Abstract
Purpose :
Constructing retinal pigment epithelium derived from induced pluripotent stem cells (iRPE) engineering sheets requires the selection of scaffold materials conducive to the maturation and functional implantation of RPE, which is challenging. In this study, we investigate the effects of using femtosecond laser intrastromal lenticule (FLI-lenticule) as a scaffold for construction of engineered RPE sheets.
Methods :
iRPE cells were obtained by differentiating induced pluripotent stem cells (iPSC). These cells were then seeded on decellularized FLI-lenticules (dfLEN). The functionality, characterization, and oxidative stress of iRPE cultured on dfLEN were compared with those cultured on plates (TCP) using various assays such as immunofluorescence (IF), Edu, CCK8, ElLISA, DFCH-DA, and JC-1. Additionally, RNA-seq assays and electron microscope (SEM and TEM) were used to test the iRPE characteristic on engineered dfLEN.
Results :
iRPE cells grown on dfLEN demonstrated improved RPE shape of a hexagon and superior functionality compared to those grown on TCP. Specifically, dfLEN facilitated melanin production and characterization of RPE, maintaining a smaller morphology, increasing the secretion of collagen IV, and reducing proliferation, similar to native human RPE cells. Furthermore, dfLEN significantly inhibited H2O2-induced ROS production, protecting RPE cells from cell death and apoptosis, and against mitochondrial dysfunction-mediated mitochondrial membrane potential. RNA-seq on iRPE cells indicated that dfLEN promoted the epithelial morphogenesis differentiation of iRPE cells, resulting in significant alterations in the cilium-related gene profile. Additionally, dfLEN downregulates inflammatory-related genes. Apical cilium or microvilli well developed in the engineered dfLEN group were identified and confirmed again by the assays of IF, SEM, and TEM.
Conclusions :
We determine that engineered RPE sheets using dfLEN scaffolds enhance RPE characteristics and functions, and suggest that dfLEN scaffolds promote cilium process maturation and polarization of iPSC-derived epithelial cells. Such a strategy to construct iRPE sheets is a promising avenue for developing RPE cell therapy, disease models, and drug screening tools.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.