Human limbal progenitor cells expanded on intact amniotic membrane ex vivo

Arch Ophthalmol. 2002 Jun;120(6):783-90. doi: 10.1001/archopht.120.6.783.


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.

Conclusion: 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.

Clinical relevance: Intact amniotic membrane is a suitable substrate for bioengineering limbal epithelia for ocular surface reconstruction.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amnion / physiology*
  • Animals
  • Biological Dressings
  • Bromodeoxyuridine / metabolism
  • Cell Communication / physiology
  • Cell Cycle / physiology
  • Cell Division / physiology
  • Connexin 43 / metabolism
  • DNA Replication
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Epithelial Cells / transplantation
  • Fluorescent Antibody Technique, Indirect
  • Gap Junctions / physiology
  • Humans
  • Keratins / metabolism
  • Limbus Corneae / cytology*
  • Limbus Corneae / metabolism
  • Mice
  • Mice, Nude
  • Microscopy, Fluorescence
  • Stem Cell Transplantation
  • Stem Cells / cytology*
  • Stem Cells / metabolism
  • Transplantation, Heterologous


  • Connexin 43
  • Keratins
  • Bromodeoxyuridine