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Photoreceptor-like cells from reprogramming cultured mammalian RPE cells.
Mol Vis. 2013 May 30;19:1178-87. Print 2013. doi:
Yan RT, Li X, Huang J, Guidry C, Wang SZ
Abstract:
PURPOSE:
Previous studies showed that chick retinal pigment epithelium (RPE) cells can be reprogrammed by a specific gene to take on the path of photoreceptor differentiation. In this study, we tested whether this reprogramming scheme could be applied to mammalian RPE cells.
METHODS:
Human RPE cell lines ARPE-19, a spontaneously transformed line of RPE cells derived from a 19-year-old person, and hTERT-RPE1, a telomerase-immortalized RPE cell line derived from a 1-year-old person, were commercially obtained and cultured as recommended. Primary RPE cell cultures were established using RPE isolated from 3- to 6-month-old pig and postnatal day 5 mouse. Cultured cells were transduced with a virus expressing neuroD, neurogenin1 (ngn1), or ngn3, basic helix-loop-helix (bHLH) genes previously identified as capable of inducing RPE-to-photoreceptor reprogramming in the chick system. Alternatively, cells in the culture were transfected chemically or physically through electroporation with vector DNA expressing one of the three genes. The cultures were then analyzed for RPE-to-photoreceptor reprogramming with in situ hybridization and/or immunostaining for photoreceptor gene expression.
RESULTS:
Both hTERT-RPE1 and ARPE-19 cultures gave rise to cells bearing markers of photoreceptors after transduction or transfection with vehicles expressing neuroD or ngn1. The new cells expressed genes encoding photoreceptor proteins, including interphotoreceptor retinoid-binding protein IRBP), recoverin, retinal cone arrestin 3, transducin α-subunit, Cone-rod homeobox protein (Crx), and red opsin. They displayed morphologies resembling differentiating photoreceptor cells. In primary porcine and mouse RPE cell cultures, transduction with lenti virus (Lvx-IRES-ZsGreen1) expressing ngn1 or ngn3 resulted in the emergence of ZsGreen1(+) cells that exhibited morphologies reminiscent of differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1(+) cells were positive for neural marker microtubule-associated protein 2 (Map2) and photoreceptor hallmark proteins red opsin and rhodopsin.
CONCLUSIONS:
The results suggest that cells in human RPE cell lines and in primary cultures of porcine and mouse RPE respond to gene-induced reprogramming by giving rise to photoreceptor-like cells. The responsiveness of primary RPE cells, especially those from porcine cells, enhances the biologic feasibility of exploring RPE-to-photoreceptor reprogramming for in situ mammalian photoreceptor replacement without cell transplantation.
Previous studies showed that chick retinal pigment epithelium (RPE) cells can be reprogrammed by a specific gene to take on the path of photoreceptor differentiation. In this study, we tested whether this reprogramming scheme could be applied to mammalian RPE cells.
METHODS:
Human RPE cell lines ARPE-19, a spontaneously transformed line of RPE cells derived from a 19-year-old person, and hTERT-RPE1, a telomerase-immortalized RPE cell line derived from a 1-year-old person, were commercially obtained and cultured as recommended. Primary RPE cell cultures were established using RPE isolated from 3- to 6-month-old pig and postnatal day 5 mouse. Cultured cells were transduced with a virus expressing neuroD, neurogenin1 (ngn1), or ngn3, basic helix-loop-helix (bHLH) genes previously identified as capable of inducing RPE-to-photoreceptor reprogramming in the chick system. Alternatively, cells in the culture were transfected chemically or physically through electroporation with vector DNA expressing one of the three genes. The cultures were then analyzed for RPE-to-photoreceptor reprogramming with in situ hybridization and/or immunostaining for photoreceptor gene expression.
RESULTS:
Both hTERT-RPE1 and ARPE-19 cultures gave rise to cells bearing markers of photoreceptors after transduction or transfection with vehicles expressing neuroD or ngn1. The new cells expressed genes encoding photoreceptor proteins, including interphotoreceptor retinoid-binding protein IRBP), recoverin, retinal cone arrestin 3, transducin α-subunit, Cone-rod homeobox protein (Crx), and red opsin. They displayed morphologies resembling differentiating photoreceptor cells. In primary porcine and mouse RPE cell cultures, transduction with lenti virus (Lvx-IRES-ZsGreen1) expressing ngn1 or ngn3 resulted in the emergence of ZsGreen1(+) cells that exhibited morphologies reminiscent of differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1(+) cells were positive for neural marker microtubule-associated protein 2 (Map2) and photoreceptor hallmark proteins red opsin and rhodopsin.
CONCLUSIONS:
The results suggest that cells in human RPE cell lines and in primary cultures of porcine and mouse RPE respond to gene-induced reprogramming by giving rise to photoreceptor-like cells. The responsiveness of primary RPE cells, especially those from porcine cells, enhances the biologic feasibility of exploring RPE-to-photoreceptor reprogramming for in situ mammalian photoreceptor replacement without cell transplantation.
PMID: 23734087
Free Full-Text: http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23734087/
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