Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo
Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell defi...
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MDPI AG
2023-06-01
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| Series: | Biomedicines |
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| Online Access: | https://www.mdpi.com/2227-9059/11/7/1829 |
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| author | Jovana Bisevac Kirankumar Katta Goran Petrovski Morten Carstens Moe Agate Noer |
| author_facet | Jovana Bisevac Kirankumar Katta Goran Petrovski Morten Carstens Moe Agate Noer |
| author_sort | Jovana Bisevac |
| collection | DOAJ |
| description | Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (<i>TP63</i>), progenitor (<i>SOX9</i>), quiescence (<i>CEBPD</i>), and proliferation (<i>MKI67</i>, <i>PCNA</i>) genes and the upregulation of genes for differentiation (<i>CX43</i>, <i>KRT3</i>) in treated- compared to non-treated samples. The pathway activation was shown by <i>AXIN2</i> upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of <i>WNT1</i>, <i>WNT3</i>, <i>WNT5A</i>, <i>WNT6</i>, and <i>WNT11</i> gene expression and a downregulation for <i>WNT7A</i> and <i>DKK1</i> in the treated samples. No significant differences were found for <i>WNT2</i>, <i>WNT16B</i>, <i>WIF1</i>, and <i>DKK2</i> gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability. |
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| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-11T01:17:01Z |
| publishDate | 2023-06-01 |
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| series | Biomedicines |
| spelling | doaj.art-ccd454c8e03a449b97b890aa51bb50392023-11-18T18:25:42ZengMDPI AGBiomedicines2227-90592023-06-01117182910.3390/biomedicines11071829Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex VivoJovana Bisevac0Kirankumar Katta1Goran Petrovski2Morten Carstens Moe3Agate Noer4Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, NorwayDepartment of Immunology, Oslo University Hospital, Hf Rikshospitalet, 0424 Oslo, NorwayCenter for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, NorwayCenter for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, NorwayCenter for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, NorwayHuman limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (<i>TP63</i>), progenitor (<i>SOX9</i>), quiescence (<i>CEBPD</i>), and proliferation (<i>MKI67</i>, <i>PCNA</i>) genes and the upregulation of genes for differentiation (<i>CX43</i>, <i>KRT3</i>) in treated- compared to non-treated samples. The pathway activation was shown by <i>AXIN2</i> upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of <i>WNT1</i>, <i>WNT3</i>, <i>WNT5A</i>, <i>WNT6</i>, and <i>WNT11</i> gene expression and a downregulation for <i>WNT7A</i> and <i>DKK1</i> in the treated samples. No significant differences were found for <i>WNT2</i>, <i>WNT16B</i>, <i>WIF1</i>, and <i>DKK2</i> gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.https://www.mdpi.com/2227-9059/11/7/1829limbal epithelial stem cellsWnt/β-catenin signalingstemnessproliferationdifferentiation |
| spellingShingle | Jovana Bisevac Kirankumar Katta Goran Petrovski Morten Carstens Moe Agate Noer Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo Biomedicines limbal epithelial stem cells Wnt/β-catenin signaling stemness proliferation differentiation |
| title | Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo |
| title_full | Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo |
| title_fullStr | Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo |
| title_full_unstemmed | Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo |
| title_short | Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo |
| title_sort | wnt β catenin signaling activation induces differentiation in human limbal epithelial stem cells cultured ex vivo |
| topic | limbal epithelial stem cells Wnt/β-catenin signaling stemness proliferation differentiation |
| url | https://www.mdpi.com/2227-9059/11/7/1829 |
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