Human limbal progenitor cells expanded on intact amniotic membrane ex vivo.

Abstract

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