Abstract
Abstract: :
Purpose: To dissect the signaling pathways involved during the ex vivo expansion of limbal epithelial stem cells on amniotic membrane (AM). Methods: Each human limbal ring was subdivided into two halves via the 12 h to 6 h meridian with each half equally subdivided to 3 pieces, each piece was cut into two explants of approximately 1x1.5x2.5 mm, and all explants were seeded on intact AM. To eliminate variations of age, sex, and race, we used explants from the same limbal position for the control receiving the vehicle of dimethyl sulphoxide and for the experimental group receiving each of small M.W. inhibitors against PI3K, MEK1/2, and p38/SAPK2. All explants were cultured in SHEM with the medium changed every other day, their outgrowth was monitored daily, and the surface area was scanned with the Adobe Photoshop 5.5 and measured by ImageJ 1.30v at day 19. Results: In the control, expansion of human limbal epithelial cells was more rapidly from the limbal area than the corneal and scleral areas during day 5 to 6 and reached confluence at day 19 of a 6 well culture plate. Compared to the control, LY294002 (PI3K inhibitor) at 50 uM and 20 uM, but not 10 uM and 5 uM, significantly decreased the surface area of expanded limbal epithelial cells on AM (p=0.0008, p=0.0007, p=0.08, and p=0.8, respectively). The outgrowth surface area was significantly decreased by U0126 (MEK1/2 inhibitor) at 10 uM (p=0.0027) but not at 5 uM (p=0.3). The outgrowth surface area was increased but not significant by 10 uM of SB203580 (p38/SAPK2 inhibitor)(p=0.7). All inhibitor–suppressed explants showed regrowth upon transferred to a new intact AM without the inhibitor in the medium. Conclusions: Signaling pathways mediated by PI3K and MEK1/2 may be involved in the promotion, while that of p38/SAPK2 may be involved in the suppression of ex vivo expansion of human limbal epithelium on AM.
Keywords: cornea: basic science • cornea: epithelium • cytokines/chemokines