Objectives: To identify the stem-cell property of the ex vivo expansion of limbal stem cells (LSCs) on amniotic membrane (AM) in culture system and after clinical transplantation. Methods: Four key factors have to be performed in the defined culture system: (1) the label-retaining cells have to be identified; (2) the cells can be serially expanded and passaged in vitro; (3) the expanded cells can be labeled by tissue-specific keratin or markers, and (3) their stem cells cannot be labeled by those keratin or markers. Results: The ex vivo-expanded LSCs on AM were positive for p63 and ABCG2 and BrdU label-retaining studies on flat mount preparation. When the ex vivo-expanded LSCs with AM were transplanted into a subcutaneous layer of nude mice, they formed multiple layers of cells. Only the basal layer of cells was positive for p63 and BrdU. The cells over the suprabasal layers were positive for K12/K3. The pathologic studies of corneal specimen of successful LSC transplantation after penetrating keratoplasty demonstrated that P63-positive cells were noticed all over the basal layer of central cornea and AM could be identified at 10 months after LSC transplantation. Conclusions: These results indicate that the AM provided the niche function for cultured LSCs and maintained the limbal-like environment for the transplanted area of cornea. The survival of cases depends on the severity of the disease entity, culture technique, and maintenance of the niche environment for LSCs in the culture and after clinical transplantation.
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