Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo
Abstract
:1. Introduction
2. Materials and Methods
2.1. Limbal Biopsies and Human Limbal Epithelial Stem Cell (hLESC) Cultures
2.2. Small Molecule LY2090314 Treatment of HLESCs
2.3. Cytotoxicity Assay
2.4. RNA Isolation and Real-Time Quantitative RT-PCR (RT-qRT-PCR)
2.5. Microarray and Data Analysis
2.6. Immunocytochemistry (ICC)
2.7. Western Blot (WB)
2.8. Edu Cell Proliferation Assay
2.9. Statistical Analysis
3. Results
3.1. Wnt/β-Catenin Signaling Activation Affects Cell Morphology in hLESC Cultures
3.2. hLESC Toxicity upon Treatment with Increasing Concentrations of Selective Small-Molecule LY2090314
3.3. Real-Time qRT-PCR Identified Wnt/β-Catenin Signaling Activation in hLESC Cultures and Upregulation of Genes for Differentiation and Downregulation of Genes for Limbal Epithelial Stemness Maintenance and Proliferation
3.4. Wnt Signaling mRNA Expression Profile in Primary hLESC Cultures upon Treatment with LY2090314
3.5. Microarray Data Analysis of Primary hLESC Cultures upon Treatment with 5 nM Concentration of LY2090314 Compared to Untreated Controls
3.6. Immunocytochemistry and Western Blot Analysis of 5 nM LY2090314-Treated hLESC Cultures Revealed Higher Levels of β-Catenin and Cx43 Protein Expression with Low Levels of Ki-67, SOX9, and p63α
3.7. Edu Proliferation Assay Demonstrates Lower Proliferation upon Wnt/β-Catenin Signaling Activation in Ex Vivo Expanded hLESC Cultures
4. Discussion
4.1. Upon Persistent Activation of Wnt/β-Catenin Signaling the Stemness and Proliferation Decrease, Whereas the Differentiation Increases in Ex Vivo hLESC Cultures Expanded from Limbal Biopsies
4.2. Regulation of Wnt Ligands upon Wnt/β-Catenin Signaling Activation in hLESC Cultures
4.3. Wnt Inhibitors in LY2090314 Treated and Wnt/β-Catenin Signaling Activated hLESCs
4.4. Differentially Expressed Genes Identified by Microarray Comparing hLESC Cultures Untreated or Treated with 5 nM LY2090314
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Symbol | Entrez Gene Name | p-Value | Fold Change |
---|---|---|---|
Differentially expressed genes that increase in the hLESCs treated with 5 nM LY2090314 | |||
ACVR1C | Activin A receptory type 1C | 3.1 × 10−3 | 2.0 |
AXIN 2 | Axin 2 | 3.9 × 10−6 | 6.36 |
FZD7 | Frizzled class receptor 7 | 1.3 × 10−4 | 2.24 |
GJA1 | Gap junction protein alpha 1 | 6.2 × 10−9 | 3.66 |
MMP7 | Matrix metallopeptidase 7 | 3.2 × 10−5 | 4.3 |
PPP2R2B | Protein phosphatase 2 regulatory subunit B beta | 6.4 × 10−5 | 4.07 |
TCF7 | Transcription factor 7 | 2.3 × 10−4 | 2.16 |
TGFB3 | Transforming growth factor beta 3 | 3.7 × 10−5 | 2.69 |
WNT5A | Wnt family member 5A | 7.4 × 10−5 | 2.28 |
Differentially expressed genes that decrease in the hLESCs treated with 5 nM LY2090314 | |||
DKK1 | Dickkopf Wnt signaling pathway inhibitor | 5.5 × 10−7 | −3.34 |
MYC | MYC proto-oncogene, bHLH transcription factor | 2.2 × 10−6 | −2.25 |
SRFP1 | Secreted frizzled related protein 1 | 2.3 × 10−4 | −3.76 |
TGFB2 | Transforming growth factor beta 2 | 1.1 × 10−3 | −2.42 |
WNT7A | Wnt family member 7A | 8.3 × 10−4 | −2.00 |
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Bisevac, J.; Katta, K.; Petrovski, G.; Moe, M.C.; Noer, A. Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo. Biomedicines 2023, 11, 1829. https://doi.org/10.3390/biomedicines11071829
Bisevac J, Katta K, Petrovski G, Moe MC, Noer A. Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo. Biomedicines. 2023; 11(7):1829. https://doi.org/10.3390/biomedicines11071829
Chicago/Turabian StyleBisevac, Jovana, Kirankumar Katta, Goran Petrovski, Morten Carstens Moe, and Agate Noer. 2023. "Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo" Biomedicines 11, no. 7: 1829. https://doi.org/10.3390/biomedicines11071829