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Kazunari Higa, Junko Higuchi, Reona Kimono, Hideyuki Miyashita, Jun Shimazaki, Kazuo Tsubota, Shigeto Shimmura; Human limbal organoid transplantation in a rabbit limbal deficiency model. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4129. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cells have a specialized microenvironment for maintaining self-renewal capacity termed the niche, and the niche of corneal epithelial stem cells exists in the limbus. We previously reported that the limbal phenotype, including stem/ niche-like structures, can be maintained in organoids derived from human limbal tissue for up to 1 month in culture with KGF+Y27632 using Matrigel (2016 ARVO). In this study, we transplanted human limbal organoids in a rabbit limbal deficiency model.
Organoids were prepared from donor limbal tissue. After the treatment with collagenase, epithelial cells and surrounding cells were scraped from limbal tissue, and plated on Matrigel.Cells were cultivated using medium containing KGF and the Rho kinase inhibitor Y27632 for 1 month. To detect the transplanted organoids, cultivated organoids were labeled by Ferucarbotran on the last day as a contrast agent. Rabbit limbal deficiency was prepared by scraping the corneal and limbal epithelium using a grinder. Pockets were made in the rabbit limbus, and organoids were transplanted using surgical tissue adhesives. After transplantation, rabbits were followed by slit-lamp, and tissue sections were observed histologically at the end of the study.
Organoids derived from the pigmented limbus retained pigments after 1 month in culture. Epithelial cells with pigments were observed in the limbus of rabbit limbal deficiency model transplanted with pigmented organoids. Ferucarbotran labeled cells derived from transplanted organoids were also observed in the transplanted sites by reacting with Berlin Blue. Immunohistochemistry against human nuclei confirmed the presence of organoid-derived cells extending on to host corneas.
Our data suggest that limbal organoids are able to engraft to the limbus of a rabbit limbal deficiency model and supply corneal epithelium.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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