May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Side Population Stem Cell Molecular Signature Reveals Role of Dual Specificity Phosphatases and G1 to S Cell Cycle Control Proteins in Stem Cell Slow Cycling
Author Affiliations & Notes
  • J. M. Wolosin
    Ophthalmology, Mount Sinai School of Medicine, New York, New York
  • M. A. M. Akinci
    Ophthalmology, Mount Sinai School of Medicine, New York, New York
  • P. S. Reinach
    Biological Sciences, SUNY State College of Optometry, New York, New York
  • Z. Wang
    Biological Sciences, SUNY State College of Optometry, New York, New York
  • A. Barash
    Ophthalmology, Mount Sinai School of Medicine, New York, New York
  • Footnotes
    Commercial Relationships  J.M. Wolosin, None; M.A.M. Akinci, None; P.S. Reinach, None; Z. Wang, None; A. Barash, None.
  • Footnotes
    Support  RO1-EY14878
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4518. doi:https://doi.org/
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      J. M. Wolosin, M. A. M. Akinci, P. S. Reinach, Z. Wang, A. Barash; Side Population Stem Cell Molecular Signature Reveals Role of Dual Specificity Phosphatases and G1 to S Cell Cycle Control Proteins in Stem Cell Slow Cycling. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4518. doi: https://doi.org/.

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

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Abstract

Purpose: : Side population (SP) cells isolated from limbus and conjunctiva derive from the in vivo slow cycling population (Budak et al., J. Cell Sci. 2005), a distinguishing stem cell feature. While SP cells display multiple stem cells features in vitro, they exhibit a poor ability to initiate clonal growth over feeder cells, negatively impinging in efforts at stem cell expansion. The purpose of this study was to identify cell signaling pathways that may underpin this phenotype.

Methods: : Overnight cultures of freshly isolated human and rabbit conjunctival and limbal epithelial cells were stained with Hoechst and cytometrically sorted into SP (~ 0.5 % of adhering cells) and non-SP (G1) cells. Human conjunctival cell RNA was used to establish a SP/G1 differential gene expression profile using microarrays (Wolosin et al, ARVO 2006). Applying robust filtering criteria (e.g., a transcript is considered over-expressed only if the SP/G1 expression ratio > 1.5 in each of all four replicate experiments) we created a differential gene list containing all transcripts that are over-expressed or under-expressed in the SP. Immunostaining or Western blots of sorted limbal and conjunctival cells and immunostaining of total overnight cultures were used for protein level validation. Data mining tools were used to identify differentially affected cell activities and pathways.

Results: : Application of the SP differentially expressed gene list to the MAP kinase cascades using GenMapp revealed that over-expressions of dual specificity phosphatases (DUSPs) that selectively dephosphorylate the ERK1/2 (DUSP4, 5 and 6), p38 (DUSP1) or JNK (DUSP1 and 4) MAP kinases, result in large decrease in total MAPKs/MAPK phosphatase activity (> 7 fold at the gene level). Consistent with these results Westem blots showed that in SP cells ERK and p38 remain essentially unphosphorylated in response to EGF stimulation, hence blocking the EGF induction of proliferation. Similar analysis of the cell cycle pathway (Kegg) revealed 3-fold unde-expression of the cyclins that promote S-phase protein synthesis and concomitant over-expression of their inhibitors (p15, p19, p27 and p 57).

Conclusions: : The semi-quiescent phenotype of ocular surface SP stem cells can be traced to intrinsic changes in the expression of DUSP and G1 to S control proteins. Manipulation of these expressions may prove critical to improve ocular surface stem cell expansion in vitro.

Keywords: cornea: basic science • cornea: epithelium • conjunctiva 
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