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：
7691491
负责人：
LOUISE T CHOW
金额：
$ 4.52万
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：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7691491
关键词：
Address Adenoviruses Adhesions Adult Affect Area Autologous Basal Cell Biological Models Bulla Burn injury Cell Lineage Cell Therapy Cells Cellular Morphology Chromosomes Clinical Trials Collaborations Collagen Type VII Collection Complementary DNA Cultured Cells DNA Dependovirus Derivation procedure Dermal Desmosomes Development Disease Disease model Ectopic Expression Embryo Epidermolysis Bullosa Epidermolysis Bullosa Simplex Epithelial Epithelial Cells Experimental Models Fibroblasts Follow-Up Studies Frequencies Gelatin Gene Mutation Gene Transfer Genes Genetic Genetic Recombination Genetic Skin Diseases Genotype Globin Healed Hematopoietic Hereditary Disease Histocompatibility Human Human Papillomavirus In Situ In Vitro Indirect Immunofluorescence Individual Infection Inherited Injection of therapeutic agent Inner Cell Mass Integrins Investigation Knowledge Lesion Malignant Neoplasms Mediating Messenger RNA Methods Mus Mutation Neonatal Neuroepithelial Cells Nude Mice Oncolytic Patients Phenotype Plasmid Cloning Vector Plasmids Property Proto-Oncogenes Protocols documentation Provirus Integration Regulation Religion and Spirituality Replicon Reporting Research Research Personnel Retroviridae Role Serum-Free Culture Media Sickle Cell Anemia Signal Transduction Skin Skin Tissue Skin graft Somatic Cell Squamous Epithelium Stem cells Subfamily lentivirinae Sum System Tail Testing Therapeutic Trans-Splicing Transgenes Tretinoin Tumor Suppressor Genes Week base blastocyst bone morphogenic protein c-myc Genes c-myc Proto-Oncogenes cellular engineering chromatin remodeling day embryonic stem cell gene correction gene therapy gene therapy clinical trial healing homologous recombination implantation improved in vitro Model in vivo Model induced pluripotent stem cell inhibitor/antagonist interest keratin 14, K14 keratin 5 keratinocyte knockout gene laminin-5 mouse model non-oncogenic novel novel therapeutics penis foreskin pluripotency progenitor research study self-renewal sickling skin disorder skin xenograft stem success tissue culture tool transcription factor transgene expression vector

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.

皮肤很容易获得，原则上是遗传性皮肤疾病基因治疗的理想靶标。但是，这并没有成为现实。该建议的项目旨在概括稀有的遗传皮肤使用角质形成细胞与人类诱导多能分化的器官组织培养物中的疾病引入了相关基因突变的茎（IP）细胞。最近有几个实验室报道了来自小鼠或人类体细胞细胞的IPS细胞的推导。这个进步创造了一个专业开发疾病模型和疗法的机会。体细胞被重编程为IPS 通过表达三个染色质复制转录因子SOX2，KLF4和OCT4的细胞。大约3周。蒂姆·汤斯（Tim Townes）的实验室已使用Genecorcor纠正成功治愈了人类镰刀疾病的小鼠 IPS细胞。目前，通过逆转录病毒或慢病毒。但是，对这些向量的诱变插入一直是一个严重的关注，这是高度的希望发展非整合向量。短期和长期特定的目的是：（1）构建非整合质粒载体表达转基因。复制子基于简单的复制人乳头瘤病毒DMA质粒的要求。转基因表达的策略正在由Townes Lab开发，并将被纳入我们的基于质粒的载体中。（2）转染向量 DNA进入新生儿包皮细胞并得出IPS细胞。（3）将IPS细胞区分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的能力将检查细胞型培养物中的上皮，并将其与原发性新生儿包皮相比角质形成细胞。（4）在体外概括EB-Simpfex皮肤模型。角蛋白5或角蛋白中的主要突变在表皮溶液中鉴定出的14个基因单纯子患者将被引入IPS细胞（或人类）通过同源重组，在衍生IPS细胞之前成纤维细胞。 IPS单元将分化进入角质形成细胞，然后将其用于在器官筏培养物中发展鳞状上皮通过原位方法检查。这些实验的成功将是IPS细胞的原理证明研究人员可以广泛使用研究遗传皮肤疾病并测试新的治疗性方法。