Abstracts for 2015th Annual Poster Presentation (Research Institute for Diseases of Old Age)

Type 2 diabetes (T2D) is a metabolic disease that affects over 340 million people worldwide.This condition can go unnoticed and undiagnosed for years, leading to a late stage where high blood sugar produces complications such as delayed wound healing .Current research has shown that eicosanoids participate in the decrease of insulin signaling and 12-HTT through its receptor BLT2 accelerates keratinocyte migration and wound healing .Aditionally, evidence has emerged about the role of nitric oxide (NO) as a proregenerative mediator that is decreased in type 2 diabetes, however there are no reports linking BLT2 and this free radical, as mechanism associated to reepithelization.Aim: to elucidate the role of keratinocyte BLT2 in response to hyperglycemia, and the participtation of NO as a mediator of cellular migration.Methods: 12 males, 9 weeks old C57BL/6J were divided in 2 groups.6 animals were fed for 5 weeks with High Fat Diet and 6 with control diet.During the protocol, the weight and blood glucose was measured weekly.At 5 th week the animals were euthanatized and the skin was analyzed.In vitro, HaCaT cells were transfected with a FLAG-tagged human BLT2-pCXN2.1 or the empty pCXN2.1 vector.Stable transfectants were selected in the presence of processes (FPs).Development of FPs was contained three morphological states, the primitive, immature, and mature FPs.Immature podocytes were columnar in shape and connected each other by junctional complex (JC), which migrated toward the basal side of the cell.When the JC was close to the basement membrane, immature podocytes started to interdigitate each other with primitive FPs under the level of JC.As the lengthening of primitive FPs, the JC moved between primitive FPs to form immature FPs.Finally, the JC was gradually replaced with the slit diaphragm, resulting that the immature FPs became the mature FPs.In conclusion, the developmental process of podocytes is now clearly understood by block-face SEM imaging. Differentiation into Inner Ear sensory Epithelia from iPS Cells Using Chick Inner Ear Cells and Application to Age Related Hearing LossIchiro Fukunaga Department of Otolaryngology Objectives: Approximately 40% of adults between the ages of 65 and 75 had diagnosis of age-related hearing loss (AHL).And, 70% of adults over 75 years old had diagnosis of AHL.Hearing aid or Cochlear implant apply to Age-related Hearing loss.However, fundamental therapeutic strategy has not been developed.Inner ear cell therapy has been expected to be an effective therapy for AHL.Recently, in vitro differentiation of ES/iPS cells into inner ear hair cell-like cells.However, these report were poorlyreproducible.In this study, we developed a new method for in vitro/in vivo differentiation of iPS cells into inner ear progenitor cells.Methods: The iPS cells were aggregated by floating culture.During the first 7 days, a series of precisely timed protein and small-molecule treatment sequentially induce epithelia that non-neural ectodermal and preplacodal ectoderm.On culture Day 7, aggregates were transfer on to feeder cells.Result: After the aggregate formation, epithelial like cells were observed at the surface of these aggregates.In a part of the aggregates, Connexin26 (Cx26) positive cells were observed and these cells showed GJP-like formations as cochlear cells.Conclusions: The Cx26 protein, which assembles to form channels between cells in the cochlear supporting cells.In this study, we demonstrate the differentiation of mouse iPS cells into Cx26 positive cells followed by Cx26-GJP formation. Generation of Induced Pluripotent Stem Cells from Connexin26 Conditional Knockout MiceToru Aoki Department of Otorhinolaryngology Objectives: Hereditary deafness affects approximately one in 1,600 children.Gjb2 gene encoding connexin26 (Cx26) plays important role in intercellular communication of cochlea Gap junction between the inner ear cells.We previously developed Cx26 knockout mice (CX26 f/f P0-Cre) by using Cre-loxP system.In this study, we generated induced pluripotent stem (iPS) cells from CX26 f/f P0-Cre mice to reproduce the pathogenesis caused by Cx26 deficiency.Our final goal is to produce the inner ear cell-like cells from the patients with GJB2 related hearing loss for drug screening and cell therapy.Methods: We dissected cochlear fibrocytes of CX26 f/f P0-Cre mice and C57BL/6 mice because Cx26 gene is specifically deleted in inner ear of this mouse.These cells were reprogramed to generate iPS cells by using Sendai-virus vector with reprogramming factors, Klf4, Oct3/4, Sox2 and c-Myc, and characterized the pluripotency by alkaline phosphatase (ALP) staining and immunostaining.Results: Cochlear fibrocytes were dissected from CX26 f/f P0-Cre mice and C57BL/6 mice.Cultured cochlea fibrocytes were infected with Sendai-virus (SeV) vector with reprograming genes.iPS cells generated CX26 f/f P0-Cre and C57BL/6 inner ear cells were stained with ALP and Oct3/4, Sox-2 and SSEA-1 antibodies as a marker of pluripotency.Conclusions: In this study, we generated iPS cells from the cochlear fibrocytes of CX26 f/f P0-Cre and C57BL/6 mice by SeV vector with reprogramming genes.The ALP staining and immunostaining suggested that the generated cells have a pluripotency as iPS cell.Disease specific iPS cells derived from mutant animal models will be powerful tool for drug screening and cell therapy targeting GJB2 related hereditary deafness. Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient MiceTakashi Anzai Research Institute for Diseases of Old Age Mutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness.The degeneration of the organ of Corti observed in Cx26 mutant-associated deafness is thought to be a through blood flow).

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