| Title:
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Isolation, Characterization and Establishment of Spontaneously Immortalized Cell Line HRPE-2S with Stem Cell Properties.
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| Author(s):
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Hoda Shams Najafabadi, Zahra-soheila Soheili, Shahram Samiei, Hamid Ahmadieh, Ehsan Ranaei Pirmardan, Maryam Masoumi
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| Presentation Type:
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Oral
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| Subject:
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Retina and Retinal Cell Biology
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| Others:
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Presenting Author:
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| Name:
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Hoda Shams najafabadi
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| Affiliation :(optional)
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1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
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| E mail:
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shams.hoda250@gmail.com
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| Phone:
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Mobile: |
90138322880
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Abstract (Max 200 words)
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| Purpose:
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The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line which was cloned from a primary culture of human RPE cells. The cells maintained in culture for more than 3 years.
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| Methods:
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HRPE-2S cell line was isolated from primary RPE cell culture of 2 days old male donor. The growth pattern of the cells in culture was determined and specific proteins expression was assessed by immunostaining. The morphological characteristics were investigated using phase-contrast and electron microscopy. The cell line was karyotyped and its mitotic index was assessed. Functional properties such as clonogenicity, tumorigenesity, metastasis and invasion were examined by colony forming efficiency, anchorage-independent growth, scratch wound healing and invasion assay. Cells viability in serum free and hypoxic culture conditions and LDH isoenzymes expression patterns of hRPE cell line were identified as well.
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| Results:
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The cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 hours. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all RPE cell line. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cell line and primary RPE cells.
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| Conclusion:
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Characteristics of HRPE-2S cell line promises it as an in vitro model for RPE stem cell-based researches.
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| Attachment:
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