Ex vivo expansion of corneal stem cells on amniotic membrane and their outcome.
OBJECTIVE
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.
Tsai
Ray Jui-Fang
RJ
Taipei Eye Center and Taipei Medical University, Taipei, Taiwan. raytsai@ms4.hinet.net
Tsai
Ryan Yao-Nien
RY
D000650:Amnion; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division; D002478:Cells, Cultured; D003315:Cornea / Q000737:chemistry / Q000166:cytology* / Q000473:pathology; D003318:Corneal Opacity / Q000473:pathology / Q000601:surgery; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000096:biosynthesis; D015948:Keratoplasty, Penetrating; D051379:Mice; D008819:Mice, Nude; D011184:Postoperative Period; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology*
D015415:Biomarkers; D007633:Keratins