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Kazunari Higa, Hideyuki Miyashita, Jun Shimazaki, Kazuo Tsubota, Shigeto Shimmura; Spheroidal cultivation of the human limbal epithelial niche from small limbal tissue. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4345.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cells have a specialized microenvironment for maintaining self-renewal and multipotent capacities, which is called as niche. The niche of corneal epithelial stem cell exists in the limbus. We previously reported that the limbal phenotype including stem/niche-like structure can be maintained in spheroids derived from the human limbus, for up to 1 month in medium with KGF+Y27632 using matrigel (2014 ARVO). To improve the cultivation method, we assumed a limited autologous tissue and performed spheroidal cultivation from human small limbal tissue.
Spheroids were prepared from donor limbal tissue 2.5 millimeters in diameter used after cornea transplantation. After the treatment with collagenase, epithelial cells and surrounding cells were scraped from limbal tissue, and spread on matrigel. Cells were cultivated using medium containing KGF and the Rho kinase inhibitor Y27632. To detect the slow cycling cells, cultivated spheroids were labeled by 5-bromo-2’-deoxyuridine (BrdU) in first 3 days. After 1 month, spheroids were observed by histochemical analysis, cell cycle analysis, and colony forming efficiency.
Uniformly small and round spheroids were formed even from small limbal tissue. Efficiency of spheroidal formation was differed in the derived site of the original limbus. Spheroids derived from the high spheroidal formation site showed the large colony forming efficiency and higher ratio of slow cycling cells. In addition, N-cadherin was infrequently-expressed in epithelial spheroid.
The limbal niche can be maintained in spheroids for up to 1 month from small limbal tissue, but spheroidal forming ability was differed in different parts of the limbus.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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