September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Photoreceptor precursor transplantation leads to exchange of cellular contents between the donor and host retina
Author Affiliations & Notes
  • Alun R Barnard
    Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Jasmin Balmer
    Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Mandeep S Singh
    Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Daniela Moralli
    Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
  • Catherine Green
    Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
  • Robert Maclaren
    Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
    Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships   Alun Barnard, None; Jasmin Balmer, None; Mandeep Singh, None; Daniela Moralli, None; Catherine Green, None; Robert Maclaren, None
  • Footnotes
    Support  The Royal College of Surgeons of Edinburgh
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Alun R Barnard, Jasmin Balmer, Mandeep S Singh, Daniela Moralli, Catherine Green, Robert Maclaren; Photoreceptor precursor transplantation leads to exchange of cellular contents between the donor and host retina. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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

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Abstract

Purpose : Ongoing experiments raised concerns over subretinal transplantation of photoreceptor precursors into non-degenerate mouse eye, related to misidentification of fluorescent-labelled cells in the host outer nuclear layer (ONL) as genuine, integrated donor cells. We used molecular-genetic techniques to confirm the identity of fluorescent-labelled cells in the ONL and further investigate this phenomenon.

Methods : Retinas of female pups (postnatal day 4-7) of the Tg(Nrl-EGFP)1Asw line were collected and dissociated. GFP-positive photoreceptor precursors were isolated by FACS. Cell suspensions were injected subretinally into male, adult, wild-type host mice. Injected eyes were collected 2 weeks later and prepared for histology. Fluorescent in situ hybridisation (FISH) was performed against the Y-chromosome. Dissociated retinas from early postnatal mice of a line combining GFP-labelled photoreceptor precursors (Tg(Nrl-EGFP)1Asw) with an allele that generates a red fluorescent protein in a Cre-sensitive manner (Gt(ROSA)26Sortm9(CAG-tdTomato)Hze) were injected subretinally into adult host mice in which Cre recombinase is driven by the cone-rod homeobox gene promoter (Tg(Crx-cre)1Tfur). Injected eyes were collected 2 weeks later and prepared cryosections examined using confocal microscopy.

Results : Sex-mismatching experiments showed cells in the host ONL that were GFP-positive but that also clearly contained a Y-positive nucleus. As donor GFP-cells were exclusively of female origin (confirmed by absence of Y-positive nuclei in subretinal cell mass), GFP-positive ONL cells could only be explained by transfer of cellular contents from donor to host photoreceptors. Cre-Lox recombination experiments identified cells in the ONL that were positive for GFP and also the Cre-sensitive reporter protein (tdTomato). This indicated Cre recombinase (present in the host) had been taken up by donor cells.

Conclusions : These experiments indicate there is cell fusion and bidirectional exchange of cellular contents and/or cytoplasm between transplanted photoreceptor precursor donor cells and cells in the host ONL. This suggests care should be taken in the interpretation of apparently well-formed and integrated donor photoreceptor cells in the ONL of transplanted eyes. Conversely, these results also raise the possibility of donor-host cell fusion as a potential therapeutic strategy in the outer retina.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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