A Newt Ex Vivo System To Study RPE Transdifferentiation

Abstract

Purpose : The newt is one of a handful of vertebrates that has the capability of regenerating tissue as an adult. In the eye, if the retina is removed, the cells of the retina pigmented epithelium (RPE) reprogram to produce a fully functional retina while retaining the RPE layer. Understanding the mechanism of this process could potentially result in therapies for retinal diseases. To elucidate the RPE reprograming mechanisms involved in the newt, we established an ex vivo culture system, providing a controlled environment to test possible molecules involved in retina regeneration.

Methods : To establish an ex vivo model system, retina-less eyecups (RLEC) were created from enucleated eyes where the anterior portion of the eye along with the retina was removed. The remaining portion of the eye consisting of the RPE, choroid, and sclera were attached to a filter membrane and cultured with matrigel added over each RLEC. The media was supplemented with FGF2 combined with IGF1, SB431542 (SB, Activin A inhibitor), and/or XAV939 (XAV, Wnt inhibitor). Samples were collected at 2, 4, 6, and 10 weeks of culture with EdU added 24hrs prior to collection. RLECs were processed and embedded in paraffin and analyzed for H&E staining, EdU labeling and immunohistochemistry.

Results : All three conditions induced the cells of the RPE to depigment and proliferate (EdU positive) by 4 weeks in culture. By 6 weeks, FGF2+SB and FGF2+IGF1 conditions induced multilayer thick non-pigmented tissue that was positive for RX, an early retinal marker. In addition to the non-pigmented tissue, FGF2+SB treated RLECs also had lentoid structures that were β-crystallin positive. Without the addition of factors, there was no response from the RPE.

Conclusions : RLECs are potentially an ideal ex vivo model system to elucidate specific factors involved in RPE reprograming into various tissues of the eye.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.