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RJ Tsai, DF K Ma; The Selection and Expansion of Human Limbal Stem Cells on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1625.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The limbal zone is the exclusive site of corneal epithelial stem cells. We have established an innovative method for bioengineer corneas tissue by ex vivo expansion of limbal stem cells on amniotic membrane. From our clinic results that the transplantation of the ex vivo expansion of the limbal stem cells on amniotic membrane can be restored and maintained the function for corneal clarity more than 2 years. Therefore, we speculate that the amniotic membrane may play an as-yet-unrecognized role of serving as a "niches" for limbal stem cells proliferation and differentiation. Methods: The explant cultures of human limbus on amniotic membrane (AM) were studied by the proliferative ability relate to the location of limbus; donor age and preservation period; and the cell-cycle kinetics of cells outgrowth from explant by BrdU labeling, PCNA and p63. The proliferative function of limbal stem cells on AM also were studied by serial passage of cultures. Results: The human limbal rings from 20 donor corneas were cut into 20 pieces and cultured on AM individually for studying the proliferative ability relate to limbal locations. During 2 weeks period, all limbal tissues were growth on AM. The proliferation ability of limbus was no any relation for the localization of limbal area. The age (60.5+15.2 y/o, n=20) and date of death to culture (7+1.8 D) were also no related to the culture rate. The BrdU labeling detected a high labeling index in the margin of cell-outgrowth from the explant. This phenomenon also confirmed by p63 and PCNA staining. The cells over the margin of outgrowth with AM was cut into 3x3 mm in size and passaged on the new AM. Every three weeks of growth, the procedure was repeated for serial passages studied. Up to 4 passages the cells preserved the same characteristic functions of primary explant culture on AM. Conclusion: These data demonstrate that the AM cultures select and expand limbal epithelial stem cells for ex vivo culture system. Within 2 weeks preserved period and any clock hour position of donor corneas can be cultured in this system. This result allows us to further study the adult stem cell function in ex vivo culture system.
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