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Human Induced Pluripotent Stem Cells To Investigate Inherited Skin Diseases

人类诱导多能干细胞研究遗传性皮肤病

基本信息

批准号：
7677167
负责人：
LOUISE T CHOW
金额：
$ 5.41万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7677167
关键词：
3&apos Splice Site 5&apos Splice Site Accounting Acute Adverse effects Affect Air Aliquot Alkaline Phosphatase Antibiotics Biological Assay Biology Bulla CD15 Antigens Cell Cycle Cell division Cells Cellular Morphology Chromosomes Code Collaborations Collagen Coupled Cyclin B Cytomegalovirus Cytoplasm DNA DNA amplification Derivation procedure Dermal Detection Differentiation Antigens Disease Disease model Drug resistance Epidermolysis Bullosa Simplex Epithelial Excision Fibroblasts Figs - dietary Fluorescence Fluorescence Microscopy Formalin Gene Mutation Generalized Epidermolysis Bullosa Simplex Genes Genetic Recombination Genetic Skin Diseases Globin Hand Healed Histology Human Human Papillomavirus Human papillomavirus 16 E1 protein Human papillomavirus 18 Immunoglobulins In Situ In Vitro Indirect Immunofluorescence Inherited Internal Ribosome Entry Site Ker10 protein Keratin Knock-in Mouse Length Location Longevity Male Circumcision Mediating Mesenchymal Stem Cells Messenger RNA Methods Monitor Mus Mutation Neonatal Open Reading Frames Operon Paraffin Embedding Patient Care Patients Plasmid Cloning Vector Plasmids Primates Process Production Property Proteins Protocols documentation Publications RNA Splicing Relative (related person)Replicon Reporting Research Research Personnel Residual state Retroviridae Role Series Site Skin Skin Transplantation Skin graft Somatic Cell Source Southern Blotting Squamous Epithelium Staining method Stains Subfamily lentivirinae System Testing Therapeutic Time Tissues Transfection Transgenes Tretinoin Viral Virus Work antibiotic G 418 bacterial vector base bone morphogenic protein cell type chromatin remodeling design embryonic stem cell experience experimental analysis expression vector gene therapy healing homologous recombination human embryonic stem cell line in vitro Model in vivo induced pluripotent stem cell involucrin keratin 5 keratinocyte mutant novel therapeutic intervention penis foreskin pluripotency prevent programs promoter protein expression recombinase research study sickling skin disorder skin lesion stage-specific embryonic antigen 4 stem success tissue culture transcription factor transgene expression vector viral DNA

项目摘要

Skin is easily accessible and in principle is an ideal target for gene therapy of inherited skin disorders. However this has not become a reality. This proposed project intends to recapitulate rare inherited skin diseases in organotypic tissue cultures using keratinocytes differentiated from human induced pluripotent stem (iPS) cells into which relevant gene mutations have been introduced. Several labs have recently reported the derivation of iPS cells from mouse or human somatic cells. This advance creates a major opportunity for developing disease models and therapies. Somatic cells are reprogrammed to become iPS cells by expressing three chromatin-remodeling transcription factors, Sox2, KLF4, and Oct4 over a period of about 3 weeks. Tim Townes' lab has successfully cured mice with human sickle ceil disease using genecorrected iPS cells. Presently, the transgenes are introduced into the somatic cells via retroviruses or lentiviruses. However, mutagenic insertion of these vectors has been a serious concern and it is highly desirable to develop a non-integrating vector. The short and long term Specific Aims are: (1) To construct a non-integrating plasmid vector to express the transgenes. The replicon is based on the simple replication requirements of the human papillomavirus DMA plasmid. The strategy for transgene expression is being developed by the Townes lab and will be incorporated into our plasmid-based vectors. (2) To transfect vector DNA into neonatal foreskin fibroblasts and derive iPS cells. (3) To differentiate the iPS cells into the keratinocyte lineage. The properties of these keratinocytes and their ability to differentiate into squamous epithelium in organotypic cultures will be examined and compared to those of primary neonatal foreskin keratinocytes. (4) To recapitulate EB-simpfex skin models in vitro. Dominant mutations in keratin 5 or keratin 14 genes identified in epidermolysis bullosa simplex patients will be introduced into iPS cells (or human fibroblasts prior to derivation of iPS cells) by homologous recombination. The iPS cells will be differentiated into keratinocytes that will then be used to develop squamous epithelium in organotypic raft cultures and examined by in situ methods. Success in these experiments would serve as proof-of-principle that iPS cells can be used widely by researchers to study genetic skin diseases and to test for new therapeutic approaches.

皮肤易于接近，原则上是遗传性皮肤病基因治疗的理想靶点。然而这并没有成为现实。这个拟议的项目旨在概括罕见的遗传性皮肤使用从人诱导多能分化的角质形成细胞的器官型组织培养物中的疾病干细胞（iPS），其中已引入相关基因突变。几个实验室最近报道了从小鼠或人类体细胞衍生iPS细胞。这一进步创造了一个重大的为开发疾病模型和疗法提供了机会。体细胞被重新编程为iPS 细胞通过表达三个染色质重塑转录因子，Sox 2，KLF 4和Oct 4，在一段时间内，大约三个星期。Tim Townes的实验室已经成功治愈了患有人类镰状细胞病的小鼠， iPS细胞目前，转基因通过逆转录病毒或逆转录酶导入体细胞。慢病毒然而，这些载体的致突变插入一直是一个严重的问题，它是高度关注的。希望开发非整合载体。近期和远期的具体目标是：（1）建立一个非整合质粒载体表达转基因。复制子是基于简单复制人乳头瘤病毒DNA质粒的要求。转基因表达的策略是由汤斯实验室开发，并将被纳入我们的质粒载体。(2)选择矢量将DNA导入新生儿包皮成纤维细胞并衍生iPS细胞。(3)为了将iPS细胞分化为角质形成细胞谱系。这些角质形成细胞的特性及其分化为鳞状细胞的能力将检查器官型培养物中的上皮细胞，并与原发性新生儿包皮的上皮细胞进行比较角质形成细胞(4)总结EB-simpfex体外皮肤模型。角蛋白5或角蛋白在单纯性大疱性表皮病患者中鉴定的14个基因将被引入iPS细胞（或人 iPS细胞衍生前的成纤维细胞）。iPS细胞将分化为转化为角质形成细胞，然后用于在器官型筏培养物中发育鳞状上皮，用原位方法检测。这些实验的成功将证明iPS细胞可以被研究人员广泛用于研究遗传性皮肤病和测试新的治疗方法。接近。