Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane.
OBJECTIVE
To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model.


METHODS
Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively.


RESULTS
Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium.


CONCLUSIONS
During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.
Du
Yiqin
Y
Peking University Stem Cell Research Center, Beijing China. yiqindu@pitt.edu
Chen
Jing
J
Funderburgh
James L
JL
Zhu
Xiuan
X
Li
Lingsong
L
D000650:Amnion / Q000637:transplantation*; D000818:Animals; D001684:Biological Dressings; D002455:Cell Division; D017690:Cell Transplantation; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D003316:Corneal Diseases / Q000378:metabolism / Q000601:surgery*; D004268:DNA-Binding Proteins; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D016332:Fetal Tissue Transplantation; D019084:Fluorescent Antibody Technique, Indirect; D016147:Genes, Tumor Suppressor; D006801:Humans; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014945:Wound Healing
D018031:Connexin 43; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D007633:Keratins