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      Modeling Keratoconus Using Induced Pluripotent Stem Cells

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          Abstract

          Purpose

          To model keratoconus (KC) using induced pluripotent stem cells (iPSC) generated from fibroblasts of both KC and normal human corneal stroma by a viral method.

          Methods

          Both normal and KC corneal fibroblasts from four human donors were reprogramed directly by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, using a single polycistronic lentiviral vector coexpressing four transcription factors (Oct 4, Sox2, Klf4, and Myc) to yield iPSC. These iPS cells were characterized by immunofluorescence detection using of stem cell markers (SSEA4, Oct4, and Sox2). The mRNA sequencing was performed and the datasets were analyzed using ingenuity pathways analysis (IPA) software.

          Results

          The generated stem cell-like clones expressed the pluripotency markers, SSEA4, Oct4, Sox2, Tra-1-60, and also expressed pax6. Our transcriptome analysis showed 4300 genes, which had 2-fold change and 870 genes with a q-value of <0.05 in keratoconus iPSC compared to normal iPSC. One of the genes that showed difference in KC iPSC was FGFR2 (down-regulated by 2.4 fold), an upstream target of Pi3-Kinase pathway, was further validated in keratoconus corneal sections and also KC iPSC-derived keratocytes (down regulated by 2.0-fold). Both normal and KC-derived keratocytes expressed keratocan, signature marker for keratocytes. KC iPSC-derived keratocytes showed adverse growth and proliferation and was further confirmed by using Ly2924002, a PI3k inhibitor, which severely affected the growth and differentiation in normal iPSC.

          Conclusions

          Based on our result, we propose a model for KC in which inhibition FGFR2-Pi3-Kinase pathway affects the AKT phosphorylation, and thus affecting the keratocytes survival signals. This inhibition of the survival signals could be a potential mechanism for the KC-specific decreased cell survival and apoptosis of keratocytes.

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          Most cited references46

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          The transcriptional landscape of the yeast genome defined by RNA sequencing.

          The identification of untranslated regions, introns, and coding regions within an organism remains challenging. We developed a quantitative sequencing-based method called RNA-Seq for mapping transcribed regions, in which complementary DNA fragments are subjected to high-throughput sequencing and mapped to the genome. We applied RNA-Seq to generate a high-resolution transcriptome map of the yeast genome and demonstrated that most (74.5%) of the nonrepetitive sequence of the yeast genome is transcribed. We confirmed many known and predicted introns and demonstrated that others are not actively used. Alternative initiation codons and upstream open reading frames also were identified for many yeast genes. We also found unexpected 3'-end heterogeneity and the presence of many overlapping genes. These results indicate that the yeast transcriptome is more complex than previously appreciated.
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            Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.

            G Martin (1981)
            This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.
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              In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.

              Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.
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                Author and article information

                Journal
                Invest Ophthalmol Vis Sci
                Invest. Ophthalmol. Vis. Sci
                iovs
                Invest Ophthalmol Vis Sci
                iovs
                Investigative Ophthalmology & Visual Science
                The Association for Research in Vision and Ophthalmology
                0146-0404
                1552-5783
                July 2016
                : 57
                : 8
                : 3685-3697
                Affiliations
                [1 ]Department of Optometry and Vision Science University of Alabama at Birmingham, Birmingham, Alabama, United States
                [2 ]Eye Research Laboratory, Eye Research Foundation, Birmingham, Alabama, United States
                Author notes
                Correspondence: Om P. Srivastava, Department of Vision Sciences, 924 18th Street South, University of Alabama at Birmingham, Birmingham, AL 35294-4390, USA; srivasta@ 123456uab.edu .
                Article
                iovs-57-07-62 IOVS-16-19105
                10.1167/iovs.16-19105
                5996875
                27403997
                b7e9ab2b-0679-4d5e-afd7-26011b5bc9ba

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

                History
                : 8 January 2016
                : 2 June 2016
                Categories
                Cornea

                keratoconus,induced pluripotent stem cells,fgfr2,disease modeling

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