Direct Conversion of Mouse Fibroblasts into Photoreceptor-like Cells

The purpose of our study is to directly convert mouse fibroblasts into photoreceptor-like cells through an adenoviral gene delivery system. The mouse cDNAs of Ascl, Crx, Ngn1, Nrl, and Otx2 were cloned into a modified commercial adenoviral vector. Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos, and mouse postnatal fibroblasts (MPFs) were isolated from three-day-old mice. A pool of adenoviruses containing five genes was prepared to infect MEFs once daily for two days. Next, half of the neural medium supplemented with forskolin was changed every two days. After 7 or 14 days, the photoreceptor-like cells were assayed via immunofluorescence or polymerase chain reaction with reverse transcription (RT–PCR). The photoreceptor-like cells were then transplanted into adult C57BL/6 mouse retina and were assessed by immunofluorescence 14 days following transplantation. Screening from a pool of five candidate genes, we reported that a combination of only three factors—Crx, Nrl, and Otx2—was sufficient to convert mouse embryonic and postnatal fibroblasts into functional photoreceptors. The induced photoreceptor-like cells ex-pressed photoreceptor-specific proteins such as Recoverin, Rhodopsin, and Opsin and integrated into the outer nuclear layer of the retina following transplantation. This study demonstrates that the photoreceptor-like cells converted by defined factors from fibroblasts can provide a source of photoreceptor transplantation and feasible future treatment for retinal repair.