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We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin(+) cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP(+) embryonic cones and postnatal Nrl(-/-) 'cods' into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.
Retinal degenerative diseases, due to the lack of regeneration systems and self-renewable cells, often lead to visual impairment. Pax6 is a pleiotropic transcription factor and its expression level determines self-renewal status or differentiation of retinal cells. Here, we investigated the fate of simultaneous induction of retinal ganglion cell death and Pax6 overexpression in retro-differentiation of retinal cells and their commitment to re-enter into the cell cycle. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. Recombinant AAV2 virus harboring PAX6 cDNA and reporter gene was injected into untreated and model mouse eyes. Histological analyses, including IHC and retinal flatmounts immunostaining were performed. The number of Ki67+ cells was clearly increased in model mice, presumably due to NMDA treatment and regardless of Pax6 over-expression. Unlike previous studies, Ki67+ cells were found in GCL layer and interestingly ONL cells expressed Sox2 stemness marker after NMDA cytotoxicity. The potential of retinal cells for robust Ki67 expression, after injury, and expression of Sox2, confirmed their intrinsic plasticity and made a vivid prospect for retinal regenerative medicine.
Cell-based therapies have recently been the focus of much research to enhance skin wound healing. An important challenge will be to develop vehicles for cell delivery that promote survival and uniform distribution of cells across the wound bed. These systems should be stiff enough to facilitate handling, whilst soft enough to limit damage to newly synthesized wound tissue and minimize patient discomfort. Herein, we developed several novel modifiable nanofibre scaffolds comprised of Poly (ε-caprolactone) (PCL) and gelatin (GE). We asked whether they could be used as a functional receptacle for adult human Skin-derived Precursor Cells (hSKPs) and how naked scaffolds impact endogenous skin wound healing. PCL and GE were electrospun in a single facile solvent to create composite scaffolds and displayed unique morphological and mechanical properties. After seeding with adult hSKPs, deposition of extracellular matrix proteins and sulphated glycosaminoglycans was found to be enhanced in composite grafts. Moreover, composite scaffolds exhibited significantly higher cell proliferation, greater cell spreading and integration within the nanofiber mats. Transplantation of acellular scaffolds into wounds revealed scaffolds exhibited improvement in dermal-epidermal thickness, axonal density and collagen deposition. These results demonstrate that PCL-based nanofiber scaffolds show promise as a cell delivery system for wound healing.
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