July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Levels of Wnt6 differentially regulate human limbal stem/progenitor cells
Author Affiliations & Notes
  • Denise Oh
    Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States
  • Hua Mei
    University of North Carolina Chapel Hill, North Carolina, United States
  • Sophie Xiaohui Deng
    Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Denise Oh, None; Hua Mei, None; Sophie Deng, None
  • Footnotes
    Support  National Eye Institute 1R01EY021797
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5095. doi:
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      Denise Oh, Hua Mei, Sophie Xiaohui Deng; Levels of Wnt6 differentially regulate human limbal stem/progenitor cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5095.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To investigate the mechanism and role of Wnt6 in the in vitro proliferation and maintenance of human limbal stem/progenitor cells (LSCs).

Methods : Freshly isolated limbal epithelial cells (LECs) were cultured on 3T3 cell lines for 10-12 days and treated with Wnt6 conditioned media to evaluate signaling mechanisms of Wnt. The expression of the canonical factor, β-catenin, and the non-canonical factors, RhoA and CamKII were analyzed after exposure to Wnt6 for time intervals under 1 hour. LECs were also raised under continuous exposure (10-12 days) to Wnt6 on 3T3 cell lines overexpressing low, medium, and high levels of Wnt6 (Wnt6-3T3). These cells were characterized for colony forming efficiency (CFE), proliferation, cell size, and expression of the p63α and K14 LSC markers, and the K12 differentiation marker.

Results : Western blot analysis revealed the activation of both canonical and non-canonical signaling factors upon exposure to Wnt6. Signaling was detected within minutes but the level of activation within specific pathways varied with Wnt6 concentration and colony density. Characterization of long term Wnt6-LSC cultures showed that although CFEs remained similar between all groups, high levels of Wnt6 increased proliferation of LSCs by 17.1% (p<0.05). Medium and high levels of Wnt6 also increased the percentage of small cells by 10.8% and 20.4%, respectively. In parallel, the percentage of cells expressing K14 was between 91.4% and 92.1%, however, the expression of K12 was reduced by 40.6% and 54.6%, in medium and high Wnt6 cultures.

Conclusions : Wnt6 control of LSCs appears to demonstrate dynamic regulation of both canonical and non-canonical pathways as the concentration of Wnt6 appears to have varying effects on human LECs in vitro. High levels of Wnt6 support increased proliferation and better maintains the stem/progenitor phenotype while low levels tend to differentiate LECs. Wnt6 regulation appears to require tight control of both canonical and non-canonical pathways to regulate LSC expansion and phenotype.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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