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Sarah Colanesi, Barbara Käsmann-Kellner, Tanja Stachon, Nóra Szentmáry, Berthold Seitz; Efficacy of different protocols for ex vivo expansion of limbal epithelial stem cell. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3473.
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© ARVO (1962-2015); The Authors (2016-present)
Aniridia-related keratopathy is a serious defect affecting 90% of aniridia patients. It is characterized by progressive recurrent erosions, fibrotic scarring and vascularization, i.e. loss of regenerative capacity of the cornea due to limbal stem cell deficiency (LSCD). Here we present a comparative study for reliable ex vivo expansion of limbal epithelial stem cells (LESC) with the aim to restore the regenerative capacity of the cornea and to reduce the symptoms of aniridia-related keratopathy.
Human LESC were extracted from corneoscleral rings of donor corneas using Migration, Dispase or Collagenase Assay in combination with a commercial serum-free medium (KSFM, Gibco). Single corneas were used for Migration and Dispase Assay, pooled extracts from 2-3 corneas were used for the Collagenase Assay. No feeder cells or other animal products were used in our experiments. For all three assays, efficient isolation and enrichment of LESC was assessed based on morphology in the primary cultures. For the Collagenase Assay, expression of stem cell markers ABCG2, p63 and differentiation marker CK3 was analyzed in early and late passages (P1-P5).
Morphologically, the success rate of the Collagenase Assay (N=10) was 90.0%, of Migration Assay (N=65) 20.0%, of Dispase Assay (N=11) 27.3%. Successfully expanded cells had small sizes and large nuclei typical for epithelial precursor and stem cells. With Collagenase Assay 100%, with Migration Assay 76.9% and with Dispase Assay 9.1% could be subcultured over up to 7 rounds of passaging. In the Collagenase Assay, expression of ABCG2 was on average 20.0 % ± 10.3 in P1, which gradually decreased in later passages. This was inversely correlated to expression of CK3 (13.4% ± 2.6 in P1) which gradually increased in later passages. LESC morphology and proliferation was fully restored after cryopreservation of early passages obtained from Collagenase Assays. Expression of ABCG2 but not CK3 was lost after cryopreservation.
For LESC expansion, the Collagenase Assay provides a better protocol than Migration or Dispase Assay. Nonetheless, the strong variability in marker expression reflects persisting difficulties for enriching the stem cell population in our primary cultures.
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