title: Derivation, characterization and retinal differentiation of induced pluripotent stem cells.
abstract: Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.
authors: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Prof Brien Holden Eye Research Centre,  Hyderabad Eye Research Foundation, C-TRACER, L.V. Prasad Eye Institute, Hyderabad, India.
authors: Subba Rao
authors: SR
authors: Mekala
authors: Vasundhara
authors: V
authors: Vauhini
authors: Usha
authors: U
authors: Nagarajan
authors: Savitri
authors: S
authors: Maddileti
authors: Subhash
authors: S
authors: Gaddipati
authors: Indumathi
authors: I
authors: Mariappan
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D019175:DNA Methylation; D058732:Embryoid Bodies / Q000166:cytology* / Q000378:metabolism; D044127:Epigenesis, Genetic; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005136:Eye Proteins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489428:Myc protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse
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