April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Small Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Pluripotent Stem Cells
Author Affiliations & Notes
  • Alexandra Mikhailova
    Institute of Biomedical Technology, University of Tampere, Tampere, Finland
    BioMediTech, Tampere, Finland
  • Tanja Ilmarinen
    Institute of Biomedical Technology, University of Tampere, Tampere, Finland
    BioMediTech, Tampere, Finland
  • Hannu M T Uusitalo
    SILK, Department of Ophthalmology, University of Tampere, Tampere, Finland
    TAUH Eye Center, Tampere University Hospital, Tampere, Finland
  • Heli Skottman
    Institute of Biomedical Technology, University of Tampere, Tampere, Finland
    BioMediTech, Tampere, Finland
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5502. doi:
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      Alexandra Mikhailova, Tanja Ilmarinen, Hannu M T Uusitalo, Heli Skottman; Small Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5502.

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

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Abstract

Purpose: Limbal stem cells are known to be responsible for maintaining corneal epithelium. Transplantation of these tissue-specific stem cells has been used to treat severe ocular surface disorders, such as limbal stem cell deficiency (LSCD). However, this is only possible if enough healthy limbal tissue is available, making it necessary to search for alternative cell sources. Human pluripotent stem cells (hPSCs) provide unique opportunities for differentiation of limbal and corneal epithelial cells for cell transplantations. In this study, we developed an efficient and directed method for differentiating hPSCs towards corneal epithelial progenitor cells capable of terminal differentiation towards mature corneal epithelial-like cells.

Methods: Differentiation of hPSCs was initiated in suspension culture, using a serum-free induction medium containing two small molecule inhibitors. After four days, the cell aggregates were plated onto collagen IV-coated substrate, and maintained as adherent cultures in a serum-free corneal epithelium medium for 40 days. The extent of differentiation was evaluated by following cell morphology, and analyzing expression of key markers using immunocytochemistry and qPCR at several time-points.

Results: Small molecule induction down-regulated pluripotency marker expression, while up-regulating the transcription factors active during ocular development. Protein expression of the limbal stem cell marker p63 was greatly enhanced, with up to 95% of cells being p63-positive after five weeks of differentiation. Finally, after a total of six weeks in differentiation culture, the two proteins specific to terminally-differentiated corneal epithelium, cytokeratins 3 and 12, were expressed in an average of 35% and 71% of cells, respectively.

Conclusions: In this study, we aimed to replicate signaling cues active during ocular surface ectoderm development in order to improve the efficiency and reproducibility of hPSC differentiation towards corneal epithelial-like cells. In contrast to all earlier studies, differentiation was carried out in serum-free culture conditions without the use of amniotic membrane or other undefined culture substrates. This highly efficient differentiation method could potentially be used for treating severe ocular surface disorders, or in disease modeling.

Keywords: 482 cornea: epithelium • 721 stem cells • 500 differentiation  
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