Greater Growth Potential of p63-positive Epithelial Cell Clusters
Maintained in Human Limbal Epithelial Sheets
Tetsuya Kawakita 1*,
Shigeto Shimmura 2,
Kazunari Higa 3,
Edgar M. Espana 4,
Hua He 4,
Jun Shimazaki 5,
Kazuo Tsubota 2,
and
Scheffer C. G. Tseng 4
1 Ophthalmology, Keio University, 35 @Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan
2 Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
3 Department of Ophthalmology, Tokyo Dental College, Ichikawa, Japan
4 Ocular Surface Research & Education Foundation, Miami, Florida, United States
5 Ophthalmology, Tokyo Dental College, Chiba, Japan
* To whom correspondence should be addressed. E-mail: kawatetsu{at}gmail.com.
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Abstract |
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OBJECTIVE: To determine the growth potential of p63-positive cell clusters maintained in human limbal epithelial sheets.
METHODS: Intact human limbal epithelial sheets were isolated from corneoscleral rims and cultured with or without being rendered into single cells by trypsinization on either plastic or 3T3 fibroblast feeder layers. Clonal growth on 3T3 fibroblast feeder layers was compared between monolayer derived by sheets or single cells and between areas with or without laser-microdissected p63-enriched cell clusters. Immunostaining, immunoblot analysis and reverse transcription-polymerase chain reaction of such differentiation markers as keratins 3 and 12 and such progenitor markers as p63, ABCG2, and MDR-1 were also compared.
RESULTS: Clusters of small p63-positive cells were enriched in limbal palisades of dispase-isolated epithelial sheets immediately or following brief cultivation on plastic in SHEM. Clonal growth of p63-rich cell clusters was higher than areas without clusters (p<0.001). Clonal growth of epithelial monolayers derived from sheets was also higher than that derived from single cells (p<0.01). Although expression of ABCG2 and MDR-1 transcripts was similar, cells from epithelial sheets expressed higher protein levels of p63 and lower protein levels of K3 and K12 keratins than single cells regardless cultured on plastic or 3T3 fibroblast feeder layers.
CONCLUSION: Limbal palisades contain clusters of p63-rich progenitor cells. Maintenance of such cluster architecture during ex vivo expansion yields higher growth potential than being dispersed into single cells.
Key Words:
corneal epithelium, cell culture, cell proliferation, stem cell niche
