June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Proteomic analysis of secreted factors produced by human limbal epithelial cell cultures during in-vitro growth and expansion
Author Affiliations & Notes
  • Enrique Salero
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States
    Interdisciplinary Stem Cell Institute, Miami, Florida, United States
  • Maitee Urbieta
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States
    Interdisciplinary Stem Cell Institute, Miami, Florida, United States
  • Alfonso L. Sabater
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States
    Interdisciplinary Stem Cell Institute, Miami, Florida, United States
  • Maria del Carmen Piqueras
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States
  • Sanjoy K Bhattacharya
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States
  • Victor L Perez
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States
    Interdisciplinary Stem Cell Institute, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Enrique Salero, None; Maitee Urbieta, None; Alfonso Sabater, None; Maria del Carmen Piqueras, None; Sanjoy Bhattacharya, None; Victor Perez, Allergan, Inc (C), Baush & Lomb (C), Eleven Biotherapeutics (C), EyeGate Pharma (C), Genentech (C), Parion Sciences (C), Rigel (C)
  • Footnotes
    Support  NIH/NEI NIH: R01EY024484, NIH/Core: P30EY014801, Department of Defense: W81XWH-13-1-0048
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2603. doi:
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      Enrique Salero, Maitee Urbieta, Alfonso L. Sabater, Maria del Carmen Piqueras, Sanjoy K Bhattacharya, Victor L Perez; Proteomic analysis of secreted factors produced by human limbal epithelial cell cultures during in-vitro growth and expansion
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):2603.

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

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Abstract

Purpose : In-vitro growth and expansion of human limbal epithelium using various culture modalities has been extensively reported in the literature. However, little is known about the secreted factors released by these cells during their growth and expansion in culture or the impact of such factors on cell limbal epithelial cell differentiation and function. We hypothesize that, in-vitro cultured limbal epithelial cells may produce secreted factors involved in regulation of their phenotype and function irrespective of the use exogenous supplements

Methods : Limbal epithelial tissue of cadaveric origin was obtained from the Florida Lions Eye Bank, processed, and digested overnight in collagenase at 37°C. Cell cultures were started at a density of 1x105 cells/mL/well using stem cell media supplemented with 2% BSA. Culture supernatant was collected at days 7, 15, 20, and 27 of culture and a proteomic profile was generated by mass spectroscopy. Data was analyzed using functional protein association network software (STRING) and a protein classification system based on protein analysis through evolutionary relationships (PANTHER)

Results : We found that in-vitro cultured limbal epithelial cells release a plethora of factors within the first 27 days of growth/expansion. Overlap of secreted factors is observed across all time points analyzed; however, day 7 cultures appeared to be the most metabolically active, secreting over 300 gene products involved in dozens of biological processes including; extracellular matrix organization and disassembly, cell activation and adhesion, and wound healing among others. Data analysis suggests that at this level of enrichment proteins are at least partially biologically connected as a group

Conclusions : In-vitro cultured limbal epithelial cells release a multitude of biologically relevant factors into their culture medium. These factors may be produced as part of their normal physiology or as a response to stress. Negative or positive regulation exerted by these factors, such as changes in concentration and availability to cells, may determine the fate, phenotype and function(s) of these cells after culture in-vitro. Addition or removal of secreted factors may enable manipulation of cultured cell phenotype for potential use in the clinic, particularly in restoring ocular surface defects

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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