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REGENERATIVE MEDICINE

Vision Science Division, Krembil


Research Institute, University Health
Network and 2Department of Ophthalmology and Vision Sciences, and
3
Department of Laboratory Medicine
and Pathobiology University of Toronto
Address for correspondence: Valerie
Wallace, PhD, KRI, 60 Leonard St.
Toronto ON M5T 2S8, Phone: 416603-5800
x
7378,
vwallace@uhnresearch.ca; Funding: E.
L.S. Tsai and M. Bergeret are recipients of Vision Science Research Program studentships and A. OrtinMartinez is a recipient of a postdoctoral fellowship from the McEwen
Center for Regenerative Medicine.
This work was supported by operating grants to V. A. Wallace from Brain
Canada, Foundation Fighting Blindness, Ontario Institute of Regenerative Medicine and Krembil Foundation.; * co-first authors; Summary
Statement: We report a novel observation of material exchange between
donor to host cells in subretinal photoreceptor transplantation
Received September 02, 2016; accepted for publication November 04,
2016; available online without subscription through the open access
option.
AlphaMed Press
1066-5099/2016/$30.00/0
This article has been accepted for
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Please cite this article as doi:
10.1002/stem.2552

A Re-interpretation of Cell Transplantation:


GFP Transfer from Donor to Host Photoreceptors
ARTURO ORTIN-MARTINEZ1*, EN LEH SAMUEL TSAI1,3*, PHILIP E.
NICKERSON1*, MIRIAM BERGERET1,3, YAO LU3, SHEILA SMILEY1,
LACRIMIOARA COMANITA1 AND VALERIE A. WALLACE1,2,3
Key words. GFP photoreceptors transplantation fusion

ABSTRACT
The utilization of fluorescent reporter transgenes to discriminate donor
versus host cells has been a mainstay of photoreceptor transplantation
research, the assumption being that the presence of reporter+ cells in outer nuclear layer (ONL) of transplant recipients represents the integration of
donor photoreceptors. We previously reported that GFP+ cells in the ONL
of cone-GFP transplanted retinas exhibited rod-like characteristics, raising
the possibility that GFP signal in recipient tissue may not be a consequence
of cell integration. To investigate the basis for this mismatch, we performed a series of transplantations using multiple transgenic donor and
recipient models, and assessed cell identity using nuclear architecture,
immunocytochemistry and DNA pre-labeling. Our results indicate that GFP+
cells in the ONL fail to exhibit hallmark elements of donor cells, including
nuclear hetero/euchromatin architecture. Furthermore, GFP signal does
not appear to be a consequence of classic donor/host cell fusion or transfating post-transplant, but is most likely due to material exchange between
donor and host photoreceptors. This transfer can be mediated by rods and
cones, is bidirectional between donor and host cells, requires viable photoreceptors, occurs preferentially at sites of outer limiting membrane disruption and can be detected in 2nd order retinal neurons and Mller glia. Collectively, these data warrant re-evaluation of the use of lineage tracing
fluorescent reporters in the eye and other CNS tissues. Furthermore, the
re-interpretation of previous functional rescue data, based on material
exchange, rather than cell integration, may offer a novel approach to vision
rescue. STEM CELLS 2016; 00:000000

SIGNIFICANCE STATEMENT
Improvements in vision following transplantation of GFP-labeled photoreceptors are associated with the presence of GFP+ cells in the recipient retina. Here we show that the origin of the GFP signal is due to transfer from
donor photoreceptors rather than integration of donor cells. These findings
warrant a re-evaluation of the mechanism of photoreceptor mediated vision
rescue.

INTRODUCTION
Transplantation of photoreceptors is reported to partially rescue vision in blind animal models [1-5], an efSTEM CELLS 2016;00:00-00 www.StemCells.com

fect that has been attributed to the physical integration


of donor photoreceptors. Reporter transgenes have
garnered in this interpretation, as the extent of rescue
correlates with the number of GFP+ cells in the outer
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nuclear layer (ONL) of host retinas [1-5]. We described
the utility of cone and cone/rod hybrid precursor cell
(termed cods) transplantation through generation of
Ccdc136GFP/+ gene trap, and Nrl/;Ccdc136GFP/+ compound mutant mice, respectively [6]. Although we observed GFP+ photoreceptors in the recipient ONL following transplants, we reported an unexpected mismatch
in nuclear morphology between donor and integrated
cells [6]. Here, we examined the relationship between
GFP+ cells the ONL of transplant recipients and donor
cell identity. Rather than representing classic cell fusion
or donor cell trans-fating, our results implicate transfer
of cellular contents, including GFP, between donor and
host photoreceptors.

MATERIALS AND METHODS


Animals and Genotyping
All experiments were approved by the University Health
Network Research Ethics Board and adhered to the
guidelines of the Canadian Council on Animal Care. Animal husbandry was in accordance with the Association
for Research in Vision and Ophthalmology (ARVO)
Statement for the Use of Animals in Ophthalmic and
Vision Research. Animals were maintained under standard laboratory conditions and all procedures were performed in conformity with the University Health Network Animal Care Committee (protocol 3499.10).
Mouse strains are summarized in Table S1. C57BL/6J
mice (Charles River), Crx-/- [7] and Nrl-/- [8] of both sexes
were used as transplantation recipients, all of them
being 6-14 weeks at the time of transplantation. NrlGFP [9], Ccdc136GFP/+ and Nrl-/-;Ccdc136GFP/+ [6] and
ROSAmT/mG mice were used as photoreceptor donors at
postnatal P3 - P5. For homozygous lines, genotyping
was performed by extracting genomic DNA from ear clip
samples through incubation in 200 l alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA pH 8.0) for 1 hour at 95
C. Samples were neutralized with 200 l neutralization
buffer (40 mM Tris-HCl) and genotyped by PCR using
primer sets indicated in Table S2.

Donor cell preparation, labeling and fluorescence activated cell sorting


To prepare donor cells for transplantation, retinas from
Ccdc136GFP/+, Nrl-/-;Ccdc136GFP/+ and Nrl-GFP were harvested in CO2 independent media (Fisher Scientific) and
dissociated with papain (Worthington Biochemical, UK)
according to the manufacturers directions. Cells were
washed in Ca2+/Mg2+ free PBS and counted using 0.4%
Trypan blue (Thermo Fisher) as a viability counter stain
before being re-suspended in 5% BSA 2 mM EDTA 25
mM HEPES 0.005% DNAse in Ca2+/Mg2+ free PBS, and
passed through a cell strainer (40 m) into FACS tubes
(BD Falcon). Cells were then sorted for GFP fluorescence using an Aria III (BD Biosciences) equipped with a
488 nm laser and collected in 10% BSA in Ca2+/Mg2+ free
PBS. The sort was performed using an 85 m nozzle at
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GFP transfer to recipient photoreceptors


45 psi, and a flow rate that allowed collecting at 5000
events/second. The data acquisition, analysis, and image preparation were carried out using the instrument
software FACSDiva (BD Biosciences). The GFP gating
histograms are shown in Figure S1A. Sorted GFP+ and
unsorted cells were re-suspended at a concentration of
200,000 cells/l in 0.005% DNAse, Earls Balanced Salt
Solution (EBSS) and kept on ice prior to transplantation.
To label donor photoreceptor nuclei, P1 Nrl-GFP pups
received 3 intra-peritoneal injections separated by 4
hours (summarized in Figure S1B) with 5-ethynyl-2'deoxyuridine (50 mg/kg, 5 mg/ml stock concentration,
Life Technologies EdU). The retinas were dissociated at
P4 and GFP+ photoreceptors were enriched by FACS.
EdU labeling in the GFP+ donor cell population was confirmed at 40% EdU coverage of postmitotic donor cells
(Figure S1C). EdU detection was performed using an
Alexa Fluor 647 Click-iT (azides/alkyne) reaction as per
manufacturers instructions.

Subretinal injections

Adult recipient C57BL/6J, Nrl-GFP, Crx-/-, and Nrl-/- mice


(6-8 weeks old) were anesthetized using a mixture of
ketamine (100 mg/ml, Vetalar) at 50mg/kg and medetomidine (1 mg/ml, Dexdomitor, Zoetis) at 1mg/kg in
sterile 0.9% NaCl administered intraperitoneally. Eyes
were dilated using 1% tropicamide (Mydriacyl, Alcon)
drops, followed by application of a 0.2% hydromellose
gel (Genteal, Novartis) to maintain proper lubrication of
the eyes. All injections were performed in the left eye.
The eye was gently prolapsed and then immobilized
using a latex dam customized to allow free blood circulation. Injections were performed using a nano-injector
(Harvard Apparatus) and two surgical microscopes. The
retina was visualized for confirmation of cell deposit
using a glass coverslip under a dissection microscope. A
microscopy-guided microinjection was performed by a
second surgeon located orthogonal to the viewing microscope. A scleral incision was made in the dorsal side,
posterior to the limbus using a 30-gauge needle. Next, a
blunt 32-gauge needle (Hamilton) was inserted tangentially into the subretinal space (SRS) and advanced in
the SRS under the guidance of the person visualizing the
fundus. Once the needle was located in the SRS, a small
incision was made in the cornea to relieve the intraocular pressure. 1.0 l of cell suspension (dose varying between 50,000 to 200,000 donor cells) was injected over
30 seconds, followed by a 60 second pause to allow for
equilibration of pressure and to prevent efflux. The
needle was then slowly retracted and the animal anesthesia reversed using an intraperitoneal injection of
1mg/kg atipamezole (5mg/mL Revertor, Zoetis). Animals were kept on a heating pad until fully recovered.

Tissue Processing, Histology and Immunocytochemistry


Mice were harvested 21 days post-surgery by exsanguination with phosphate buffered saline (PBS) (0.14 M
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NaCl, 2.5 mM KCl, 0.2 M Na2HPO4, 0.2 M KH2PO4) and
transcardially perfused with 4% paraformaldehyde
(PFA). Eyes were then marked with a silver nitrate stick
on the dorsal part of the cornea. Tissues were fixed for
an additional 30 minutes in 4% PFA on ice and then cryoprotected overnight in 30% sucrose at 4oC in PBS. After
overnight cryoprotection, tissues were equilibrated in
50:50 30% sucrose in PBS: OCT (Tissue-Tek) for 1 hour
and subsequently oriented and embedded in plastic
molds. Tissue blocks were stored in -80 C. Tissue was
sectioned at 20 m thickness onto Superfrost Plus slides
(Fisher Scientific) on a Leica cryostat and air-dried for 1
hour before being stored in a slide box with desiccant at
-20 C. For immunocytochemistry the sections were
screened and selected to exclude the injection site. Sections were blocked with 10% donkey serum (DS) (Sigma
Aldrich) 0.3% Triton-X in PBS for 1 hour at room temperature. Primary antibodies (Table S3) were diluted in
5% DS 0.15% Triton-X in PBS for overnight staining of
retina sections at 4 C. After three washes with PBS,
sections were incubated with fluorescent secondary
antibody diluted in 5% DS 0.15% Triton-X in PBS for 1
hour at room temperature in a light protected humidified box. Nuclei were counterstained with fluorescent
DNA-binding dye, Hoechst 33342 (Life Technologies).
Slides were washed and glass coverslips were mounted
with DAKO mounting media.

Confocal Imaging, Cell Counting and Statistics


Wide-field fluorescent images were acquired using
an Axioimager M1 (Carl Zeiss Inc.). 2048 x 2048 resolution confocal images were acquired using a LSM 780
(Carl Zeiss Inc.) with 2% laser intensities and no greater
than 800 gain, with a minimum of 4x averaging. Objectives (20x, 40x water immersion, 63x oil immersion) and
a 1.0 Airy Unit pinhole determined voxel depth on zstacks. Image acquisition parameters were maintained
for all comparative images. Images were processed using Photoshop CS4 or CS6 and any adjustments were
made to the entire image and equally for all comparative images. The number of GFP+ cells in the ONL was
quantified by counting all cells with the entire cell body
located in the host ONL. The total number of GFP+ cells
per eye was determined by extrapolation, based on
quantification of every 4th section. Nuclear mismatching and co-localization values were generated from
counts in alternate sections that were visualized with
40x and 63x objectives. Orthogonal images were generated using Zeiss Zen software. Animals devoid of any
bolus cells in the subretinal space were excluded from
the analysis. All data are presented as mean standard
error of the mean (S.E.M.).

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GFP transfer to recipient photoreceptors

RESULTS AND DISCUSSION


Mismatch between GFP+ donor and recipient
cells

To investigate the identity of GFP+ cells located the ONL


(ONL-GFP) of photoreceptor transplant recipients, we
exploited the distinct nuclear architecture of rods and
cones in vivo ([10], Fig. S2A). Consistent with previous
reports [1, 3], ONL-GFP cells in C57BL/6J (wildtype) recipients transplanted with P3-P5 Nrl-GFP rod (rod-GFP)
precursors [9] were rods, based on nuclear architecture
and morphology (Fig. 1A). However, we also detected a
GFP+ cell with cone-like morphological and cytochemical
features (Fig. 1B), suggesting a fate mismatch between
donor rods and ONL-GFP cells in recipients. To investigate whether this donor/host mismatch extends to
cones, we transplanted P3-P5 Nrl/;Ccdc136GFP/+ cods
(cod-GFP) or Ccdc136GFP/+ cones (cone-GFP) into roddominant wildtype recipients. Cod and cone cells in the
subretinal space (SRS) exhibited cone-like nuclei and
marker expression (Fig. 1C, D, S2B-B), whereas >99%
ONL-GFP cells were rod-like and negative for cone
markers (Fig. 1C,D,F). Similarly, in rod-GFP transplanted
cod-only Nrl-/- retinas, ~80% of ONL-GFP cells had a
cone nuclear morphology (Fig. 1E,F), and all coexpressed Cone arrestin (CAr), a cone marker (Fig. 1E),
indicating that rod transplantation generates a cod
identity in Nrl-/- recipients. The extent of mismatch was
not a function of the number of GFP+ cells in the recipient ONL, although notably, these cells were abundant in
Nrl-/- recipients (Fig. 1G), which is consistent with previous reports [2]. In contrast, transplantation of cod-GFP
and cone-GFP cells into Crx-/- mice, a mouse model of
photoreceptor degeneration [7], appeared consistent
with bona fide cell integration (Fig. S3), as evidenced by
GFP/CAr co-localization and donor-matched nuclear
architecture in ONL-GFP cells. These data provide evidence that GFP labeling observed in recipient retinas
following photoreceptor transplantation favors the
most abundant cell type present in recipient retinas,
with the exception of that observed in the degenerating
retina.

GFP+ cells in transplant recipients may arise


from GFP transfer
The mismatch between donor and ONL-GFP cells could
arise from classic cell fusion, a cell fate switch, or previously uncharacterized transfer. Fusion of GFP-labelled
hematopoietic cells results in bi-nucleated cerebellar
neurons [11, 12]. Orthogonal-examination of ONL-GFP
cells in all recipients failed to identify a bi-nucleate
event (not shown). Rod photoreceptors originate from
the S-cone lineage [13] , raising the possibility that a
fate switch from rod to cone could account for the GFP
labeling in cones in rod transplanted retinas. To address
this possibility, we transplanted P4 rod-GFP cells that
were pre-labelled in vivo with the thymidine-analogue
EdU (Fig. S1B-C), into Nrl-/- recipients. This labeling parAlphaMed Press 2016

GFP transfer to recipient photoreceptors

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adigm serves as an indelible lineage-independent DNA
marker, which should be detectable if donor cells switch
fate after integrating into the recipient retina. We utilized Nrl-/- recipients, which exhibit high rates of ONLGFP cells post-transplantation (Fig. 1F), to maximally
identify EdU+ cells in the recipient ONL. Although we
detected EdU+/GFP+ cells in the SRS, we did not detect a
single EdU+ nucleus in the ONL (>7800 ONL-GFP cells
examined - Fig. 2A-C) arguing strongly against donor cell
integration as the source of ONL-GFP cells. Fusion or
transfer has not been reported in experiments where
rod-GFP cells were transplanted into cytoplasmic cyan
fluorescent reporter recipients [1]. Utilizing a different
approach, we transplanted retinal dissociates from P3P5 mice expressing membrane-tethered tdTomato
(ROSAmT/mG, Fig. S4A-E) into rod-GFP recipients (Fig.
2D-E). We identified tdTomato/GFP co-labelled cells in
the ONL, demonstrating transfer of a membranetethered reporter (Fig. 2D, S4A-E). Furthermore, we
observed transfer of GFP (Fig. 2E) and CAr (Fig. S4F-G),
but not S-Opsin (Fig. S2C), which are expressed in the
recipient retinas, to the donor cells in the SRS, suggesting that host-to-donor cell transfer is also possible for
some proteins.
We next investigated the microenvironment surrounding donor/host interaction. We were unable detect tdTomato/NRL-GFP exchange in vitro using cocultures, suggesting that in vivo elements are required
for donor/host exchange. Strikingly, 452/456 in vivo
columns of ONL-GFP cells in Nrl-/- recipients were associated with brightly GFP-labelled, donor cells located at
the outer limiting membrane (OLM) (Fig. 3A). We also
observed what appears to be physical contact between
donor/host cells in transplanted retinas (Fig. 3B). Previously, disruption of the OLM was associated with increased numbers of ONL-GFP cells in recipients [14, 15].
Staining for the OLM marker Zo1 in control and Nrl/
retinas revealed mislocalization at points of putative
GFP transfer (Fig.3C-D), suggesting that OLM breaks
could facilitate cell-cell contact or GFP transfer via noncontact-mediated mechanisms. Comparison of uninjected, sham-injected, Nrl/, and Crx/ recipients (Fig.
S6) identified variability in OLM integrity across all
transplant contexts, suggesting that graded OLM disruptions may be variably permissive to GFP transfer.

GFP transfer to the INL


We next considered the possibility of transfer beyond
the ONL by screening for low-level GFP in INL cells,
which would represent 2nd order transfer. Screening
revealed low-level signal in INL cells (Fig. 4A), situated
below areas of high ONL-GFP expression in rod-GFP to
Nrl/ transplants. Co-immunolabeling for Vsx2/GFP (Fig.
4C, S5A) and GS/GFP (Fig. 4E, S5B) identified GFP signal
in bipolars and Mllers, respectively, whereas Calbindin-D28k (Fig. 4D) failed to detect GFP in horizontal
cells. Imaging of Rod-GFP donor retinas using the same

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imaging parameters identified similar GFP signal the


INL, but not in wildtype and control regions (Fig. S5D-E).
GFP/Iba1 staining failed to identify GFP in phagocytic
microglia (Fig. S5C).

CONCLUSION
We suggest that GFP transfer, rather than cell integration, explains GFP-labeling in recipient retinas transplanted with GFP+ cone and rod photoreceptors, an
interpretation that is in agreement with recent findings
from other research groups [16, 17]. Transfer requires
intact photoreceptors, is associated with cell contact
and OLM disruption, and is bidirectional (summarized in
Figure S7). The putative transfer of cone proteins from
host to donor photoreceptors suggests that the exchange of other functionally important photoreceptor
proteins is likely, which could explain vision rescue reported in blind mice transplanted with photoreceptors
[1, 2]. Consequently, data pertaining to donor/recipient
age [1], OLM disruption [14], and marker-based stratification [18] describe the conditions for GFP transfer rather than cell integration per se. Donor/host DNA and
mitochondiral transfer [19][20], and intercellular exchange via microvesicle/exovesicle GFP [21] [222], tunneling nanotubes [23] have been described in various
systems, offering prospective cellular transfer mechanisms. While intercellular transfer of material raises
safety concerns for cell-based therapies in the eye and
beyond, it also has the potential to be exploited for
therapeutic applications.

ACKNOWLEDGMENTS
We thank Drs. van der Kooy, Bremner, Monnier and
Schuurmans for helpful discussion and Drs. van der
Kooy and Hui for sharing reagents.

COMPETING INTERESTS
No competing interests declared.

AUTHOR CONTRIBUTIONS
A O M: Concept and design, collection and assembly of
data, Data analysis and interpretation; E L S T: Concept
and design, collection and assembly of data, Data analysis and interpretation; P E. N: Concept and design, collection and assembly of data, data analysis and interpretation, manuscript writing; Y L: collection and assembly of data; M B: Collection and assembly of data,
data analysis and interpretation; S S: Collection and
assembly of data, data analysis and interpretation; L C:
Concept and design, collection and assembly of data,
data analysis and interpretation; V A. W ; Concept and
design, financial support, data analysis and interpretation, manuscript writing

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GFP transfer to recipient photoreceptors

Figure 1: GFP-labeled cells in the ONL of photoreceptor transplant recipients exhibit nuclear and morphological mismatch to donor cells.
All mice were sacrificed at 21 days post-transplant. (A-E) Immunohistochemistry for GFP, Cone arrestin (CAr) and nuclei in retinal sections of wildtype recipients transplanted with rod-GFP (A, B), cod-GFP (C) cone-GFP cells (D) and Nrl-/recipients transplanted with rod-GFP cells (E). (A, C, D) Irrespective of the donor cell nuclear morphology in the subretinal space (SRS), GFP+ cells in the recipient ONL have a rod identity based on nuclear morphology (bottom right
panel) and the presence of GFP+ spherules (bottom left panels). The only exception is (B) where in a rod-GFP transplanted wildtype retina we observed a single GFP+ in the outer nuclear layer with a cone nuclear morphology (lower
right panel), a cone pedicle (lower left panel) and co-localization with CAr (middle panels). (E) A cod-like nuclear morphology (lower right panel) and cod pedicle (lower left panel) is observed in the ONL of the Nrl-/- recipients when donor cells were GFP+ rods. (F) Quantification of nuclear mismatch between donor and recipient GFP-labeled photoreceptors. Included are the number of animals (n) quantified and the number of cells analyzed. Error bars represent
S.E.M., ** P < 0.01, Students t-test. (G) Quantification of GFP-labeled cells in the recipient ONL.Y-axis is interrupted
due to robust labeling in Nrl-/- recipients. Error bars represent S.E.M., ** P < 0.01, Students t-test. Scale bars: yellow
2m, white, 10 m.

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GFP transfer to recipient photoreceptors

Figure 2. Membrane-tethered tdTomato transfers from donor to host photoreceptors, while donor nuclei fail to migrate into recipient retinas.
(A) Maximum intensity projection of immunostaining for GFP, EdU and nuclei in retinal sections of Nrl-/- recipients, 21
days after transplantation of EdU-labeled rod-GFP cells into the SRS. (B) Inset single plane image showing GFP+/EdU+
cells in the SRS. Note the presence of GFP+/EdU+ cells exclusively in the SRS, while in the ONL, only GFP+/EdU- cells
were observed. (C) Histogram showing the proportion of EdU+ rod-GFP cells pre and post-transplant in the SRS and
ONL. (D) Immunostaining showing the co-localization of GFP and tdTomato in an ONL cell following transplantation of
rod-GFP recipients with ROSAmT/mG retinal cells. (E) GFP staining of donor cell bolus located in the SRS of retinas transplanted as in (D). SRS: subretinal space, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer,
IPL: inner plexiform layer, GCL: ganglion cell layer. Scale bar: 50 m

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GFP transfer to recipient photoreceptors

Figure 3. Enhanced GFP transfer observed when donor and recipient cells are in close contact and in the proximity of
disrupted OLM.
(A) Donor cells located directly above columns of GFP+ cells in the ONL of Nrl-/- retinas transplanted with rod-GFP
cells. (B) Examples of contact between donor photoreceptors in the SRS and GFP+ cells in the host ONL. (C) GFP+ cells
localize in the SRS and ONL to sites of OLM disruption, marked by Zo1 staining. (D) Off angle 3D rendering of OLM
breaches, through which GFP+ cells reside. SRS: subretinal space, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer. Scale bar: 10 m

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GFP transfer to recipient photoreceptors

Figure 4. GFP transfer is observed in the inner nuclear layer of recipient retinas.
(A) Presence of GFP in the INL of an Nrl-/- recipient following transplant with rod-GFP cells. (B) Histogram showing the
proportion of GFP+ cell subtypes in the INL. (C) A subset of GFP labeled cells in the INL are co-labeled with Vsx2. (D)
No co-labeling of GFP and Calbindin was observed. (E) Majority of GFP+ cells in the INL co-label with glutamine synthetase (GS). SRS: subretinal space, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL:
inner plexiform layer, GCL: ganglion cell layer. Scale bar: 20 m

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GFP transfer to recipient photoreceptors

Graphical Abstract
GFP-expressing photoreceptors derived from transgenic reporter mice are dissected, dissociated, FACS purified, and
injected into the subretinal space of recipient mice. Historically, the presence of GFP-positive cells in the outer nuclear layer was interpreted as a migratory event, and integration of these cells into recipient tissue, as detailed under
the Hypothesis heading (middle panel). The work reported here identifies that donor cells do not integrate into recipient retinas, but rather, transfer GFP reporter signal to endogenous photoreceptors, revealing a novel property of GFP
kinetics in retinal cell transplantation, as detailed under the Results heading. The efficiency of this transfer is dramatically increased in the cone-only (NRL knockout) retina compared to wildtype recipients. DNA pre-labeling (EdU), nuclear architecture, dual fluorescent donor/host reporter combinations and immunocytochemical screening techniques were used to demonstrate reporter transfer in this context.

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AlphaMed Press 2016