Transplanted photoreceptor precursors transfer proteins to host photoreceptors by a mechanism of cytoplasmic fusion

Mandeep S Singh, Jasmin Balmer, Alun R Barnard, Sher A Aslam, Daniela Moralli, Catherine M Green, Alona Barnea-Cramer, Isabel Duncan, Robert E MacLaren, Mandeep S Singh, Jasmin Balmer, Alun R Barnard, Sher A Aslam, Daniela Moralli, Catherine M Green, Alona Barnea-Cramer, Isabel Duncan, Robert E MacLaren

Abstract

Photoreceptor transplantation is a potential future treatment for blindness caused by retinal degeneration. Photoreceptor transplantation restores visual responses in end-stage retinal degeneration, but has also been assessed in non-degenerate retinas. In the latter scenario, subretinal transplantation places donor cells beneath an intact host outer nuclear layer (ONL) containing host photoreceptors. Here we show that host cells are labelled with the donor marker through cytoplasmic transfer-94±4.1% of apparently well-integrated donor cells containing both donor and host markers. We detect the occurrence of Cre-Lox recombination between donor and host photoreceptors, and we confirm the findings through FISH analysis of X and Y chromosomes in sex-discordant transplants. We do not find evidence of nuclear fusion of donor and host cells. The artefactual appearance of integrated donor cells in host retinas following transplantation is most commonly due to material transfer from donor cells. Understanding this novel mechanism may provide alternate therapeutic strategies at earlier stages of retinal degeneration.

Figures

Figure 1. The majority of putative donor…
Figure 1. The majority of putative donor cells (GFP positive) in the host outer nuclear layer also contain a fluorescent marker that is present in the host (DsRed).
Postnatal day 1–3 (P1–3) Nrl-GFP donor photoreceptor precursors were transplanted into the subretinal space of adult CAG-DsRed mice without retinal degeneration and after 3 weeks, numerous GFP-positive photoreceptors were found in the host outer nuclear layer. (a) Shows merged GFP, DsRed and DAPI channels, (b) GFP only and (c) DsRed only. GFP and DsRed were co-distributed in cytoplasm in the (d) rod spherule synapse, (e–h) perinuclear cytoplasm and (i) inner segments. (j) By comparison, the majority of donor photoreceptors cells in the subretinal space contained GFP but were negative for DsRed. (k) The mean Mander's overlap coefficient (MOC) of DsRed and GFP in cells located in the ONL was 0.9. The horizontal lines indicate the medians, the boxes extend from the 25th to 75th percentiles and the whiskers indicate the minimum and maximum values. (l) A GFP+ cell located outside the host outer nuclear layer (ONL) with low DsRed and GFP co-localization (MOC=0.059). (m) A cell located in the host ONL with a high degree of co-localization (MOC=0.96).
Figure 2. The nuclei of GFP-positive donor…
Figure 2. The nuclei of GFP-positive donor cells in the host ONL contain the Y chromosome that is only present in the host.
(a,b) Projection images showing donor photoreceptor precursor cells from P3 female Nrl.GFP mice that were transplanted into the subretinal space of an adult male wild-type host.The asterisk is a separation of the subretinal donor cells (SDCs) from the ONL due to a histological artefact. GFP-positive cells in the male host photoreceptor layer can clearly be seen to contain Y-positive nuclei (c–f). (g,h) GFP-positive cells in the subretinal donor cell mass contain only X chromosomes.
Figure 3. Further evidence of cytoplasmic transfer…
Figure 3. Further evidence of cytoplasmic transfer between donor and host.
(a–d) Donor cells collected from P1–3 DsRed mice, which were also homozygous for the rd1 mutation were transplanted into adult Nrl-GFP mice in which rods are labelled with GFP. Three weeks after transplantation, a DsRed and GFP-positive photoreceptor cell bodies were seen. (e–h) To confirm cytoplasmic transfer of proteins using a Cre-lox recombination, donor cells (CAG-LSL-tdTomato, Nrl-GFP) at P7 were transplanted into adult Crx-Cre host mice. Three days post transplantation, donor cells in the subretinal space are GFP positive as expected, whereas in the host ONL (white box) a double-labelled (GFP and tdTomato) cell body is visible, confirming that Cre had passed from host photoreceptor to donor cell to activate tdTomato, which then retrogradely labelled the host photoreceptor together with GFP.

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