Human pluripotent stem cells

人类多能干细胞

• 批准号: 10263065
• 负责人: Pamela Robey
• 金额: $ 181.78万
• 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/10263065
• 关键词: 3-Dimensional; Area; Basal lamina; Biological Assay; Biological Process; CRISPR/Cas technology; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell membrane; Cells; Clinic; Clinical; Clone Cells; Collaborations; Communities; Craniosynostosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Sequence Alteration; Defect; Derivation procedure; Diabetes Mellitus; Diagnosis; Disease; Disease model; ECM receptor; Educational process of instructing; Electron Microscopy; Embryonic Development; Endoderm; Epithelial; Epithelium; Event; Extracellular Matrix; Eye diseases; FGFR3 gene; Family; Fluorescence Microscopy; Functional disorder; Future; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genetic Heterogeneity; Genetic Polymorphism; Genetic Transcription; Genomic Instability; Genotype; Glial Fibrillary Acidic Protein; Goals; Human; Hypothyroidism; In Vitro; Informal Social Control; Karyotype determination procedure; Knock-in; Knock-out; Link; Maintenance; Manuscripts; Mentors; Metabolism; Methods; Methylation; Mission; Monitor; Morphology; Muenke Syndrome; Mus; Muscular Dystrophies; Mutation; Mutation Analysis; National Institute of Environmental Health Sciences; Netherlands; Neural Crest; Neuroglia; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Receptors; Organ; Organoids; Pathogenicity; Pathologic; Pathway interactions; Patients; Peripheral; Pharmaceutical Preparations; Pharmacogenomics; Phenotype; Pluripotent Stem Cells; Preclinical Drug Development; Preparation; Process; Protein Kinase; Proteins; Protocols documentation; Publications; Quality Control; Regenerative Medicine; Regulator Genes; Reporting; Research Personnel; Role; Sampling; Side; Structure; Structure of retinal pigment epithelium; System; TP53 gene; Technology; Teratoma; Tissues; United States National Institutes of Health; Update; VX-770; VX-809; Variant; Work; X Chromosome; Zinc Fingers; alpha Dystroglycan; base; blood glucose regulation; bone; brain malformation; cell community; cell growth; cell type; clinical center; clinical phenotype; cohort; design; diabetes risk; drug discovery; dystroglycanopathy; effective therapy; gain of function mutation; gastrulation; genome wide association study; genome-wide; human disease; human embryoid body; human embryonic stem cell; human pluripotent stem cell; induced pluripotent stem cell; interest; large datasets; member; mutant; nerve stem cell; pluripotency; response; scaffold; self-renewal; small molecule; stem cell model; stem cell technology; stem cells; tool; transcription factor; web site

During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below. We have been investigating in-depth the concept of naive pluripotency, which could be important in studying diseases related to the X chromosome. We are currently analyzing large datasets related to gene expression, metabolism and methylation status and a manuscript on this work should be submitted in the coming fiscal year. Traceable markers in human pluripotent stem cell are a valuable tool to study biological processes. To generate various human pluripotent stem cell clones expressing tagged proteins, CRISPR technology has been adapted to introduce GFP or RFP cassettes to the C-termini of endogenous proteins. We are currently generating hPSC lines which express CNS cell type-specific markers for use by the scientific community. Specifically, fluorescent protein tags are being added to the C-termini of glia-specific markers such as GFAP, OLIG2 and CX3CR1. Once constructed, cell lines are differentiated to confirm correct gene targeting. We continue to collaborate with Dr. Curtis Harris (NCI), on genomic instability events using a cohort of well-controlled hESC samples. These samples are being examined with various genome-wide technologies and we expect to finish these analyses in the coming fiscal year. These comprehensive analyses will likely provide important information regarding the effect of genomic alterations on human pluripotent stem cell growth under long-term cell culture conditions. Our collaboration with Dr. Ettore Appella (NCI), investigating the role of p53 in the regulation of the self-renewal and differentiation of neural precursors or neural stem cells, is also reaching completion. A manuscript on this work is currently in preparation. In collaboration with Dr. Wei Zheng (NCATS) and Dr. Jeffrey Beekman (Regenerative Medicine Center Utrecht, The Netherlands) we considered the use of hPSCs and derivative organoids in drug discovery, in order to define a pathway to implement hPSC-based drug discovery (hPDD). In a recent publication, we reviewed the pharmacogenomics of diverse mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Targeting specific CFTR genotypes based on small molecules has been hindered because of the substantial genetic heterogeneity of CFTR mutations in patients. There are broadly four classes (e.g., II, III, and IV) of CF genotypes that differentially respond to current CF drugs (e.g., VX-770 and VX-809). There is an emerging role for stem cell-based organoids in predicting the CF drug response based on the mechanisms underlying the differential CF drug response. Use of stem cell based organoid assays will facilitate the design of safer and more effective therapies. (Chen et al, Drug Discov Today, 2019). Following on from a previous collaboration with Dr. Anton Jetten (NIEHS), the role of GLI-similar 3 (GLIS3). GLIS3 is a member of the GLIS family of zinc finger transcription factors. Deficiency of GLIS3 in mice and humans cause pathological conditions such as hypothyroidism and diabetes. The link of GLIS3 and diabetes was further supported by GWAS, which linked variants of GLIS3 to increased risk of diabetes. JAZF1 is another zinc finger protein and acts as a transcriptional co-regulator. JAZF1 protein is known to interact with a range of nuclear receptor protein kinases, and other transcription factors. GWAS has shown that JAZF1 polymorphisms are closely associated with type 2 diabetes. Recently the interaction of JAZF1 and GLIS3 has been suggested by Dr. Jetten at NIEHS. We have generated hESC lines with GLIS3 knockout and HA-tagged GLIS3 knock-in to study the role of GLIS3 in glucose regulation. To extend the study into the interaction between GLIS3 and JAZF1, we are generating hESC lines with JAZF1 knockout and HA knock-in. This year, studies were completed on alpha-dystroglycanopathies, which are caused by dysfunction of the ECM receptor, alpha-dystroglycan, and are characterized by eye disease, muscular dystrophies and specific types of brain malformation. Disruption of the basal lamina and its functions is central to these disease processes. The basal lamina is a specialized sheet of dense extracellular matrix (ECM) linked to the plasma membrane of specific cell types in their tissue context, which serves as a structural scaffold for organ genesis and maintenance. However, opportunities to study the basal lamina in various human disease tissues are restricted owing to its limited accessibility. We have reported the generation of embryoid bodies from human induced pluripotent stem cells that model the basal lamina. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In -dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy reveal ultrastructural basal lamina defects and reduced ECM accumulation. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in the -dystroglycanopathies. We applied this system to evaluate an experimental ribitol supplement therapy on genetically diverse -dystroglycanopathy patient samples (Nickolls et al, DisModel Mech, 2020). The Unit also participated in studies on Muenke syndrome, caused by a p.Pro250Arg (c.749C>G) gain-of-function mutation in the FGFR3 gene. It is the leading genetic cause of craniosynostosis and results in a variety of disabling clinical phenotypes. To model the disease and study the pathogenic mechanisms, a human induced pluripotent stem cell (hiPSC) line was generated from a patient diagnosed with Muenke syndrome. Successful reprogramming was validated by morphological features, karyotyping, loss of reprogramming factors, expression of pluripotency markers, mutation analysis and teratoma formation. These cells will be used in future studies to study the differentiation of the mutant iPSCs into neural crest-derived bone and uncover pathogenetic mechanism that result in the craniosynostosis phenotype (Mui et al, Stem Cell Rep, 2020). In terms of bringing pluripotent stem cells to the clinic, we have provided advice on assays and culture of iPSCs related to Dr. Kapil Bharti (NEI) clinical initiative regarding iPSC derivation and differentiation into retinal pigmented epithelial cells. We continued to mentor and teach, both standard and feeder-free, pluripotent stem cell cultures, provided assistance and advice on the generation of human induced pluripotent stem cells (hiPSCs) from collaborators samples, as well as assistance and advice on differentiation strategies as requested. As always, we continue to update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.