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Tissue-engineering of Limbus epithelial Stem-cells on Amniotic Membrane as a Biological Substrate
1Grüterich M., 2Tseng S.C. G.,
1Ludwig-Maximilians-Universität München, Klinikum Innenstadt, Augenklinik (München)
2Ocular Surface Research and Education Foundation (Miami)
Purpose: The stem cell (SC)-containing limbal basal epithelium does not express k3 keratin, connexin (Cx)43 and Cx50. In contrast, the transient amplifying cell-containing corneal basal epithelium expresses Cx43 and K3, and the corneal suprabasal epithelium expresses Cx50 and K3. Amniotic membrane (AM) is an ideal substrate for ex vivo expansion of limbal epithelium. We would like to analyze the AM culturing conditions that may preferentially promote limbal SC expansion.
Method: Human limbal epithelium (HLE) was expanded by explant cultures on intact and epithelially denuded AM with or without a 3T3 fibroblast feeder layer. Monolayers for each condition were analyzed for K3, Cx43 and Cx50 expression using Western blot analysis. Xenotransplantation to NIH-nu-xid-bg-mice was performed to investigate the epithelial phenotype after stratification.
Results: Western blot analysis revealed that the levels of Cx43 and Cx50 proteins by HLE on intact AM was less than those on denuded AM. Addition of a 3T3 feeder layer to denuded AM increased the level of Cx43 and decreased that of Cx50. After xenotransplantation the basal epithelium of HLE on intact AM did not express K3, Cx43 or Cx50, while that on denuded AM expressed all three markers. The addition of 3T3 fibroblasts resulted in positive staining of Cx43 and K3 but negative staining of Cx50 in the basal epithelium.
Conclusions: HLE on intact AM retains a limbal basal epithelial phenotype, while HLE on denuded AM differentiates into a corneal phenotype. The addition of a 3T3 feeder layer slows but does not prevent differentiation of HLE on denuded AM.