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Jovana Bisevac, Goran Petrovski, Morten C. Moe, Agate Noer; Induced Wnt/β-catenin signaling enhances differentiation in Human Limbal Epithelial Stem Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5101.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal epithelial stem cells (LESCs) found in special niches in limbal epithelial crypts are responsible for maintenance of the cornea epithelium. LESCs can undergo asymmetric division and give rise to transient amplifying cells, which can further differentiate into human limbal epithelial cells (HLECs) and replenish lost upper corneal layers of the central cornea. In addition, the quality of autologous ex vivo expanded human corneal epithelial cells, as well as the abundance of stem/progenitor markers is important for long-term success of transplantation in patients with limbal epithelial stem cell deficiency (LSCD). The intracellular processes that mediate the quiescence and stemness state, as well as differentiation in LESCs, are less known. Thus, we aimed to explore the role of canonical Wnt/β-catenin signaling in HLECs and LESCs.
All tissue collection complied with the Guidelines of Helsinki Declaration and was approved by Local Ethical Committee (No 2017/418). LESCs/HLECs derived from human limbal rings were expanded in vitro and cultured in SHEM medium on amniotic membrane and/or plastic plates. The cultures of LESCs/HLECs were maintained in a humidified 5% CO2, 95% air incubator at 37oC. Thereafter, different concentrations of Wnt/β-catenin activator (LY2090314) were added to the culture medium for 1-2 weeks. Gene expression comparison of non-treated and treated LESCs/HLECs was done by qRT-PCR. In addition, LESCs/HLECs were fixed in 10% formalin for 24 hours and processed further for immunohistochemistry (IHC). Fluorescent images were obtained by ZEISS Axio Imager M1 fluorescence microscope (ZEISS, Oberkochen, Germany).
Gene expression comparison of the LESCs/HLECs treated by a Wnt/β-catenin pathway activator showed upregulation of markers for differentiation (KRT3, GJA1) compared to non-treated LESCs/HLECs control, whereas the markers for stemness (TP63, ABCG2, SOX9), proliferation (MKI67) and quiescence (CEBPD) were downregulated. IHC confirmed the PCR findings.
Activation of the canonical Wnt/β-catenin pathway in LESCs most probably guides the cell fate toward differentiation, whereas the inhibition of the respective pathway may lead to the stem cell state maintenance, which remains to be elaborated in further research.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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