Using neurogenin to reprogram chick RPE to produce photoreceptor-like neurons.
OBJECTIVE
One potential therapy for vision loss from photoreceptor degeneration is cell replacement, but this approach presents a need for photoreceptor cells. This study explores whether the retinal pigment epithelium (RPE) could be a convenient source of developing photoreceptors.


METHODS
The RPE of chick embryos was subjected to reprogramming by proneural genes neurogenin (ngn)1 and ngn3. The genes were introduced into the RPE through retrovirus RCAS-mediated transduction, with the virus microinjected into the eye or added to retinal pigment epithelial explant culture. The retinal pigment epithelia were then analyzed for photoreceptor traits.


RESULTS
In chick embryos infected with retrovirus RCAS-expressing ngn3 (RCAS-ngn3), the photoreceptor gene visinin (the equivalent of mammalian recoverin) was expressed in cells of the retinal pigment epithelial layer. When isolated and cultured as explants, retinal pigment epithelial tissues from embryos infected with RCAS-ngn3 or RCAS-ngn1 gave rise to layers of visinin-positive cells. These reprogrammed cells expressed genes of phototransduction and synapses, such as red opsin, the alpha-subunit of cone transducin, SNAP-25, and PSD-95. Reprogramming occurred with retinal pigment epithelial explants derived from virally infected embryos and with retinal pigment epithelial explants derived from normal embryos, with the recombinant viruses added at the onset of the explant culture. In addition, reprogramming took place in retinal pigment epithelial explants from both young and old embryos, from embryonic day (E)6 to E18, when the visual system becomes functional in the chick.


CONCLUSIONS
The results support the prospect of exploring the RPE as a convenient source of developing photoreceptors for in situ cell replacement.
Li
Xiumei
X
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Ma
Wenxin
W
Zhuo
Yehong
Y
Yan
Run-Tao
RT
Wang
Shu-Zhen
SZ
D000818:Animals; D030161:Avian Proteins / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D002642:Chick Embryo; D055358:Cone Opsins / Q000235:genetics / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D005822:Genetic Vectors; D017403:In Situ Hybridization; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000378:metabolism; D055351:Retinal Neurons / Q000166:cytology* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D015293:Transducin / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D030161:Avian Proteins; D051792:Basic Helix-Loop-Helix Proteins; D055358:Cone Opsins; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D050825:Synaptosomal-Associated Protein 25; D015293:Transducin; C045140:visinin