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Characterisation of Human Limbal Side Population Cells Isolated Using an Optimised Protocol From an Immortalised Epithelial Cell Line and Primary Limbal Cultures

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Abstract

The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63—a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).

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Acknowledgments

Supported by grants from the Ministry of Higher Education, Malaysia and partially by Universiti Sains Malaysia, Pulau Pinang, Malaysia. The authors thank Lisa Hodgson and the staff at Flow cytometry Core Facility Unit, Newcastle University, Institute of Genetic Medicine, International Centre for Life, Newcastle Upon-Tyne, United Kingdom, for their assistance throughout this study.

Conflict of interest

Authors do not have actual or potential conflict of interest including any financial, personal or other relationships with other people or organisations that could inappropriately influence, or be perceived to influence our work.

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Authors and Affiliations

  1. Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle Upon Tyne, NE1 3BZ, UK

    Bakiah Shaharuddin, Ian Harvey & Annette Meeson

  2. Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200, Pulau Pinang, Malaysia

    Bakiah Shaharuddin

  3. St Paul’s Eye Unit, Royal Liverpool University Hospital, Prescot Street, Liverpool, L7 8XP, UK

    Sajjad Ahmad

  4. Department of Eye and Vision Sciences, Institute of Ageing and Chronic Disease, University of Liverpool, 4th Floor UCD Building, Daulby Street, Liverpool, L69 3GA, UK

    Sajjad Ahmad

  5. Institute of Cellular Medicine, Newcastle University, William Leech Building, Newcastle Upon Tyne, NE1 7RU, UK

    Simi Ali

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  1. Bakiah Shaharuddin
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Correspondence to Bakiah Shaharuddin.

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Authors’ Contribution

1. Bakiah Shaharuddin: Conception and design, Collection and/or assembly of data, Manuscript writing

2. Sajjad Ahmad: Conception and design

3. Ian Harvey: Conception and design, Collection and/or assembly of data, Manuscript writing

4. Simi Ali: Conception and design

5. Annette Meeson: Conception and design, Collection and/or assembly of data, Manuscript writing, Final approval of manuscript

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Shaharuddin, B., Harvey, I., Ahmad, S. et al. Characterisation of Human Limbal Side Population Cells Isolated Using an Optimised Protocol From an Immortalised Epithelial Cell Line and Primary Limbal Cultures. Stem Cell Rev and Rep 10, 240–250 (2014). https://doi.org/10.1007/s12015-013-9481-0

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