Regulation of limbal stem cells via Wnt signaling in the
treatment of limbal stem cell deficiency

doi:10.3969/j.issn.2095-4344.2028

Abstract

Abstract:

BACKGROUND: Wnt signaling pathway plays an important role in the regulation of stem cells, but its regulatory effect on limbal stem cells is not well defined.

OBJECTIVE: To investigate the regulation of limbal stem cells via Wnt signaling pathway and its function in the treatment of limbal stem cell deficiency.

METHODS: Rat limbal segments were digested with Dispase and trypsin/EDTA and then single limbal stem cells were seeded in 3D-Matrigel. In experimental group LiCl (500 μmol/L) was added to the culture system. Control group received no LiCl. The expression levels of p63α, CK12, CEBPδ and Ki67 in limbal stem cells were detected by qRT-PCR on the 7^th day of culture. The expression level of β-catenin in limbal stem cells was detected by immunofluorescence staining. The rat model of limbal stem cell deficiency was made by alkali burn method. Rats in treatment group were treated with Wnt-activated limbal stem cells by subconjunctival injection. Rats in control group were treated with PBS. Rats were checked by slit lamp every day. On the 4^th day after treatment, the immunofluorescence staining and hematoxylin-eosin staining were applied to evaluate the repair of limbus.

RESULTS AND CONCLUSION: (1) The limbal stem cells aggregated in 3D-Matrigel. β-Catenin was negative in the control group. β-Catenin was found in the cytoplasm and nucleus of the limbal stem cells in the experimental group. (2) qRT-PCR results showed there was no significant difference in the levels of p63α, CK12 or CEBPδ between control and experimental groups (P > 0.05). The Ki67 level in the experimental group was significantly higher than that in the control group (P < 0.05). (3) The rat models of limbal stem cell deficiency were established. The degree of corneal opacity in the treatment group was significantly lower than that in the control group (P < 0.05). Hematoxylin-eosin staining results observed that the corneal epithelial cells in the treatment group were neatly arranged, the cell size was uniform, and repair was good. Immunofluorescence staining showed that β-catenin was found in the cytoplasm and nucleus of most corneal epithelial cells in the treatment group. While β-catenin was only weakly positive in the control group, and was invisible in the cytoplasm or nucleus. (4) These results indicate that activation of Wnt signaling pathway in limbal stem cells enhances their proliferation and keeps them in an undifferentiated self-renewal state. Wnt-activated limbal stem cells can promote the regeneration of corneal epithelium and reduce the degree of corneal opacity in the treatment of limbal stem cell deficiency. The regulation of limbal stem cells via Wnt signaling pathway is expected to provide new ideas for the treatment of limbal stem cell deficiency.

Key words: Wnt signaling pathway, limbal stem cells, limbal stem cell deficiency, self-renewal

CLC Number:

Han Bo, Yang Zhe, Li Jing, Zhang Mingchang . Regulation of limbal stem cells via Wnt signaling in the treatment of limbal stem cell deficiency[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(7): 1057-1062.

Figures/Tables 4

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