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).

在上一财政年度，NIH SCCF在以下强调的一些领域取得了进展。