June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Modulation of mitochondrial respiration in hPSC-RPE
Author Affiliations & Notes
  • Divya Sinha
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Gurugirijha Rathnasamy
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Kimberly A Toops
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Molly M Wilson
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Rasa Valiauga
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Li Xuan Tan
    Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Janis T Eells
    Department of Biomedical Sciences, University of Wisconsin-Milwaukee, Madison, Wisconsin, United States
  • Aparna Lakkaraju
    Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • David M Gamm
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Divya Sinha, None; Gurugirijha Rathnasamy, None; Kimberly Toops, None; Molly Wilson, None; Rasa Valiauga, None; Li Xuan Tan, None; Janis Eells, None; Aparna Lakkaraju, None; David Gamm, None
  • Footnotes
    Support  Research to Prevent Blindness Catalyst Award, International Retina Research Foundation, Foundation Fighting Blindness, NIH R01EY024588, Retinal Research Foundation Emmett A. Humble Distinguished Directorship, McPherson Eye Research Institute (Sandra Lemke Trout Chair), Carl and Mildred Reeves Foundation, NIH P30HD03352, Muskingum County Community Foundation, SIG 1S10OD018039-01 for Seahorse XFe96 analyzer, Support Grant P30 CA014520 to SMSSF (core service of the University of Wisconsin Carbone Cancer Center)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3025. doi:
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    • Get Citation

      Divya Sinha, Gurugirijha Rathnasamy, Kimberly A Toops, Molly M Wilson, Rasa Valiauga, Li Xuan Tan, Janis T Eells, Aparna Lakkaraju, David M Gamm; Modulation of mitochondrial respiration in hPSC-RPE. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3025.

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

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Abstract

Purpose : Efficient mitochondrial activity is required to meet the energy demands of RPE, which is a highly metabolically active monolayer of cells possessing multiple critical roles in the outer retina. Given the interest in human pluripotent stem cell-derived RPE (hPSC-RPE) as a source for cell replacement therapies, it is important to investigate mitochondrial health and behavior in hPSC-RPE under relevant environmental conditions, which may in turn improve transplantation strategies.

Methods : Monolayers of RPE were derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) using methods established by our lab. hPSC-RPE cells were treated with 10 µM A2E (a retinoid in lipofuscin) for 6 h followed by incubation in fresh culture medium for 48 hours prior to live imaging, and for 72 hours for extracellular flux (XF) assay. Alternatively, cultures were exposed to 50 µM resveratrol for 24 hours directly followed by live imaging and XF analysis. Live-cell imaging and XF assays were used to image mitochondrial structure and measure mitochondrial respiration, respectively. Data analysis was performed using Imaris (Bitplane) for live imaging and Wave (Agilent Technologies) for XF assay.

Results : Mitochondria in hPSC-RPE exist as interconnected networks with readily measurable oxygen consumption rates (OCR) and spare respiratory capacity (SRC). A2E exposure leads to mitochondrial fragmentation, indicative of cellular stress, and increased basal OCR in hPSC-RPE cells (115.97±6.49 pmol/min) compared to untreated hPSC-RPE (94.40±6.98 pmol/min). hPSC-RPE cells treated with resveratrol show highly networked mitochondrial structure compared to control cells. Additionally, resveratrol treatment enhances spare respiratory capacity in hPSC-RPE by up to 1.8-fold compared to untreated cells.

Conclusions : We show that exposure to certain compounds can modulate mitochondrial structure and function in cultured hPSC-RPE. Rigorously monitoring such changes provides a means to study the response of RPE cells to conditions of oxidative stress and to screen for interventions that might enhance mitochondrial activity. In particular, enhanced SRC is critical for meeting increased energy demands under conditions of cellular stress and has the potential to enhance cell survival post-transplant.

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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