| Summary: | Retinal degeneration is the leading cause of blindness worldwide. Subretinal implantation of human retinal progenitor cells (hRPCs) has shown great promise in models of retinal degenerations for restoration of vision, but is limited by extremely low integration into the retina. Successful integration of implanted cells requires their migration from the site of implantation and into the degenerating retina. Little is known about what cues promote RPC migration. We utilized a high-throughput assay to study migration of hRPCs in three-dimensional hydrogel matrices of varying chemical composition and stiffness, and with exposure to different soluble factors, with the goal of identifying cues important for hRPC migration and associated cell signaling events driving migration. Collagen type 1 (8 mg/ml, 4 mg/ml and 2 mg/ml), collagen type 1 methacrylate (8 mg/ml, 4 mg/ml and 2mg/ml), and hyaluronic acid glycidyl methacrylate (8 mg/ml and 32 mg/ml) gels were developed with variable stiffness by changing concentration of gel components and/or photo cross-linking (collagen type 1 methacrylate and hyaluronic acid glycidyl methacrylate). The storage moduli (stiffness, measured at 1% strain) of the collagen gels were determined using a rheometer. The impact of growth factors important in development, neural regeneration, and cell migration: epidermal growth factor (EGF), fibroblast growth factor (FGF), stromal derived factor (SDF), and hepatocyte derived factor (HGF) was studied using hRPCs in 2 mg/ml collagen type 1 gels. The mean migration distance from the culture plate bottom of hRPCs within gels was measured after 48 hours. Migration of the hRPCs varied significantly in gels of varying stiffness, with higher levels of migration in higher shear storage moduli gels. In addition, the presence of EGF (10 ng/ml), FGF (10 ng/ml), SDF (10,100 ng/ml), and HGF (10,100 ng/ml) in collagen gel increased hRPC migration compared to media alone. The signaling pathways governing migration were explored using Bio-plex Pro bead-based multiplex ELISA (9-plex MAPK and 8-plex AKT panel), and two phosphoproteins in the MAPK signaling pathway critical for migration (ERK1/2, STAT3) were revealed. These results strongly motivate the exploration of material stiffness and co-delivery of soluble factors as important design parameters in cell delivery vehicles to promote transplanted hRPC migration and successful integration into degenerating retina.
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