The Construction of Retinal Pigment Epithelium Sheets with Enhanced Characteristics and Cilium Assembly Using iPS Conditioned Medium and Small Incision Lenticule Extraction Derived Lenticules.

In vitro generation of a functional retinal pigment epithelium (RPE) monolayer sheet is useful and promising for RPE cell therapy. Here, for the first time, we used induced pluripotent stem (iPS) supernatant as the conditioned medium (iPS-CM) and femtosecond laser intrastromal lenticule (FLI-lenticule) as a scaffold to construct an engineered RPE sheet. There are significant enhancements in RPE cell density, transepithelial electrical resistance (TER) and inhibitions of ultraviolet C (UVC)-irradiated apoptosis when RPE cells are cultured in iPS supernatant/Dulbecco's modified Eagle's medium (DMEM)-F12 of 1/2 (iPS-CM) compared with those in normal medium (NM, DMEM-F12). Using the assay of a panel of cytokines, combined with transcriptome and protein analyses, we discover that iPS-CM contains high levels of platelet-derived growth factor AA (PDGF-AA), insulin-like growth factor binding protein (IGFBP)-2, transforming growth factor (TGF)-α and IGFBP-6, which are responsible for the upregulation of gene and protein markers with RPE phenotypes and downregulation of gene and protein markers with epithelial-mesenchymal transition (EMT) phenotypes for RPE cells in iPS-CM when compared to those in NM. Moreover, compared to cultures on tissue culture plates (TCP), RPE cells on FLI-lenticule display more microvilli and cilium in accordance with the results in terms of RNA-Seq data, quantitative polymerase chain reaction (qPCR) expression, immunofluorescence staining, and western blot assays. Furthermore, acellular FLI-lenticule exhibits biocompatibility after rabbit subretinal implantation by 30 days through electroretinography and histological examination. Thus, we determined that engineered RPE sheets treated by iPS-CM in conjunction with FLI-lenticule scaffold aid in enhanced RPE characteristics and cilium assembly. Such a strategy to construct RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools. STATEMENT OF SIGNIFICANCE: In vitro generation of a functional RPE monolayer sheet is useful and promising for RPE cell therapy. Here, we constructed engineered RPE sheets treated by iPS-CM in conjunction with FLI-lenticule scaffolds to help in enhanced RPE characteristics and cilium assembly. Such a strategy to generate RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools.