Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency.
OBJECTIVE
To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD).


METHODS
Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization.


RESULTS
Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects.


CONCLUSIONS
The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.
Ang
Leonard P K
LP
The Eye and Cornea Transplant Centre, Singapore.
Tanioka
Hidetoshi
H
Kawasaki
Satoshi
S
Ang
Leslie P S
LP
Yamasaki
Kenta
K
Do
Tien Phuc
TP
Thein
Zaw M
ZM
Koizumi
Noriko
N
Nakamura
Takahiro
T
Yokoi
Norihiko
N
Komuro
Aoi
A
Inatomi
Tsutomu
T
Nakatsukasa
Mina
M
Kinoshita
Shigeru
S
D000650:Amnion; D000818:Animals; D017690:Cell Transplantation; D002478:Cells, Cultured; D018920:Coculture Techniques; D003228:Conjunctiva / Q000166:cytology*; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D004847:Epithelial Cells / Q000378:metabolism / Q000637:transplantation* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D053551:Keratin-12 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D053527:Keratin-4 / Q000378:metabolism; D016850:Limbus Corneae / Q000473:pathology*; D055271:Mucin 5AC / Q000378:metabolism; D011817:Rabbits; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000473:pathology*; D014183:Transplantation, Heterologous
D053551:Keratin-12; D053558:Keratin-3; D053527:Keratin-4; C524678:MUC5AC protein, human; D055271:Mucin 5AC