June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Different Regions of Amniotic Membrane Denuded with EDTA or Thermolysin Equally Support the in vitro Expansion of Limbal Stem/progenitor Cells
Author Affiliations & Notes
  • Hua Mei
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Luxia Chen
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Elfren Baclagon
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Sophie Xiaohui Deng
    Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Hua Mei, None; Luxia Chen, None; Elfren Baclagon, None; Sophie Deng, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2608. doi:
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      Hua Mei, Luxia Chen, Elfren Baclagon, Sophie Xiaohui Deng; Different Regions of Amniotic Membrane Denuded with EDTA or Thermolysin Equally Support the in vitro Expansion of Limbal Stem/progenitor Cells
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):2608.

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

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Abstract

Purpose : This study was to investigate the capability of the amniotic membrane (AM) denuded with thermolysin on supporting the proliferation and stem-cell properties of limbal stem/progenitor cells (LSCs) propagated in vitro compared to the AM denuded with the standard non-enzymatic protocol using EDTA. In addition, whether different regions of AM equally support the growth of LSCs was studied.

Methods : AMs were divided into distal, middle, and proximal regions according to their relative distances to the umbilical cord. Each region of AM from the same donor was divided into two denudation groups: EDTA and thermolysin. The integrity of basement membrane was examined by immunohistochemistry for collagen IV, laminin V, and integrin α6, and scanning electron microscopy (SEM). Human limbal explant pieces were cultured on different regions of EDTA- or thermolysin- denuded AMs for around 14 days. Cell doubling and stem-cell properties of the expanded LSCs including small cells, expressions of putative stem cell markers (p63α, cytokeratin(K) 14, vimentin), expression of epithelial marker (pancytokeratin), and expression of maturation marker (K12) were compared between different regions of the two AM denudation groups.

Results : AMs denuded with thermolysin retained comparable levels of collagen IV, laminin V, and Integrin α6 in the basement membrane compared to those denuded with EDTA. The comparable integrity of basement membrane between the two denudation groups was further confirmed by SEM. No apparent compositional difference was observed among different regions of AMs. AMs denuded with thermolysin showed a comparable level of cell doubling and similar percentages of small cells, p63α-bright cells, K14+, vimentin+, and pancytokeratin+ cells to AM denuded with EDTA. Proximal AMs denuded with thermolysin generated a slightly higher percentage of K12+ cells, However, middle and distal AMs supported similar levels of K12+ cells between the two denudation groups. Proximal, middle and distal regions of AMs supported the growth and stem-cell properties of LSCs at a comparable level.

Conclusions : AMs denuded with thermolysin supported the in vitro expansion of LSCs at a similar potency compared to AMs denuded with EDTA. Different regions of AMs showed no difference on supporting the growth of LSCs.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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