June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Rabbit limbal stem cells in vitro observation and their transplantation to rabbits
Author Affiliations & Notes
  • Julia Khorolskaya
    Institute of Cytology of the Russian Academy of Science, St-Petersburg, Russian Federation
  • Olga Aleksandrova
    Institute of Cytology of the Russian Academy of Science, St-Petersburg, Russian Federation
  • Daria Perepletchikova
    Institute of Cytology of the Russian Academy of Science, St-Petersburg, Russian Federation
  • Kirill Zhurenkov
    Institute of Cytology of the Russian Academy of Science, St-Petersburg, Russian Federation
  • Ilya Gavrilyik
    S. M. Kirov Military Medical Academy, St-Petersburg, Russian Federation
  • Natalia Mikhailova
    Institute of Cytology of the Russian Academy of Science, St-Petersburg, Russian Federation
  • Miralda Blinova
    Institute of Cytology of the Russian Academy of Science, St-Petersburg, Russian Federation
  • Footnotes
    Commercial Relationships   Julia Khorolskaya, None; Olga Aleksandrova, None; Daria Perepletchikova, None; Kirill Zhurenkov, None; Ilya Gavrilyik, None; Natalia Mikhailova, None; Miralda Blinova, None
  • Footnotes
    Support  The reported study was funded by RFBR, project number 19-34-90146; the Ministry of Science and Higher Education Russian Federation (grant № 075-15-2020-773).
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 765. doi:
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      Julia Khorolskaya, Olga Aleksandrova, Daria Perepletchikova, Kirill Zhurenkov, Ilya Gavrilyik, Natalia Mikhailova, Miralda Blinova; Rabbit limbal stem cells in vitro observation and their transplantation to rabbits. Invest. Ophthalmol. Vis. Sci. 2021;62(8):765.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Cell technology is one of the most promising approaches to reconstruct the ocular surface and restore the integrity and transparency of the damaged cornea. In the present study, we investigated rabbit limbal stem cells (LSCs) in vitro during several passages in different conditions and used them as a component of the cornea substitute in an animal model of limbal stem cell deficiency (LSCD). The goal was to find a highly proliferative population that can be used for cornea recovery.

Methods : Rabbit LSCs were isolated from rabbit corneoscleral rims using the Dispase II, Collagenase I, and trypsin. Cells were cultured under standard conditions in different media: epithelium growth medium and in DMEM/F12 with serum. LSCs cultured in different media were compared by the main stem markers and markers of differentiation (PAX6, ALDH3A1, ABCB5, p63𝛼, cytokeratins 3/12,14, 15, 19). LSCs at the 6th passage were subjected to air-lifting, cells were cultured in cell culture inserts for 14 days in the epithelial medium. Then cytokeratin 14 and actin were observed. For in vivo study rabbits with previously formed limbal stem cell deficiency were used. LSCs were transplanted inside collagen hydrogel. As a control collagen hydrogel without cells was used. The process of corneal restoration was examined by histological analysis.

Results : It was shown that in DMEM/F12 LSCs have high proliferative potential and after several passages change their morphology and mainly mesenchymal-like cells are observed. In mesenchymal-like LSCs some markers such as ΔNp63α and PAX6 are present, but change their localization from nucleus to cytoplasm. Even though the population has a mesenchymal-like phenotype, the presence of various cytokeratins was observed in the cytoplasm. Also, it was shown that in LSCs that were subjected to air-lifting, the expression of cytokeratin 14 increased. On the 90th day after transplantation mesenchymal-like LSCs to rabbits with previously formed LSCD, normal corneal epithelium was restored, vascularization, and goblet cells were absent. In the control group opacity and neovascularization of the stroma was observed.

Conclusions : During in vitro cultivation, LSCs may undergo an epithelial-mesenchymal transition. It was shown that mesenchymal-like LSCs are a highly proliferative population and are able to restore corneal epithelium in rabbits.

This is a 2021 ARVO Annual Meeting abstract.

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