。经典的CLN3症状异常出现，视力丧失发生在幼儿园前年龄，神经发育高原和7-8岁左右的下降，以及中期青少年左右的运动和心脏功能障碍。当前，没有可靠的模型可用于研究CLN3。我们使用重编程的HIPSC线来检验以下假设：CLN3表达在不同发育阶段在不同阶段影响细胞途径。我们正在这些HIPSC分化模型中进行RNAi筛选，以识别与CLN3相关的潜在靶标和抑制剂。马德拉斯运动神经元疾病 Madras运动神经元疾病（MMND）的特征是四肢萎缩和萎缩，多个下颅神经麻痹和感觉神经性听力损失。为了研究MMND的病理生理学，使用仙台病毒将两名患者的两名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 - 威尔逊综合症索尔 - 威尔逊综合征（SWS）是一种遗传疾病，其特征是身材矮小和其他骨骼异常。 SWS是由COG4基因突变引起的。该基因提供了制作一组蛋白质的指令，称为保守的低聚物高尔基（COG）复合物。 NHGRI的Carlos Ferreira博士从患有SWS患者的患者中产生了人类IPSC系列。为了分析突变细胞的细胞表型，必须在相同的遗传背景（等源性对照）中具有对照细胞系没有突变的对照细胞系。使用CRISPR/CAS9基因靶向技术，通过将突变序列转换回正常，从SWS患者HIPSC产生了同基因控制HIPSC系。 Pink1缺乏小鼠 PTEN诱导的激酶1是由PINK1基因编码的线粒体丝氨酸/苏氨酸 - 蛋白激酶。 PINK1活性导致帕克蛋白蛋白与去极化的线粒体结合，以诱导这些线粒体的自噬。已经发现PINK1蛋白的突变导致许多帕金森氏症患者在线粒体中折叠不当的蛋白质的积累。为了生成用于研究PINK1诱导的帕金森氏病的细胞模型系统，使用CRISPR/CAS9基因组操纵技术将PINK1基因淘汰。 3。记者细胞线生成胶质标记我们一直在开发可靠的方案，以将HIPSC区分为神经胶质细胞。作为其中的一部分，三个内源性神经胶质特异性蛋白GFAP，OLIG2和CX3CR1 - 已用GFP标记。适当分化后，正在评估GFP的表达。神经元亚型哺乳动物新皮质是一种复杂的高度组织结构，包含数百种不同的神经元细胞类型。可以通过操纵基本发展原理来实现HPSC的功能专业神经亚型的产生。但是，由于缺乏跟踪分化过程的工具，对这些发育原理的微调妨碍了产生每种神经元亚型的微调。为了生成这样的工具，已选定的内源性神经元特异性蛋白被GFP标记。迄今为止已经标记了以下标记：MAP2（神经元特异性微管相关蛋白2），Rho（Rhodopsin），SLC6A3（多巴胺转运蛋白），SLC6A4（5-羟色胺转运蛋白），SLC18A3，SLC18A3（乙酰胆碱转运器）和SLC322A2A（GABA）。