Abstract
Purpose :
ARPE-19 cells have been used as a model system to study retinal pigment epithelium (RPE) behaviour, however, with increasing passage, these cells undergo epithelial-mesenchymal transition (EMT), losing RPE morphology, pigmentation and marker expression. Previous studies show that ARPE-19 can re-establish RPE characteristics by adapting the culture conditions. Here, we have cultured cells in X-VIVO, a media which re-establishes aspects of the RPE phenotype in ARPE-19 cells, and investigated pathways associated with EMT reversal and wound healing mechanisms.
Methods :
ARPE-19 cells were cultured in X-VIVO or control DMEM medium for 6 weeks. Morphology and transepithelial resistance (TER) were recorded weekly, and cells were collected to measure changes in gene and protein expression. Transcripts were analysed over the 6-week period using RNAseq. Ingenuity Pathway analysis identified upstream pathways associated with reversal of EMT. The effects of 11 potential regulatory molecules were examined in culture by assessing their effects on PMEL17 expression. Wound healing responses were assessed in DMEM and X-VIVO cultured cells at 6 weeks using DNA and actin staining and live cell imaging.
Results :
After 6 week culture in X-VIVO media, ARPE-19 cells developed cobblestone-like RPE morphology, pigmentation, and increased TER compared to cells cultured in DMEM. RNAseq analysis showed a temporal increase in RPE cell gene and protein markers. Peak expression of PMEL17 was seen at Week 3. Pathway transcriptome analysis identified revealed 11 upstream regulators of ARPE-19 differentiation. Dose response curves to these molecules confirmed the involvement of the RAS/MEK/ERK cascade and TGFβ-RI. The timing of wound closure was the same in X-VIVO and DMEM, however, wound closure was driven primarily by cell migration in X-VIVO and cell proliferation in DMEM.
Conclusions :
Here, we have demonstrated that ARPE-19 cells cultured in X-VIVO re-establish an early RPE phenotype. We have identified molecular pathways associated with the reversal of EMT, which could enable long term culture and passage of RPE. Responses to wound healing suggest that ARPE-19 behave differently depending on their culture condition. Although a full RPE transcription profile comparable to fetal RPE was not achieved under the culture conditions investigated, ARPE-19 remain a useful cell model for investigating RPE cell differentiation and potential pathological changes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.