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Jianing Gu, Yini Wang, Zekai Cui, Hong Li, Jiansu Chen, Shibo Tang; The Construction of Bioengineered RPE Sheets with Enhanced RPE Cilium Assembly Using SMILE-Derived Lenticule. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2894.
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© ARVO (1962-2015); The Authors (2016-present)
Developing a suitable tissue engineering (TE) sheet of human retinal pigment epithelium (hRPE) is difficult. The property of a TE RPE sheet fundamentally determines the success of hRPE cell transplantation. Here we explore the effects of femtosecond laser lntrastromal lenticule (FLI-lenticule) as scaffold on hRPE cells.
Primary hRPE cells were isolated from human eyes balls and cultured. The characteristics of decellularization and the integrity of FLI-lenticule by our hypertonic saline plus nuclease procedure were investigated by H&E staining. Then hRPE cells were seeded on decellularized FLI-lenticules compared to culture on tissue culture plates (TCP). And RPE changes were assayed by immunofluorescence (IF), SEM, qPCR, western blot (WB) and RNA-seq. Rabbit subretinal transplantation of acellular FLI-lenticule was also taken.
Primary hRPE cells positively expressed RPE cell markers such as RPE65, MITF, EMMPRIN. H&E staining showed that cells were eliminated in FLI-lenticules treated with hypertonic saline plus nuclease, and corneal acellular ECM structure integrity could maintain. hRPE cells grown on FLI-lenticule exhibited a correctly orientated monolayer sheet with a polygonal cell shape and abundant microvilli on their apical surfaces (Fig.1). Interestingly, RNA-seq displayed some cilium-related genes were up-regulated, which were selected to obtain heatmaps. Cilium-related proteins acetyl-α-tublin and β-tublin were up-regulated by IF and WB assays. According to the DEGs, GO terms and literature, the cilium-related diagram associated with this experiment was plotted (FDR < 0.05) (Fig. 2). Moreover, FLI-lenticule exhibits biocompatibility after rabbit subretinal transplantation by 2 weeks through electroretinography and histological examination.
We determine that TE RPE sheets using FLI-lenticule scaffolds aid in enhanced RPE cilium assembly. Such strategy to construct RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Fig.1 (A) H&E staining images of FLI-lenticule. (B) Heatmaps of some differentially expressed genes.
Fig.2 (A) IF staining of RPE cilium and basal body markers. (B) WB analysis of cilium-relatived proteins.
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