May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Profiling of Extracellular Matrix Molecules Expressed by Mouse Feeder Layer Cells Screened by Limbal Epithelial Cell Colony Forming Assay
Author Affiliations & Notes
  • R. Takagi
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • M. Yamato
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • A. Kushida
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • K. Nishida
    Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
    Department of Ophthalmology, Osaka, Japan
  • J. Yang
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • M. Utsumi
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • C. Kohno
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • Y. Tano
    Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
    Department of Ophthalmology, Osaka, Japan
  • T. Okano
    Institute of Advanced Biomedical engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
    Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan
  • Footnotes
    Commercial Relationships  R. Takagi, None; M. Yamato, None; A. Kushida, None; K. Nishida, None; J. Yang, None; M. Utsumi, None; C. Kohno, None; Y. Tano, None; T. Okano, None.
  • Footnotes
    Support  21COE, HRC, Core–to–Core Programs from MEXT in Japan
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 2711. doi:
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      R. Takagi, M. Yamato, A. Kushida, K. Nishida, J. Yang, M. Utsumi, C. Kohno, Y. Tano, T. Okano; Profiling of Extracellular Matrix Molecules Expressed by Mouse Feeder Layer Cells Screened by Limbal Epithelial Cell Colony Forming Assay . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2711.

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

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Abstract

Introduction: : Mouse 3T3 feeder layer has been utilized for epidermal and corneal epithelial cell culture to promote tissue–like cell stratification. However, the molecular mechanism underlying the epithelial–feeder layer interactions remains poorly understood. Here, we examined feeder layer activity of six different mouse cell lines in terms of colony forming efficiency of primary limbal epithelial cells including corneal epithelial stem/progenitor cells.

Methods: : Six mouse fibroblasts lines were examined as feeder layers, and colony forming assay with primary rabbit limbal epithelial cells was performed. Expression of mRNA for twenty nine extracellular matrix molecule genes was quantified by TaqMan PCR after reverse transcription for two highest and two lowest cell lines in colony forming efficiency.

Results: : When epithelial cells and feeder layers were separated by culture inserts, colony forming efficiency was significantly lower than that when both cells were cultured on the same dish surfaces, implying that direct contacts between these cells and/or pericellular extracellular matrix deposition by feeder layers have an important role in feeder layer activity. With TaqMan PCR assay, significant difference in expression correlated with colony forming efficiency was observed for some extracellular matrix molecules including tenascin C, type XII collagen alpha1 chain, and type XVII collagen alpha1 chain.

Conclusions: : Decreased colony forming efficiency under the condition where epithelial cells and feeder layers were separated by culture inserts suggests that direct contacts and/or efficient extracellular matrix deposition is essential for the feeder layer activity. Correspondingly, mouse cell lines showed varied colony forming efficiency for limbal epithelial cells, and significantly different profiles of extracellular matrix proteins.

Keywords: cornea: epithelium • gene/expression • extracellular matrix 
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