June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Single human retinal pigment epithelial (hRPE) cell analysis under oxidative stress reveals differential expression of pro-inflammatory and apoptosis related genes.
Author Affiliations & Notes
  • Aram Asatryan
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Marie-Audrey Ines Kautzmann
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Jessica Heap
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Nicolas Guillermo Bazan
    Neuroscience, LSUHSC, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Aram Asatryan, None; Marie-Audrey Kautzmann, None; Jessica Heap, None; Nicolas Bazan, None
  • Footnotes
    Support  Supported by NEI grant EY005121, NIGMS grant GM103340, the Eye, Ear, Nose and Throat Foundation (NGB) and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1849. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Aram Asatryan, Marie-Audrey Ines Kautzmann, Jessica Heap, Nicolas Guillermo Bazan; Single human retinal pigment epithelial (hRPE) cell analysis under oxidative stress reveals differential expression of pro-inflammatory and apoptosis related genes.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1849.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Activation of innate immunity in conjunction with photo-oxidative stress are linked to Age-Related Macular degeneration (AMD), that involves RPE and photoreceptor cell death. Previously we showed a robust induction of both pro- and anti-apoptotic genes by uncompensated oxidative stress (UOS). Single cell gene expression assay is a powerful tool to dissect heterogeneous cellular populations. The goal of this study was to define apoptosis related genes in response to UOS on a single RPE cell to open new avenues in research in ophthalmology.

Methods : hRPE cells were grown and maintained in MEM medium supplemented with 10% FBS, 5 % NCS, non-essential amino-acids and incubated at 37°C with a constant supply of 5% CO2. hRPE cells were subjected to UOS (600μM H2O2/10ng/ml TNFα) for four hours then dissociated by trypsin were loaded on a medium size C1 chip (Fluidigm technologies). Briefly: RPE cells were separated in automated C1 system which utilizes microfluidics technologies. Cells were lysed in micro-chambers and pre-amplified with human apoptosis and inflammation panel genes. Pre-amplified samples were further transferred into Biomark HD 96x96 chip for a RT-PCR amplification. Results were analyzed using Fluidigm Real-Time PCR anylysis Software.

Results : As predicted UOS induced the expression of apoptotic and pro-inflammatory genes including BIRC3, BIRC5, RELB, IL8 and others. PCA analysis showed two distinct clusters of genes between treated and control groups. Surprisingly, most RPE cells had two distinct populations of cells with varying gene expression. APAF1, TRAF5, CASP8, CASP7 and TAB1 show two distinct modes of gene expression on violin plots indicating a subpopulation with either high expression or low. Addition of UOS shifted the bimodal distribution of these genes towards the high end of expression.

Conclusions : Our results indicate that a hRPE cell population in culture is diverse with very distinct gene expression profile. Moreover, the heterogeneity of cells expressing genes at different levels changes with treatment indicating that a portion of cells responds to treatment, which suggests that unveiling different sub-populations in cell cultures is critical. The innovative technology of simultaneous measurement of 96 genes on a single cell allows us to have a better approach to understanding disease mechanisms.

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

×
×

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.

×