Human Induced Pluripotent Stem Cells To Investigate Inherited Skin Diseases

皮肤很容易接触，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。这个提议的项目打算重述罕见的遗传皮肤。 人诱导多能性角质形成细胞在器官型组织培养中的疾病 已引入相关基因突变的干细胞(IPS)。几个实验室最近 报道了从小鼠或人体细胞衍生的诱导性多能性细胞。这一进步创造了一个重大的 开发疾病模型和治疗方法的机会。体细胞被重新编程为iPS 通过在一段时间内表达三种染色质重塑转录因子Sox2、KLF4和Oct4 大约3周。蒂姆·汤斯的实验室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 慢病毒。然而，这些载体的突变性插入一直是一个严重的问题，它高度 需要开发一个非积分向量。近期和长期的具体目标是：(1)建设 非整合质粒载体表达转基因。复制图标基于简单复制 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，并将被整合到我们的基于质粒的载体中。(2)转染载体 将DNA导入新生儿包皮成纤维细胞，获得iPS细胞。(3)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 器官培养中的上皮细胞将被检查，并与初级新生儿包皮的上皮细胞进行比较。 角质形成细胞。(4)建立EB-simpfex体外皮肤模型。角蛋白5或角蛋白的显性突变 在单纯性大疱性表皮松解症患者中发现的14个基因将被导入iPS细胞(或人类 IPS细胞衍生之前的成纤维细胞)。IPS细胞将被分化 角质形成细胞，然后用来在器官移植培养中发育鳞状上皮 采用原位方法进行检测。这些实验的成功将证明iPS细胞 可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 接近了。

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.