Purchase this article with an account.
X. Li, R.-T. Yan, S.-Z. Wang; Reprogramming Cultured Human RPE Cells to Express Photoreceptor Genes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5244.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Previous studies using the chick system provide compelling evidence for gene-directed reprogramming of RPE progeny cells to produce differentiating photoreceptors. This study tests human RPE cells (APRE-19 and hTERT-RPE1) for their capacity to express photoreceptor genes under the induction of neurogenin1 (ngn1), a bHLH proneural gene with pro-photoreceptor activity during retinal neurogenesis and can direct as high as 80% of cells in a chick RPE cell culture to express photoreceptor markers.
To construct ngn1 expression cassettes, human ngn1 was RT-PCR amplified and cloned into pGEM-T. After sequence verification, the DNA was subcloned into retroviral vector pMSCV (Clontech) and AAV vector pAAV (Stratagene) modified to include the CAG sequence for high level of gene expression. Cultures of ARPE-19 and hTERT-RPE1 were transfected with the recombinant DNA by Fugene 6 or electroporation. The cultures were then examined for the presence of cells displaying photoreceptor properties.
Cells exhibiting neuronal morphologies were present in an ARPE-19 culture 3 days after transfection with AAV-ngn1. Cells in the AAV-eGFP control maintained their typical appearance. Immunostaining detected cells positive for photoreceptor proteins arrestin, recoverin, and transducin α-subunit in ARPE-19 cultures electroporated with AAV-ngn1, but not in the control electroporated with AAV-eGFP. Morphologically, these cells bore resemblance to developing photoreceptor cells. Transfection of hTERT-RPE1 cells with MSCV-ngn1 DNA by electroporation produced cells displaying photoreceptor-like morphologies and expressing photoreceptor proteins arrestin, recoverin, or red opsin. These cells were absent in the control electroporated with MSCV-eGFP.
These results suggest that the human cells are amendable to reprogramming by ngn1 to express photoreceptor genes.
This PDF is available to Subscribers Only