September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Inhibition of Notch Signaling Pathway Appears to Help Maintain the Undifferentiated State of Human Limbal Epithelial Stem/Progenitor Cells in vitro
Author Affiliations & Notes
  • Sheyla Gonzalez
    Ophthalmology, Stein Eye Institute UCLA, Los Angeles, California, United States
  • Elfren Ray Baclagon
    Ophthalmology, Stein Eye Institute UCLA, Los Angeles, California, United States
  • Sophie Xiaohui Deng
    Ophthalmology, Stein Eye Institute UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Sheyla Gonzalez, None; Elfren Baclagon, None; Sophie Deng, None
  • Footnotes
    Support  NEI 5P30EY000331 and 1R01EY021797; CIRM TR2-01768; Research to Prevent Blindnes �
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4907. doi:
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      Sheyla Gonzalez, Elfren Ray Baclagon, Sophie Xiaohui Deng; Inhibition of Notch Signaling Pathway Appears to Help Maintain the Undifferentiated State of Human Limbal Epithelial Stem/Progenitor Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4907.

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

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Abstract

Purpose : Controversy exits regarding the role of Notch signaling on limbal epithelial stem/progenitor cells (LSCs). We investigated the inhibition of Notch signaling pathway on the phenotype of LSCs in vitro.

Methods : Primary human LSCs were cultured as single cells on a monolayer of 3T3 feeder cells in SHEM medium. Notch signaling was inhibited using the small molecule DAPT to prevent the release of the active Notch intracellular domain (NICD). DAPT titration at 1, 5, 10 and 20 µM was performed. Notch inhibition was confirmed by the absence of the NICD in the nucleus by immunocytochemistry. The phenotype of cultured LSCs was analyzed by assessing cell morphology, colony forming efficiency (CFE), cell expansion rate, cell size and expression level of putative LSC markers both at the mRNA and protein level. Differences in stratification and differentiation in the presence of DAPT were also analyzed after air-lifting induction.

Results : Morphology and CFE of LSCs cultured in the presence of DAPT were similar to those in the control. The cell expansion rate was reduced at high concentrations of DAPT (10 and 20µM, p<0.05). An increase in the percentage of small cells (≤12 µm) was detected in the presence of DAPT at 20µM (p<0.05). The percentage of p63α bright cells was comparable to that in the control (p>0.05). The percentage of K12+ cells was significantly reduced at high concentrations of DAPT (10 and 20µM, p<0.05). Upon air-lifting induction, LSCs in the presence of DAPT showed no significant differences in the stratification and differentiation capacity compared to the control.

Conclusions : Inhibition of Notch signaling using DAPT appears to help maintain the LSC phenotype in vitro.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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