52 Cell Therapy Improved the Lifespan of Murine Models of Accelerated Aging with Defective Proliferation and Myogenic Differentiation Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © Th[.]
STEM CELL THERAPIES in the ES or iPS cells Furthermore, gene defects with unknown sites of mutation and those involving large deletions cannot be restored by homologous recombination Conventional gene transfer techniques can also insert DNA randomly into the host genome, possibly even resulting in cancer The use of human artificial chromosomes (HACs) as a vector for gene therapy may solve these problems, because HACs exhibit several important characteristics for an ideal gene therapy vector, including stable episomal maintenance and the capacity to carry large genomic loci with regulatory elements, thus allowing the physiological regulation of the introduced gene similar to the native chromosome Duchenne muscular dystrophy (DMD) is caused by dysfunction in dystrophin gene Although several DMD gene therapy vector have been developed, no episomal vector containing the entire dystrophin genomic region has been reported, due to its extremely large size (2.4Mb) As a proof of concept, we herein report the correction of a genetic deficiency in iPS cells derived from DMD model (mdx) mice and human DMD patient, using a HAC with a entire genomic dystrophin (Dys-HAC) which we have recently developed The Dys-HAC was transferred to the mdx- or DMD patient-specific iPS cells via microcell-mediated chromosome transfer (MMCT) FISH and multiplex PCR analyses showed that the Dys-HAC was present independently in the iPS cells and the transferred Dys-HAC corrected the deletion of dystrophin in the iPS cells, respectively Next, the iPS cells containing the Dys-HAC were injected into nude mice to assess the capacity of the differentiation and the expression in the teratoma The transplanted mdx-iPS (Dys-HAC) cells gave rise to differentiate to three germ layers and the human dystrophin expression was detected in the tissues Furthermore, chimeric mice from the mdx-iPS (Dys-HAC) were produced to assess the capacity of differentiation and human dystrophin expression in vivo The Dys-HAC was detected in the all tissues examined and the dystrophin was detected in the sarcolemmal membrene of the chimeric muscle Therefore, the combination of patient-specific iPS cells and HAC containing defective gene(s) provides a powerful tool for gene and cell therapies 51 Contribution of PDGFRa-Positive Bone Marrow Cells for Epithelial Regeneration in Genetic Blistering Skin Disease, RDEB Katsuto Tamai, Takehiko Yamazaki, Takenao Chino, Yasufumi Kaneda Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Physiologic homeostasis of epithelial structures is maintained by resident stem cells residing in the epithelia themselves In the skin of patients with recessive dystrophic epidermolysis bullosa (RDEB), the epidermal stem cell pool is continually depleted as a result of detachment of full-thickness epidermis from dermis due to genetic dysfunction of the dermo-epidermal basement membrane zone Nevertheless, the epidermal regeneration mechanisms in RDEB still adequately maintain functional epithelium in the skin, suggesting epithelial stem/progenitor cell-supplementation from extracutaneous sources In this study, we demonstrate that detached RDEB epithelia release a 25kDa protein, designated hear as KOIKOI (KOI2; come on come on in Japanese), to recruit platelet-derived growth factor receptor alpha (PDGFRa)-positive bone marrow cells via circulation Separated RDEB mouse epithelia immediately releases KOI2 in the blister fluid within 10 to 20 seconds Flow cytometry analysis indicated that intravenous inoculation of KOI2 induced mobilization of PDGFRa-positive cells in mouse circulation, resulting in robust elevation of the PDGFRa-positive cell population in the blood To elucidate the role of the mobilized PDGFRa-positive bone marrow cells in the regeneration of RDEB skin in vivo, we combined PDGFRa-positive/GFP-positive bone marrow cells (P/GBMCs) with wild-type bone marrow cells, and transplanted those Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy cells to the lethally irradiated mice to generate P/G-BMT mouse, followed by engraftment of RDEB mouse skin on the back of the P/G-BMT mouse In weeks after the RDEB skin engraftment, significant numbers of GFP-positive cells were observed in both mesenchymal and epithelial tissues of the engrafted RDEB skin The GFP-positive epithelial cells were then shown to express keratin in the regenerating RDEB epithelia, clearly demonstrating that the PDGFRa-positive bone marrow cells contain particular population to provide epithelial cells in the RDEB skin Collectively, our data suggest that KOI2, which is rapidly released from the separated RDEB epithelia of the blisters, contributes to mobilize PDGFRa-positive bone marrow cells in circulation, and accelerates regeneration of the skin lesions by recruiting those cells to raise marrow-derived mesenchymal and epithelial cells in the RDEB skin Finally we intravenously administered KOI2 to the mouse with full-thickness wound on the back of the skin KOI2 administration significantly accelerated wound closure, and more extraordinary, inhibited scar formation by recruiting circulating mesenchymal cells in the dermis of the regenerating skin Appropriate application of KOI2 protein or the expressing DNA vector may provide a novel therapeutic strategy to recruit PDGFRa-positive stem/progenitor cells of both mesenchymal and epithelial lineages from bone marrow to the damaged tissues for accelerating damaged tissue regeneration 52 Cell Therapy Improved the Lifespan of Murine Models of Accelerated Aging with Defective Proliferation and Myogenic Differentiation Mitra Lavasani,1 Aiping Lu,1 Joseph Feduska,1 Andria R Robinson,2 Laura J Niedernhofer,2 Johnny Huard.1 Department of Orthopaedic Surgery, Stem Cell Research Center, Pittsburgh, PA; 2Department of Microbiology and Molecular Genetics, University of Pittsburgh Cancer Institute, Pittsburgh, PA Genetic depletion of ERCC1 (Excision repair crosscomplementation-1) or XPF (complementation group F) in humans or mice causes accelerated aging of multiple organ systems including the musculoskeletal, nervous, hepatobiliary, renal and hematopoietic ERCC1-XPF-deficient (Ercc1-/-and Ercc1-/∆) mice express a unique phenotype that mimics human progeria (or symptoms of accelerated aging) XPF levels in Ercc1-/∆ mice are 10% that of wild type (WT) mice, whereas Ercc1-/- mice have 0% of the normal level of XPF Ercc1-/∆ mice have maximum lifespan of months while Ercc1-/mice only live for month Here we found that the number of slow adhering cells (SAC) isolated from progeroid Ercc1-/∆ mice using a modified preplate technique was substantially lower than that yielded from control littermates (Ercc1+/∆), indicating a loss of stem cells with aging In vitro Ercc1+/∆ cells were mitotically active and proliferate in a similar fashion as the WT-SAC previously isolated and characterized in our lab, while the Ercc1-/∆ cells showed much reduced mitotic activity and proliferated poorly Cells derived from Ercc1+/∆ mice fused to form significantly more and larger multi-nucleated myotubes than their Ercc1-/∆ counterparts (68.4% versus 21.9% respectively, p