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Yini Wang, Jianing Gu, Zekai Cui, Shibo Tang, Jiansu Chen; Enhancement of RPE Characteristics and Anti-EMT by iPS Conditioned Medium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3846.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) cell therapy requires abundant high viability and RPE property cells. As serial passages however, RPE cells tend to form epithelial mesenchymal transition (EMT). Here we investigate to use induced pluripotent stem cell conditioned medium (iPS-CM) for the effects on anti-EMT of RPE cells.
Primary adult RPE cells were isolated from human eye balls and cultured. The optimum concentration of iPS-CM for the viability of RPE was screened by CCK-8 assay. Then cell morphology, proliferation, apoptosis and barrier function were compared between iPS-CM and normal medium (NM, DMEM-F12 medium) in RPE cells by immunofluorescence (IF), Edu, flow cytometry, transepithelial electrical resistance (TER) and permeability analysis. Growth factor array and RNA-seq assays test the RPE changes after iPS-CM treatment.
Primary RPE cells positively expressed RPE cell markers such as RPE65, MITF, EMMPRIN. The optimum concentration of iPS-CM (iPS supernatant ratio DMEM-F12) was 1:2. There was statistically significant enhancement in RPE proliferation and barrier function as well as inhibition of apoptosis in iPS-CM (p<0.05)(Fig.1). Using assay of a panel of cytokines, combined with transcriptome and protein analyses, we discovered that iPS-CM contained high levels of PDGF-AA, IGFBP-2, TGF-α and IGFBP6, which had responsibility with the increase of RPE markers RPE65, MITF and BEST1 as well as the decrease of EMT markers FN1, ACTA2 and CTNNB1 in iPS-CM group (Fig.2). Our bioinformatic analysis also presented that PI3K/AKT and TGF-β signaling pathways were implicated in regulating RPE cells in iPS-CM.
We illustrate that high levels of PDGF-AA, IGFBP-2, TGF-α and IGFBP6 in iPS-CM not only promote RPE proliferation but also inhibit EMT, which are favorable for RPE therapy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Fig.1 (A) RPE inverted contrast microscope images. (B) RPE IF images. Epithelial barrier function by TER (C) and permeability assays (D) (*, p<0.05; **, p<0.01; ***, p<0.001).
Fig.2 (A) Heatmaps of some differentially expressed genes. (B) qPCR and western blot results of RPE-related markers. (C) qPCR and western blot results of EMT-related markers with or without TGF-β1 treatment(#, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001).
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