June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Non-invasive retinal imaging of fluorescent hESC-derived photoreceptor precursors in the living primate
Author Affiliations & Notes
  • Juliette E. McGregor
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • M Joseph Phillips
    McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States
  • Sarah Walters
    Center for Visual Science, University of Rochester, Rochester, New York, United States
    Institute of Optics, University of Rochester, Rochester, New York, United States
  • Jie Zhang
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Jennifer Strazzeri
    Flaum Eye Institute, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • David DiLoreto
    Flaum Eye Institute, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Amber Walker
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • William S. Fischer
    Flaum Eye Institute, University of Rochester, Rochester, New York, United States
  • Qiang Yang
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Louis DiVincenti
    Division of Comparative Medicine, University of Rochester, Rochester, New York, United States
  • David M Gamm
    McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, New York, United States
  • David R Williams
    Center for Visual Science, University of Rochester, Rochester, New York, United States
    Institute of Optics, University of Rochester, Rochester, New York, United States
  • Jennifer J Hunter
    Flaum Eye Institute, University of Rochester, Rochester, New York, United States
    Center for Visual Science, University of Rochester, Rochester, New York, United States
  • Merigan H William
    Center for Visual Science, University of Rochester, Rochester, New York, United States
    Flaum Eye Institute, University of Rochester, Rochester, New York, United States
  • Footnotes
    Commercial Relationships   Juliette McGregor, None; M Phillips, Opsis therapeutics (I), Opsis therapeutics (P), Opsis therapeutics (S); Sarah Walters, None; Jie Zhang, Canon Inc. (F), Canon Inc. (P), University of Rochester (P); Jennifer Strazzeri, None; David DiLoreto, None; Amber Walker, None; William S. Fischer, None; Qiang Yang, Canon Inc. (F), Canon Inc. (P), Montana State University (P), University of Rochester (P); Louis DiVincenti, None; David Gamm, Opsis therapeutics (I), Opsis therapeutics (P), Opsis therapeutics (S); David Williams, Canon Inc. (F), Canon Inc. (R), University of Rochester (P); Jennifer Hunter, University of Rochester (P); Merigan William, None
  • Footnotes
    Support  Research reported in this publication was supported by the National Eye Institute of the National Institutes of Health under Awards No. U01 EY025497 and No. P30 EY001319 and by an Unrestricted Grant to the University of Rochester Department of Ophthalmology from Research to Prevent Blindness, New York, New York. Additional funding was provided by a National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1419118. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the National Science Foundation or Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4576. doi:
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    • Get Citation

      Juliette E. McGregor, M Joseph Phillips, Sarah Walters, Jie Zhang, Jennifer Strazzeri, David DiLoreto, Amber Walker, William S. Fischer, Qiang Yang, Louis DiVincenti, David M Gamm, David R Williams, Jennifer J Hunter, Merigan H William; Non-invasive retinal imaging of fluorescent hESC-derived photoreceptor precursors in the living primate. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4576.

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

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Abstract

Purpose : The efficacy of stem cell injections into the retina to treat vision loss is conventionally evaluated post-mortem using histological methods in animal models. We explore the use of optical coherence tomography (OCT) and high resolution adaptive optics scanning light ophthalmoscopy (AOSLO) as non-invasive imaging methods to longitudinally monitor the spatial distribution, longevity and structural remodeling of photoreceptor precursor cells in the living macaque. To allow the specific identification of transplanted photoreceptor precursors in vivo we created a human embryonic stem cell (hESC)-derived CRX reporter line expressing the fluorescent protein tdTomato.

Methods : Gene targeting via homologous recombination was used to create the hESC CRX:tdTomato line, and 3D optic vesicle-like structures were generated using previously published protocols established by the Gamm lab. Photoreceptor precursor cells were dissociated and suspended in HBSS before subretinal injection into a 15-year-old male macaque (Macaca fascicularis) using a 41-gauge needle and a 20 psi pressure pulse. Immune suppression was provided by daily subcutaneous injection of cyclosporine (17.5 mg). Reflectance (796nm) and fluorescence (excitation 561 nm, emission 584-676 nm) AOSLO images were collected over a period of three months post-transplant. OCT (Heidelberg Spectralis) was conducted in regions expressing Tdtomato.

Results : Photoreceptor precursors expressed tdTomato for more than three months post transplantation, implying cell survival during that period. Precursor cell aggregates continued to remodel during that time with OCT data indicating a two-fold increase in aggregate thickness between day 52 and day 114 post-transplant. AOSLO imaging showed ring-like groupings of cells with median diameter 95 µm, similar to ‘rosettes’ previously seen in histological studies. Locations associated with photoreceptor precursors were hyper-reflective.

Conclusions : Survival, distribution and remodelling of fluorescently-labelled photoreceptor precursors injected into the subretinal space can be tracked non-invasively using AOSLO and OCT. These non-invasive methods can be used for long-term in vivo monitoring of stem cell transplants at a cellular spatial scale, reducing the need to sacrifice animals at a range of time points. This approach could be especially valuable applied to human therapy.

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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