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Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures.

ABSTRACT: This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial side down into 12 Wells. As soon as the outgrowing cells had reached confluence, they were harvested. mRNA expression of corneal differentiation markers KRT12, KRT3, DSG1, PAX6, ADH7 and ALDH1A1, conjunctival markers KRT19, KRT13 and stem cell marker ABCG2 were measured via qPCR. KRT12 and PAX6 protein expressions were evaluated using Western Blot. Results suggested that KRT12 mRNA expression was significantly higher in outgrowing cells from the corneal side of the biopsies as in those from the conjunctival side (p?=?0.0043). There was no significant difference in mRNA expression of other analyzed markers comparing with marker expression of outgrown cells from both limbal biopsies (p?>?0.13). KRT12 and PAX6 Western Blot analysis showed no difference in cells harvested from both sides. In conclusion, KRT12 mRNA might be a marker to measure corneal origin of cells from limbal biopsies with unknown composition of corneal and conjunctival progenitor cells. KRT3, DSG1, PAX6, ADH7, ALDH1A1, KRT19, KRT13 and ABCG2 mRNA as well as KRT12 and PAX6 protein expression could not contribute to differentiate corneal from conjunctival cell identity from limbal biopsies.

SUBMITTER: Shi L

PROVIDER: S-EPMC7192984 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures.

Shi Lei L Stachon Tanja T Käsmann-Kellner Barbara B Seitz Berthold B Szentmáry Nóra N Latta Lorenz L

Cytotechnology 20200203 2

This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial sid ...[more]

PMID: 32016711

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Project description:Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion.Limbal explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2'-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63?), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence.Explants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ?0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63?, CK(3+12) and E-cadherin.Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric explants <0.5 mm2 had poor growth potential. However, larger explants (? 0.5 mm2) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation. This study provides a novel clinical strategy for enhancing efficacy of the limbal transplantation surgery and opens the probability of even using the cadaveric tissue by considering the size of explant.

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Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures.

Publications

Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures.

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