Human Induced Pluripotent Stem Cells To Investigate Inherited Skin Diseases

皮肤很容易接近，原则上是遗传性皮肤疾病基因治疗的理想目标。

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.