April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Influence of Substrate on Gene Expression Associated with Stemness in Human Limbal Epithelial Cell Graft
Author Affiliations & Notes
  • Aboulghassem Shahdadfar
    Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    Ophthalmology, Center for Eye Research, Oslo, Norway
  • Hans C Dalsbotten Aass
    Department of Clinical Chemistry, Oslo, Norway
    Oslo University Hospital, Oslo, Norway
  • Eli W Gulliksen
    Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    Ophthalmology, Center for Eye Research, Oslo, Norway
  • Kristiane H Berg
    Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    Ophthalmology, Center for Eye Research, Oslo, Norway
  • Morten Carstens Moe
    Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    Ophthalmology, Center for Eye Research, Oslo, Norway
  • Liv K Drolsum
    Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    Ophthalmology, Center for Eye Research, Oslo, Norway
  • Katerina Jirsová
    Laboratory of the Biology and Pathology of the Eye, Prague, Czech Republic
    Charles University and General University Hospital, Prague, Czech Republic
  • Vinagolu Rajasekhar
    Memorial Sloan-Kettering Cancer Center, New York, NY
    Cancer Center, New York, New York, NY
  • Bjorn Nicolaissen
    Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
    Ophthalmology, Center for Eye Research, Oslo, Norway
  • Footnotes
    Commercial Relationships Aboulghassem Shahdadfar, None; Hans Dalsbotten Aass, None; Eli Gulliksen, None; Kristiane Berg, None; Morten Carstens Moe, None; Liv Drolsum, None; Katerina Jirsová, None; Vinagolu Rajasekhar, None; Bjorn Nicolaissen, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5177. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Aboulghassem Shahdadfar, Hans C Dalsbotten Aass, Eli W Gulliksen, Kristiane H Berg, Morten Carstens Moe, Liv K Drolsum, Katerina Jirsová, Vinagolu Rajasekhar, Bjorn Nicolaissen, ; Influence of Substrate on Gene Expression Associated with Stemness in Human Limbal Epithelial Cell Graft. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5177.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: Protocols for generation of grafts from human limbal epithelial cells (HLECs) differ to some extent between clinics. Initiation of the cultures is performed using the explant culture technique or a suspension of dissociated cells, and a commonly used substrate is the human amniotic membrane (HAM). Outcome after transplantation is linked to various factors, including to content of cells presenting qualities associated with stemness. In contrast to HAM, cell culture coated plastic inserts (PIs) provide a readily accessible and standardized substrate. We here expanded HLECs on HAM and on PIs and examined the generated grafts for selected markers associated with stemness.

Methods: Limbal tissues were obtained after removal of the central tissue for transplant purposes. The corneo-limbal ring was divided in samples and placed epithelial side down on either HAM or on PIs in 6 well plates, and cultivated in complex medium (DMEM/F12 supplemented with 5% FBS, DMSO, human EGF, insulin, transferrin, selenium, hydrocortisone, cholera toxin, gentamicin and amphotericin B) for 3 weeks. Samples were processed for analysis using Flow Cytometry and qRT-PCR.

Results: After 3 weeks, we have previously reported expression of genes associated with stemness such as ABCG2, P63, OCT4, SOX2, KRT3, and OCLN to be similar in cultures maintained on the two types of substrate. Using FACS sorting, we here detected that expression of P63 was increased in the side population obtained using PIs compared with HAM. In contrast, the expression of ABCG2 was increased in the side population obtained when using HAM as a substrate.

Conclusions: Our findings show that genes related to stemness may be similarly expressed in grafts engineered using different substrates. However, we further demonstrate that the expression of such genes in the side population may differ considerably in grafts engineered on different substrates.

Keywords: 482 cornea: epithelium • 721 stem cells • 654 proliferation  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×