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Direct conversion of adult human retinal pigmented epithelium cells to neurons with photoreceptor properties.

ABSTRACT: Degeneration of photoreceptors is a major cause of blindness. Identifying new methods for the generation of photoreceptors offers valuable options for a cell replacement therapy of blindness. Here, we show that primary adult human retinal pigmented epithelium (hRPE) cells were directly converted to postmitotic neurons with various properties of photoreceptors by the neurogenic transcription factor ASCL1 and microRNA124. At Day 8 after the induction of ASCL1 and miRNA124 expression in hRPE cells, 91% of all cells were Tuj1⁺, and 83% of all cells were MAP2⁺ neurons. The cone photoreceptor marker L/M-opsin, the rod photoreceptor marker rhodopsin, and the generic photoreceptor marker recoverin were expressed in 76%, 86%, and 92% of all cells, respectively. Real-time quantitative PCR measurements showed significant and continuous increases in the expression of photoreceptor markers phosducin and recoverin, rod cell markers phosphodiesterases 6 b and arrestin S-antigen, and cone cell markers L/M-opsin and S-opsin in three independent lines of primary hRPE cells at different days of transdifferentiation. Transmission electron microscopy of converted neurons showed disc-like structures similar to those found in photoreceptors. While the converted neurons had voltage-dependent Na⁺, K⁺, and Ca²⁺ currents, light-induced change in membrane potential was not detected. The study demonstrates the feasibility of rapid and efficient transdifferentiation of adult hPRE cells to neurons with many properties of photoreceptors. It opens up a new possibility in cell replacement therapy of blindness caused by photoreceptor degeneration.

SUBMITTER: Li B

PROVIDER: S-EPMC7871113 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:One of the promises in regenerative medicine is to regenerate or replace damaged tissues. The embryonic chick can regenerate its retina by transdifferentiation of the retinal pigmented epithelium (RPE) and by activation of stem/progenitor cells present in the ciliary margin. These two ways of regeneration occur concomitantly when an external source of fibroblast growth factor 2 (FGF2) is present after injury (retinectomy). During the process of transdifferentiation, the RPE loses its pigmentation and is reprogrammed to become neuroepithelium, which differentiates to reconstitute the different cell types of the neural retina. Somatic mammalian cells can be reprogrammed to become induced pluripotent stem cells by ectopic expression of pluripotency-inducing factors such as Oct4, Sox2, Klf4, c-Myc and in some cases Nanog and Lin-28. However, there is limited information concerning the expression of these factors during natural regenerative processes. Organisms that are able to regenerate their organs could share similar mechanisms and factors with the reprogramming process of somatic cells. Herein, we investigate the expression of pluripotency-inducing factors in the RPE after retinectomy (injury) and during transdifferentiation in the presence of FGF2.We present evidence that upon injury, the quiescent (p27(Kip1)+/BrdU-) RPE cells transiently dedifferentiate and express sox2, c-myc and klf4 along with eye field transcriptional factors and display a differential up-regulation of alternative splice variants of pax6. However, this transient process of dedifferentiation is not sustained unless FGF2 is present. We have identified lin-28 as a downstream target of FGF2 during the process of retina regeneration. Moreover, we show that overexpression of lin-28 after retinectomy was sufficient to induce transdifferentiation of the RPE in the absence of FGF2.These findings delineate in detail the molecular changes that take place in the RPE during the process of transdifferentiation in the embryonic chick, and specifically identify Lin-28 as an important factor in this process. We propose a novel model in which injury signals initiate RPE dedifferentiation, while FGF2 up-regulates Lin-28, allowing for RPE transdifferentiation to proceed.

Project description:Background:The World Health Organization (WHO) estimates that the number of individuals who lose their vision due to retinal degeneration is expected to reach 6 million annually in 2020. The retinal degenerative diseases affect the macula, which is responsible for central and detailed vision. Most macular degeneration, i.e., age-related macular degeneration (AMD) develops in the elderly; however, certain hereditary diseases, such as the Stargardt disease, also affect young people. This degeneration begins with loss of retinal pigmented epithelium (RPE) due to formation of drusen (atrophic) or abnormal vessels (exudative). In wet AMD, numerous drugs are available to successful treat the disease; however, no proven therapy currently is available to treat dry AMD or Stargardt. Since its discovery, human embryonic stem cells (hESCs) have been considered a valuable therapeutic tool. Some evidence has shown that transplantation of RPEs differentiated from hESCs cells can result in recovery of both RPE and photoreceptors and prevent visual loss. Methods:The human embryonic WA-09 stem cell lineage was cultured under current Good Manufacturing Practices (cGMP) conditions using serum-free media and supplements. The colonies were isolated manually and allowed to spontaneously differentiate into RPE cells. Results:This simple and effective protocol required minimal manipulation and yielded more than 10e8 RPE cells by the end of the differentiation and enrichment processes, with cells exhibiting a cobblestone morphology and displaying cellular markers and a gene expression profile typical of mature RPE cells. Moreover, the differentiated cells displayed phagocytic activity and only a small percentage of the total cells remained positive for the Octamer-binding transcriptions factor 4 (OCT-4) pluripotency cell marker. Conclusions:These results showed that functional RPE cells can be produced efficiently and suggested the possibility of scaling-up to aim at therapeutic protocols for retinal diseases associated with RPE degeneration.

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Direct conversion of adult human retinal pigmented epithelium cells to neurons with photoreceptor properties.

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