Human limbal progenitor cells expanded on intact amniotic membrane ex vivo.
BACKGROUND
The transplantation of human limbal epithelium on amniotic membrane as a substrate is a new treatment for limbal stem cell deficiency. Limbal epithelial stem cells are characterized by a slow cell cycle and the lack of K3 keratin and connexin 43 (Cx43), a gap junction protein. We investigated Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of limbal epithelium expanded on amniotic membrane.


METHODS
Connexin 43 expression and bromodeoxyuridine (BrdU) incorporation were determined by immunohistology. The GJIC was investigated by a scrape-loading dye transfer assay. Expression of Cx43 and K3 keratin as well as BrdU-retaining nuclei were also analyzed after xenotransplantation in nude mice.


RESULTS
Limbal epithelium showed mean +/- SD 12.4% +/- 14.5% positive units of Cx43 expression and a low BrdU labeling index of 2.4% +/- 0.9% (n = 5), of which the latter was due to slow cycling, as proved by its increase to 62.0% +/- 9.5% after continuous BrdU labeling for 5 days. Most of the expanded epithelium did not show GJIC (83%), significantly more than that grown on plastic (6%; P<.002). Basal cells of the stratified epithelium after xenotransplantation did not express Cx43 and K3 keratin, but their nuclei retained BrdU.


CONCLUSIONS
These results support the hypothesis that intact amniotic membrane preferentially preserves and expands Cx43-negative, keratin K3-negative, and GJIC-deficient limbal epithelium, a phenotype resembling that of stem cell-containing limbal basal epithelial cells in vivo.


CONCLUSIONS
Intact amniotic membrane is a suitable substrate for bioengineering limbal epithelia for ocular surface reconstruction.
Grueterich
Martin
M
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, FL, USA.
Tseng
Scheffer C G
SC
D000650:Amnion / Q000502:physiology*; D000818:Animals; D001684:Biological Dressings; D001973:Bromodeoxyuridine / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D018031:Connexin 43 / Q000378:metabolism; D004261:DNA Replication; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000502:physiology; D006801:Humans; D007633:Keratins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014183:Transplantation, Heterologous
D018031:Connexin 43; D007633:Keratins; D001973:Bromodeoxyuridine