Wnt6 plays a complex role in maintaining human limbal stem/progenitor cells

Abstract

The corneal epithelium is consistently regenerated by limbal stem/progenitor cells (LSCs), a very small population of adult stem cells residing in the limbus. Several Wnt ligands, including Wnt6, are preferentially expressed in the limbus. To investigate the role of Wnt6 in regulating proliferation and maintenance of human LSCs in an in vitro LSC expansion setting, we generated NIH-3T3 feeder cells to overexpress different levels of Wnt6. Characterization of LSCs cultured on Wnt6 expressing 3T3 cells showed that high level of Wnt6 increased proliferation of LSCs. Medium and high levels of Wnt6 also increased the percentage of small cells (diameter ≤ 12 µm), a feature of the stem cell population. Additionally, the percentage of cells expressing the differentiation marker K12 was significantly reduced in the presence of medium and high Wnt6 levels. Although Wnt6 is mostly known as a canonical Wnt ligand, our data showed that canonical and non-canonical Wnt signaling pathways were activated in the Wnt6-supplemented LSC cultures, a finding suggesting that interrelationships between both pathways are required for LSC regulation.

Limbal stem cell therapy

“…Molecular regulation of LSCs is another approach to supplement soluble factors that sustain the self-renewal of LSCs both in vitro and in vivo. Several signaling pathways such as Wnt, Notch, BMP, Shh, YAP, and TGFb play critical roles in the maintenance and differentiation of LSCs [40][41][42][43][44]. Activation of Wnt signaling and modulation of Notch signaling are shown to impact the LSC proliferation and differentiation in culture, which opens a new approach in enhancing LSC expansion efficiency and survival of LSCs upon chemical injury in culture [45].…”

Therapeutic Strategies for Restoring Perturbed Corneal Epithelial Homeostasis in Limbal Stem Cell Deficiency: Current Trends and Future Directions

“…Profiling of limbal gene expression has revealed preferential expression of Wnt2, Wnt6, Wnt11, and Wnt16b ligands [ 95 , 96 ]. In a co-culture platform with supporting 3T3 feeder cells engineered to express varying amounts of Wnt6 ligand, Bonnet et al [ 97 ] observed that LSCs exposed to high levels of Wnt6 ligand in vitro underwent increased proliferation with decreased expression of terminal differentiation markers of mature corneal epithelium. They suggested that through both canonical Wnt/ß-catenin and noncanonical signaling, Wnt6 may promote LSC self-renewal and stemness.…”

Regulation of Limbal Epithelial Stem Cells: Importance of the Niche

“…It is important to note that individual Wnt ligands can activate different pathways depending on their concentration [ 71 , 72 , 73 , 74 ]; Wnt ligands are active primarily close by to where they were secreted [ 75 ], and even Wnt ligands that typically activate non-canonical signaling can synergize to activate canonical Wnt/β-catenin signaling [ 76 ]. A balance between canonical and non-canonical Wnt signaling likely exists in LSCs, as shown by Wnt6 modulating both canonical and non-canonical Wnt signaling [ 22 , 71 ], and this balance is affected by LSC–niche interactions.…”

“…It is important to note that individual Wnt ligands can activate different pathways depending on their concentration [ 71 , 72 , 73 , 74 ]; Wnt ligands are active primarily close by to where they were secreted [ 75 ], and even Wnt ligands that typically activate non-canonical signaling can synergize to activate canonical Wnt/β-catenin signaling [ 76 ]. A balance between canonical and non-canonical Wnt signaling likely exists in LSCs, as shown by Wnt6 modulating both canonical and non-canonical Wnt signaling [ 22 , 71 ], and this balance is affected by LSC–niche interactions. The microRNA family miR103/107 targets NEDD9 for degradation to mediate E-cadherin localization to adherens junctions, p90RSK2 to arrest cells in the G0/G1 phase of the cell cycle, PTPRM to decrease gap junctions, and Wnt3a to increase proliferation in primary human LSCs cultivated on Collagen IV-coated plates [ 60 ].…”