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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. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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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