Purchase this article with an account.
Megha Mahabole, Szu-Yu Chen, Scheffer Tseng; Optimization of Ex Vivo Expansion of Limbal Epithelial Progenitors by Maintaining Native Niche Cells on Denuded Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2013;54(15):985.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPC) on denuded amniotic membrane (dAM) is an alternative solution for treating corneal blindness due to limbal SC deficiency. We reported isolation and expansion of limbal niche cells (NCs) that express embryonic SC (ESC) and angiogenic markers in serum-free modified ESC medium (MESCM). We question whether the conventional ex vivo expansion protocol of LEPC on dAM can be further optimized by maintaining limbal NCs by switching the culture medium from supplemented hormonal epithelial medium (SHEM) to MESCM.
A limbal cluster was obtained from each 1/12 piece of the human corneosclera ring and was cut 1 mm from within and beyond the anatomic limbus and further digested in 1 mg/ml collagenase A for 18 h at 37 degrees celsius, transferred to dAM and cultured in SHEM or MESCM. On Day 8-10, epithelial outgrowth sheets were removed by dispase and subjected to real-time qPCR and immunostaining for expression of corneal markers (p63α, pax6, K12) and NC markers (FLK-1, NG2, PDGFR-B, CD31 and CD34). 1000 single cells were seeded on 6-well dish containing MMC-3T3 for 12-14 days before rhodamine B staining.
qPCR of epithelial outgrowth on dAM at Day 8 in SHEM showed a significant loss of corneal and ESC markers when compare to freshly collagenase-isolated limbal clusters, suggesting that the conventional ex vivo expansion method was not optimized. Nonetheless, the resultant monolayer sheets and cell size were consistently smaller in MESCM than in SHEM on Day 8. qPCR further confirmed significant upregulation of NC markers in outgrowth sheets expanded in MESCM when compare to that in SHEM on Day 10. Cytospin preparations further revealed a significantly higher percentage of PCK-/ Vim+ cells found in MESCM (14.8%, n= 1352) than SHEM (0.6%, n=1531, p<0.05). qPCR and immunostaining showed a substantial higher expression of corneal SC markers (K15, Bmi-1, Msi-1) in MESCM than in SHEM. Although colony-forming efficiency was similar, the number of holoclones was higher in MESCM than SHEM.
These data collectively suggest that MESCM is a better culture medium for preservation of limbal native NCs to support expansion of LEPC on dAM.
This PDF is available to Subscribers Only