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Roy Joseph, Om P Srivastava, Roswell R Pfister, ; Modeling Keratoconus disease using Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2994.
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© ARVO (1962-2015); The Authors (2016-present)
To model Keratoconus disease using induced pluripotent stem cells (iPSC) generated from fibroblasts of both keratoconus and normal human corneal stroma by viral method.
Fibroblasts derived from corneal keratocytes of three normal and three keratoconus (KC) donors were reprogrammed directly to pluripotent stem cells (PSC). This was achieved by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides. A single polycistronic lentiviral vector co-expressing four transcription factors (Oct 4, Sox2, Klf4 and Myc) was used to yield induced pluripotent stem cells (iPSC). These cells were characterized by immunofluorescence detection of stem cell markers (SSEA4, Oct4 and Sox2). Total RNA preparations of iPSC derived from normal and keratoconus fibroblasts were analyzed by RNA sequencing (RNA-Seq) using the Illumina HiSeq2000 (paired end 2 × 50 bp sequencing runs with flow cells providing up to 300 Gb of sequence information per flow cell). TopHat version 2.0.13 was used to align the raw RNA-Seq fastq reads to the human mm9 reference genome using the short read aligner algorithms, Bowtie (Johns Hopkins University, Baltimore, MD, USA). The datasets were analyzed using Ingenuity Pathways Analysis (IPA) software (Ingenuity Systems).
Immunofluorescence analysis of the iPSC showed that they were positive for all the three stem cell markers (SSEA4, Oct4 and Sox2). A total of 28665 genes were analyzed by RNA-Seq, and of these, 4300 genes showed ≥ ±2 fold change with q-value < 0.05 in KC vs. normal corneas. Among several affected pathways identified by IPA search, the most significantly affected were cellular growth and proliferation, their movement, cell death and survival, cell cycle and cellular differentiation in KC corneas. Among the affected genes, FGFR2 (Fibroblasts growth factor receptor 2) was 5-fold down-regulated in KC iPSC compared to the normal iPSC, and this gene was involved in the above mentioned pathways.
We successfully reprogramed keratocytes-derived fibroblasts to iPSC from both normal and keratoconus corneas by using single polycistronic lentiviral vector. The affected genes and pathways identified in KC IPSC’s relative to normal iPSC’s by RNA-Seq analysis would be of great significance in understanding the potential mechanism of KC disease. Since these iPSC are patient-specific thus providing a powerful tool for translation medicine.
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