Abstract
Purpose:
Senescence and epithelial-to-mesenchymal transition (EMT) are often associated with cellular aging and age-related degenerative diseases. In vitro modeling of retinal pigmented epithelium (RPE) senescence and EMT can be achieved through repetitive passaging, which leads to RPE degeneration. Here we report a method employing transcription factor-mediated reprogramming to reverse these phenomena.
Methods:
Senescence and chronic EMT were induced by repetitive passage of human fetal RPE. Mesenchymal RPE were transduced using constitutive expression transcription factor vectors and a transient 7 day treatment with the TGFBR1/ACVR1B inhibitor, A-83-01. Restoration of the RPE phenotype was evaluated based on morphology, pigmentation, RNA-Seq analysis, telomere length and senescence-associated β-galactosidase (SA-β-gal) activity.
Results:
EYA2, LHX2, MYCN, OTX2, and RAX were chosen as candidate reprogramming factors based on a transcriptome analysis of differentiated RPE, mesenchymal RPE, ARPE19, and additional primary cell types. Among the combinations, co-transduction with OTX2 and MYCN and transient A-83-01 treatment resulted in restored epithelial morphology and pigmentation that could be maintained for at least 5 additional passages. Cells co-transduced with OTX2 and MYCN in the absence of A-83-01 did not pigment or acquire an epithelial morphology. At the transcriptome level, 27% (FDR adjusted p-value < 0.05) of the expressed genes were differentially expressed between mesenchymal and differentiated RPE. In contrast, OTX2/MYCN/A-83-01 restored the expression levels of >95% of the expressed genes. In addition, there was a significant increase in telomere length and the SA-β-gal activity (p-value < 0.05) was reduced to levels similar to minimally passaged RPE.
Conclusions:
Our reprogramming method not only restores the morphology and gene expression of mesenchymal RPE, but also resets the senescence markers: telomere length and SA-β-gal activity. From a mechanistic perspective, our results imply that both activation of TGFβ signaling and loss of expression of transcriptional promoters of RPE differentiation play a role in the switch to a mesenchymal phenotype and onset of senescence. In the future it will be interesting to determine if transient expression of OTX2 and MYCN is similarly effective and to determine the mechanisms by which OTX2 and MYCN reset the biological aging clock.