June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Expansion of Human Corneal Epithelial Stem/Progenitor Cells in Feeder-Free Explant Cultures
Author Affiliations & Notes
  • Sophie Deng
    Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA
  • Martin Nakatsu
    Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA
  • SHEYLA GONZALEZ
    Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA
  • Hua Mei
    Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 999. doi:
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      Sophie Deng, Martin Nakatsu, SHEYLA GONZALEZ, Hua Mei; Expansion of Human Corneal Epithelial Stem/Progenitor Cells in Feeder-Free Explant Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):999.

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

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Abstract

Purpose: To present a feeder-free culturing method of human corneal epithelial stem/progenitor cells in vitro.

Methods: Primary limbal epithelial cell (LEC) sheets isolated from human sclerocorneal tissues were trypsinized to produce single LECs and co-cultured with growth arrested 3T3-J2 feeder cells for 14 days. Additionally, the outgrowth of LECs from limbal explant pieces was also cultured for 14 days in the presence or absence of 3T3-J2 feeder. The phenotype of the cultured LECs was assessed by their mRNA expression level of putative stem cell markers and differentiation marker by qRT-PCR and immunocytochemistry. The percentage of p63 bright cells in each culture was assessed, and the cell proliferation was evaluated by the Ki67 expression and cell number.

Results: We observed no significant difference in cultured LEC morphology among each culturing method. The LEC growth rate increased over 9-fold in NF explant cultures compared to 3T3-J2 explant cultures and the adjusted growth rate between NF cultures and 3T3-J2 single LEC cultures had similar yields (p>0.05). Gene expression of putative limbal stem cell markers, ABCG2 and ΔNp63 were elevated among NF cultures compared to the gold standard and explants on 3T3-J2. We observed a 7.6-fold and 2.2-fold increase in ABCG2 and ΔNp63 expression respectively when comparing NF explant cultures to the gold standard, while there was only a 4.4-fold and 1.4-fold increase in ABCG2 and ΔNp63 expression respectively when comparing 3T3-J2 explant cultures to the gold standard. However, we did observe an increase in K12 expression in NF explant cultures when compared to the gold standard (6.2-fold). There was a large increase in Ki67 proliferation (4.0-fold) when compared to the gold standard. There was no difference in Ki67 expression between 3T3-J2 explant cultures and the gold standard. Finally, examination of p63α expression in each condition reviewed no discernable differences in the percentage of p63α bright cells between the gold standard (9.0%) and NF explant cultures (10.0%), but we did see a decrease in the 3T3-J2 explant cultures (6%).

Conclusions: 3T3 feeder cells may not be necessary for the growth of stem/progenitor cell population in the primary explant culture. The explants themselves may already contain niche factors that are required for the viability of corneal stem cells.

Keywords: 721 stem cells • 482 cornea: epithelium  
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