本帖最后由 凤凰涅盘 于 2013-1-12 14:36 编辑
这是论文的原文，希望能有专业人士帮助大家翻译理解，先谢谢了。这是美国国家科学院院刊的链接http://www.pnas.org/content/110/ ... c-82e8-3980006c1aad
Repair of the degenerate retina by
photoreceptor transplantation
Amanda C. Barber
a
, Claire Hippert
a
, Yanai Duran
a
, Emma L. West
a
, James W. B. Bainbridge
a
,
Katherine Warre-Cornish
a
, Ulrich F. O. Luhmann
a
, Jorn Lakowski
b
, Jane C. Sowden
b
,
Robin R. Ali
a,c,1
, and Rachael A. Pearson
a,1
a
Department of Genetics, University College London Institute of Ophthalmology, London EC1V 9EL, United Kingdom; and
b
Developmental Biology Unit
and
c
Molecular Immunology Unit, University College London Institute of Child Health, London WC1N 1EH, United Kingdom
Edited by Eric A. Pierce, Massachusetts Eye and Ear Infirmary, Boston, MA, and accepted by the Editorial Board November 14, 2012 (received for review
August 6, 2012)
Despite different aetiologies, age-related macular degeneration
and most inherited retinal disorders culminate in the same final
common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy
applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into
a mouse model of stationary night-blindness, raising the critical
question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present
a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but
disease type has a major impact on outcome, as assessed both by
the morphology and number of integrated rod-photoreceptors.
Integration can increase (Prph2
+/Δ307
), decrease (Crb1
rd8/rd8
,
Gnat1
−/−
, Rho
−/−
), or remain constant (PDE6β
rd1/rd1
, Prph2
rd2/rd2
)
with disease progression, depending upon the gene defect, with
no correlation with severity. Robust integration is possible even
in late-stage disease. Glial scarring and outer limiting membrane
integrity, features that change with degeneration, significantly
affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model
with an intact outer limiting membrane, strong gliotic response,
and otherwise poor transplantation outcome (Rho
−/−
), leading
to an eightfold increase in integration and restoration of visual
function. Thus, it is possible to achieve robust integration across
a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.
gliosis
| retinal degeneration | stem cells
R
etinal degeneration is a major cause of untreatable blindness.
Despite very different aetiologies, degenerative retinopathies
culminate in the same final common pathway, the loss of photoreceptors and vision. Because photoreceptors are terminally
differentiated neurons, they cannot regenerate and once lost are
not replaced. Although recently there has been considerable progress in strategies such as gene supplementation therapy (1), when
cell death has already occurred, cell replacement therapies offer
a complementary and potentially generic approach.
We have previously demonstrated proof-of-concept for rod
(2) and cone (3) photoreceptor transplantation. Most recently,
we have shown that transplanted postmitotic rod-photoreceptor
precursor cells, identified by their expression of GFP driven by
the promoter for the rod-specific transcription factor, Nrl (4), can
migrate into the retina of a model of stationary night-blindness
in numbers sufficient to restore rod-mediated vision (5). Recent
advances in stem cell technology have also demonstrated the
potential to generate suitable populations of donor cells (6–8).
These advances provide strong justification for the development
of photoreceptor replacement as a treatment for degenerative
disease. Until now, photoreceptor transplantation has been
assessed in normal animals and a few isolated models of degeneration at single points within the degenerative process (2, 3, 5, 9–
11). To determine the feasibility of photoreceptor replacement
therapy as a generic treatment for retinal disease, transplantation
must be assessed in different models and at different stages of
retinal degeneration, both to determine the potential breadth of
application and to identify therapeutic time windows.
The degenerating retinal environment is very different from
that of the normal retina and may have an adverse effect on the
ability of cells to migrate from the site of transplantation into the
recipient outer nuclear layer (ONL). CNS injury and degeneration
are often associated with a series of events that culminate in reactive gliosis and the formation of a glial scar. This scar acts as
a reservoir of inhibitory extracellular matrix molecules, such as
chondroitin sulfate proteoglycans (CSPGs), which prevent axonal
and cell migration and regeneration (12). In the retina, photoreceptor death can induce reactive gliosis in Müller glia, which may
present a physical barrier to transplanted cell migration (13, 14).
The integrity of the ONL is also lost with the death of photoreceptors. As they die, so the interphotoreceptor matrix (IPM) is
reduced and normal barrier functions are compromised. The outer
limiting membrane (OLM), located at the outer edge of the ONL,
is a diffusion barrier comprised of a series of zonula-adherens
junctions formed between photoreceptors and Müller glia. We have
previously shown that disruption of the OLM enhances photoreceptor integration in wild-type murine recipients (10, 15). Although
there are reports of OLM disruption in the degenerating retina
(16, 17), no study to date has assessed the role of OLM integrity in
transplantation outcome in the degenerating retina. Here, we
present a unique comprehensive assessment of photoreceptor transplantation efficiency across several mouse models of inherited
retinal degeneration, assessing the impact of degeneration, OLM
integrity, and gliosis on transplanted cell integration.
Results
We chose six clinically relevant murine models of inherited retinal disease that represent a range of degeneration speeds: four
models of Retinitis pigmentosa (RP) (Prph2
+/Δ307
, Prph2
rd2/rd2
,
Rho
−/−
, PDE6β
rd1/rd1
), a model of Lebers congenital amaurosis
(Crb1
rd8/rd8
), and a model of stationary night-blindness (Gnat1
−/−
).
Each model undergoes progressive loss of photoreceptors over a
period ranging from ～10% loss over 12 mo (Gnat1
−/−
) to near
complete loss of rods within 3 wk (PDE6β
rd1
) (Table S1).
Author contributions: J.C.S., R.R.A., and R.A.P. designed research; A.C.B., C.H., Y.D., E.L.W.,
J.W.B.B., K.W.-C., U.F.O.L., J.L., and R.A.P. performed research; A.C.B., C.H., E.L.W., U.F.O.L.,
and R.A.P. analyzed data; and A.C.B., J.C.S., R.R.A., and R.A.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. E.A.P. is a guest editor invited by the
Editorial Board.
Freely available online through the PNAS open access option.
1
To whom correspondence may be addressed. E-mail: rachael.pearson@ucl.ac.uk or r.ali@
ucl.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1212677110/-/DCSupplemental.
354–359 | PNAS | January 2, 2013 | vol. 110 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.1212677110Transplanted Rod-Photoreceptors Integrate with Different Efficiency
in Different Models of RP. We first examined the number of transplanted Nrl.GFP
+ve
rod-photoreceptor precursors integrating into
each of the models at a time when the recipient retina is mature
(6–8 wk) and compared these to age-matched wild-type controls.
At 3 wk posttransplantation, integration into adult Gnat1
−/−
,
Prph2
+/Δ307
, Prph2
rd2/rd2
, and PDE6β
rd1/rd1
recipients was similar to
wild-type (Fig. 1A; left axis, gray box plot). Conversely, integration
was significantly higher in the Crb1
rd8/rd8
and significantly lower
into the Rho
−/−
mouse. The differences in disease severity mean
that at 6–8 wk of age the recipients were at very different stages of
their degeneration (Fig. 1A, right axis, black plots, and Table S1),
yet we found that even models at mid- (Crb1
rd8/rd8
; Prph2
rd2/rd2
) and
late- (PDE6β
rd1/rd1
) stage degeneration showed levels of integration comparable to wild-type.
Ability of Transplanted Rod-Photoreceptors to Assume Normal
Morphology Is Significantly Affected by the Recipient Environment.
Photoreceptor survival and function are critically dependent
upon the correct formation and maintenance of synapses and
inner/outer segments; both are prerequisites for effective photoreceptor transplantation therapy. The ability of endogenous
rods to elaborate segments differs dramatically (Fig. S1); those in
the Gnat1
−/−
mouse form long segments very similar to wild-type,
but those formed by rods in the PDE6β
rd1/rd1
models, if present,
are extremely short. Such structural pathologies are likely to present very different recipient environments that may affect the maturation of transplanted cells. We therefore examined the ability
of transplanted rod precursors to form segments and synapses
within the different degenerating retinae (Fig. 2 and Table S2).
We assessed both the number of integrated rods with segments
and their morphological quality (Fig. 2A). In all models we found
the integrated wild-type rods showed correct expression of the
protein missing in the disease model (Fig. 2C). However, overall,
the morphology and frequency of segment formation by integrated rods correlated with the ability of the endogenous
photoreceptors to form outer segments. Over 70% of rods integrated within the ONL of wild-type, Gnat1
−/−
, and Crb1
rd8/rd8
recipients developed segments and adopted typical rod-like
morphologies with long segments (Fig. 2 C, i–iii) like those of the
endogenous rods in these models. In contrast, only a fifth of
integrated rods found within the Rho
−/−
recipient ONL developed segments (Fig. 2A) and these were short (Fig. 2 C, vi). At
3 wk of age, the ONL of the PDE6β
rd1/rd1
retina is reduced to
a single layer of cones; despite this, significant numbers of rods
were found within the remaining ONL. Gross morphology was
markedly different to normal rods, with enlarged cell bodies and
multiple processes (Fig. 2 C, vii), but some developed projections
orientated toward the retinal pigment epitheliumthat colocalized
with β- phosphodiesterase (PDE) (Fig. 2 A–C, vii), indicative
of rudimentary segments. Approximately half of rods integrated
within Prph2
+/Δ307
and Prph2
rd2/rd2
recipients developed segments,
although those formed by cells transplanted into Prph2
rd2/rd2
recipients were shorter than those formed in wild-type recipients.
Of note, in models where endogenous segment formation was
poor, the segments formed by transplanted cells were longer than
those formed by endogenous photoreceptors (Table S2).
Spherule presynaptic-like structures, typical of rod photoreceptors, were formed by over 65% of integrated rods in wildtype, Gnat1
−/−
, and Crb1
rd8/rd8
recipients (Fig. 2 B and D, i–iii).
Significantly fewer presynaptic-like structures were observed
following transplantation in Prph2
rd2/rd2
and Rho
−/−
recipients
(Fig. 2 B and D, v and vi). Most severely affected were cells
transplanted into PDE6β
rd1/rd1
recipients, where only a third of
integrated rods possessed processes that terminated in boutonlike structures (Fig. 2 B and D, vi). A qualitative assessment of all
models indicated that these structures typically colocalized with
the ribbon synapse protein RIBEYE (Fig. 2D). Thus, the cytoarchitecture of the recipient retina influences the ability of
transplanted rod precursors to assume mature rod-photoreceptor morphology, although all environments tested are able to
support segment and synapse formation to some degree.
Disease Progression Has a Major Impact on Transplanted Photoreceptor
Integration Efficiency. We next sought to determine how disease
progression affects transplanted rod precursor integration and for
how long the degenerative recipient retina remains permissive to
transplantation. Cells were transplanted into each model at three
stages of degeneration: early, mid, and late (Fig. 3A and Table S1).
The number of transplanted rod precursors integrating into wildtype recipients remained constant across all timepoints examined
(Fig. 3 A, i). Conversely, integration efficiency decreased in the
Gnat1
−/−
model as disease progressed (Fig. 3 A, ii) and was already
markedly lower in the Rho
−/−
model than in any other model and
continued to decline steeply over time (Fig. 3 A, vi). Integration into
the Crb1
rd8/rd8
mouse presented a bimodal pattern, first increasing
then decreasing sharply when transplanted into late-stage recipients
(Fig. 3 A, iii). Unexpectedly, integration significantly increased with
disease in the Prph2
+/Δ307
model (Fig. 3 A, iv) and remained constant
in Prph2
rds/rds
and PDE6β
rd1/rd1
, despite significant endogenous
photoreceptor loss. Thus, very different trends in integration were
observed in the different models of retinal degeneration as disease
progressed (Fig. 3A, blue lines). These data suggest that the recipient
microenvironment plays a major role in determining photoreceptor
transplantation success and that different factors may be important
in each model. We next examined aspects of the microenvironment
of each of the disease models, specifically disease severity, OLM
integrity, and glial scarring, to try to identify factors that could account for the differences observed in integration efficiency.
No. of integrated photoreceptors
Nrl.GFP
Hoechst
LNO
i,Wildtype
LNO L IN
Nrl.GFP
Hoechst
v, Prph2
rd2/rd2
LNO L IN
Nrl.GFP
Hoechst
iv, Prph2
+/Δ307
LNO L IN
Nrl.GFP
Hoechst
vi, Rho
-/-
LNO
Nrl.GFP
Hoechst
ii, Gnat1
-/-
20000
15000
10000
5000
0
Prph2
N = 9
Prph2
N = 7
Crb1
rd2/rd2
N = 9
rd8/rd8
Rho
N = 6
-/-
PDE6
N = 9
β Wildtype
rd1/rd1
N = 9
Gnat1
N = 10
-/-
P<0.001
P<0.05
20
40
60
80
ONL thickness
Integration
***
***
***
***
*
**
LNO L IN
Nrl.GFP
Hoechst
vii, PDE6β
rd1/rd1
LNO L IN
Nrl.GFP
Hoechst
iii, Crb1
rd8/rd8
A B Integration examples
Fig. 1. Photoreceptor integration is dependent upon recipient disease type.
(A, Left axis, box plots) number of integrated rods 3 wk after transplantation
into 6- to 8-wk-old models of retinal degeneration, compared with wild-type
controls. n = number of eyes. Black bars: statistical significance (ANOVA with
Tukey’s correction). Right axis (black dots), recipient ONL thickness at 6–8 wk
(n = 3 per model). Black asterisks: statistical significance. (B) Representative
images of integrated cells in each model. (Scale bar, 25 μm.) Dotted line
denotes boundary of ONL/INL, dashed line denotes boundary of ONL. ONL,
outer nuclear layer; INL, inner nuclear layer.
Barber et al. PNAS | January 2, 2013 | vol. 110 | no. 1 | 355
NEUROSCIENCERod-Photoreceptor Transplantation Success Is Independent of ONL
Thickness and Rate of Degeneration. Measurement of ONL thickness at each stage highlighted the different rates of endogenous
photoreceptor loss in each model (Fig. S2A), but there was no
correlation between the rate of degeneration and integration
efficiency. For example, in the Gnat1
−/−
degeneration is largely
stationary after 3 mo of age (Fig. S2A, black squares), yet integration declines over time (Fig. 3 A, i and D, i). Conversely,
a rapid rate of degeneration in PDE6β
rd1/rd1
recipients (Fig. S2A,
white squares) was accompanied by little change in integration
(Fig. 3 A, vii and D, vii). It has previously been suggested that
changes in recipient ONL cell density may influence transplant
outcome (18). However, few changes in cell density were observed either between early- and late-stage degeneration or between models (Fig. S2B) and none correlated with the different
trends in integration. Finally, we examined whether there is
a threshold or minimum ONL thickness that is required for integration success. The mean ONL thickness of each model at
each degenerative stage was plotted against the corresponding
mean integration (Fig. S3); we found no correlation highlighting the finding that successful photoreceptor transplantation
can be achieved even in a thinned ONL. Notably, integration
above or similar to wild-type was observed in some (Prph2
+/Δ307
,
Prph2
rds/rds
, PDE6β
rd1
), but not all models at late-stage
degeneration. Taken together, these data show that neither the
recipient ONL cytoarchitecture, nor the rate of endogenous
photoreceptor loss, are limiting factors for transplanted rod
precursor integration.
OLM Integrity and Glial Scarring Affect Photoreceptor Transplantation
Success. It has been reported that both OLM integrity (10, 15)
and glial scarring (13), particularly CSPG deposition (11, 14, 19),
can affect transplantation into the retina. We analyzed changes in
both factors between early- and late-stage degeneration in each
of the models using immunohistochemistry (Fig. 3 B and C),
Western blot (Fig. S1A), and ultrastructural analysis (Fig. S1 B
and C) to ascertain if either factor influences the ability of
transplanted photoreceptors to integrate.
In wild-type recipients, integration remained constant with age
(Fig. 3 A, i and D, i, black trend line). As expected in the absence
of degeneration, no glial scarring was observed (Fig. 3 B, i and D,
i, green trend line, and Fig. S1 A, i): GFAP expression was
minimal and CSPGs were sparsely distributed throughout the
IPM at all stages examined (Fig. 3 B, i). Similarly, there were no
changes in OLM integrity (Fig. 3 C, i and D, i, red trend line):
ZO-1 expression appeared as a continuous unbroken line (Fig.
3 C, i) and at the ultrastructural level, neatly aligned adherens
junctions of normal appearance were observed between Müller
glial and photoreceptors (Fig. S1 B, i). In Gnat1
−/−
recipients,
despite undergoing only mild degeneration, integration decreased modestly but significantly (Fig. 3 A, ii and D, ii). The
OLM remained intact throughout (Fig. 3 C, ii and D, ii, and Fig.
S1 B, ii) and there was little change in CSPG deposition (Fig.
3 B, ii). However, GFAP, which may be inhibitory to integration
(13), increased by the latest stage examined (Fig. 3 B, ii and D, ii,
and Fig. S1 A, ii). Integration also decreased with degeneration
in Rho
−/−
recipients (Fig. 3 A, vi and D, vi), although the initial
levels were much lower and the subsequent decline more pronounced. Despite rapid degeneration, OLM integrity was
maintained even in late-stage degeneration [in contrast to previous reports (16)] (Fig. 3 C, vi, and Fig. S1 B, vi). However,
degeneration is associated with a strong glial response, including
significant up-regulation of GFAP (Fig. 3 B, vi, and Fig. S1 A, vi)
and CSPG condensation at the edge of the ONL. A bimodal
pattern of integration was observed in Crb1
rd8/rd8
recipients (Fig.
3 A, iii and D, iii) (see also ref. 10), whereby increasing disruption of the OLM [permitting increased integration (10, 15)]
appears to be offset by a delayed but significant increase in glial
scarring. CRB1 is an essential component of the OLM adherens
junctional complex (17). Accordingly, significant disturbances in
OLM integrity were observed (Fig. 3 C, iii and Fig. S1 B, iii and
C, iii). GFAP expression was limited in Crb1
rd8/rd8
in early degeneration but increased significantly by late-stage (Fig. 3 B, iii,
and Fig. S1 A, iii) together with moderate CSPG condensation
(Fig. 3 B, iii). Strikingly, integration into the Prph2
+/Δ307
recipient increased with disease progression (Fig. 3 A, iv and D, iv).
In this model, the OLM undergoes some remodeling where cell
death was apparent (Fig. 3 C, iv, and Fig. S1 B, asterisks, and C,
iv), although this did not change with degeneration. However,
there was a very marked reduction in glial scarring: extensive
GFAP expression was observed throughout the retina in early
degeneration, but decreased, particularly within the ONL, by
late degeneration (Fig. 3 B, iv, and Fig. S1 A, iv). CSPG expression also decreased (Fig. 3 B, iv). Integration efficiency was
similar in the Prph2
rds/rds
recipient regardless of the stage at
which cells transplanted (Fig. 3A, v and D, v). Some disorganization of the OLM was observed, although this was similar
at both early- and late-stage degeneration (Fig. 3 D, v). Interestingly, despite an increase in GFAP expression (Fig. 3 B, v
and Fig. S1 A, v), CSPGs at the outer edge of the ONL decreased in end stage disease (Fig. 3 B, v). In PDE6β
rd1/rd1
recipients, integration efficiency was surprisingly unaffected by
disease progression (Fig. 3 A, vii and D, vii). This model
demonstrated significant glial scarring (Fig. 3 B, vii, and Fig. S1
A, vii), yet also underwent an increase in disturbances in OLM
0
20
40
60
80
100
P<0.0001
P<0.0001
P<0.001
P<0.0001
P<0.0001
A P<0.0001
% cells with synapse
B
Nrl.GFP
Hoechst
ONL
vii, PDE6β
rd1/rd1
Nrl.GFP
Hoechst
βPDE
Nrl.GFP
Hoechst
Nrl.GFP
Hoechst
rod α-transducin
ii, Gnat1
-/-
Nrl.GFP
Hoechst
vii
ONL
ONL
ONL
LNO
v, Prph2
rd2/rd2
Nrl.GFP
Hoechst
Prph2
vi, Rho
-/-
Nrl.GFP
Hoechst
Rho4D2
LNO
iv, Prph2
+/Δ307
iii, Crb1
rd8/rd8
i,Wildtype
Nrl.GFP
Hoechst
Ribeye
i,Wildtype
ii, Gnat1
-/-
iii, Crb1
rd8/rd8
iv, Prph2
+/Δ307
v, Prph2
rd2/rd2
vi, Rho
-/-
vii, PDE6β
rd1/rd1
D. Synapse morphology C. Segment morphology
Prph2
+/∆307
Prph2
rd2/rd2
Crb1
rd8/rd8
Rho
-/-
PDE6β
rd1/rd1
Wildtype
Gnat1
-/-
0
20
40
60
80
100
Prph2
+/∆307
Prph2
rd2/rd2
Crb1
rd8/rd8
Rho
-/-
PDE6β
rd1/rd1
Wildtype
Gnat1
-/-
% cells with segments
Fig. 2. Morphology of integrated rods is influenced by recipient retinal
environment. (A and B) Percentage of integrated rods that develop outer
segments (A) and presynaptic-like structures (B) (n = 3 or more per model;
ANOVA with Tukey’s correction). (C and D) Typical morphology, outer-segment length (C) and presynaptic-bouton formation (D) of integrated cells.
(C) Integrated cells expressed the rod-specific transcription factor Nrl
(green), rod α-transducin (C, ii), peripherin-2 (C, v), rhodopsin (C, vi), or β-PDE
(C, vii) (red), as appropriate; such markers are absent in the respective endogenous photoreceptors. (D) Most (arrowheads) but not all colocalized with
RIBEYE (red). Dotted line in vii denotes ONL/INL boundary. (Scale bar, 25 μm.)
356 | www.pnas.org/cgi/doi/10.1073/pnas.1212677110 Barber et al.integrity in late degeneration (Fig. 3 C, vii, and Fig. S1 B, vii and C,
vii). Of note, ultrastructural analysis revealed that although
adherens junctions were present, they were larger in size and
fewer in number than in wild-type and the majority were formed
between Müller glial cells, rather than Müller glia and photoreceptors, indicating a significant degree of remodeling.
Integration in early, mid
& late stage disease
EARLY LATE
Trends
Glial scarring
EARLY LATE
OLM integrity
OLM
10,000
8000
0
Early
Late
6000
4000
2000
vii, PDE6β
rd1/rd1
N=8
N=11
No. of integrated photoreceptors
10 days (immature) 3 weeks 10 days (immature) 3 weeks
OLM
Gliosis
Integration
r2=0.1093
P value=0.1669
5 10 15 20
Time (days)
Time (weeks)
i, Wildtype
No. of integrated photoreceptors
Early
Mid
Late
10,000
8000
0
6000
4000
2000
N=9
N=9
N=8
6 weeks 12 months
GFAP
Hoechst
CS-56
Integration Trend
Gliosis Trend
6 weeks
Hoechst
12 months
OLM Trend
ZO-1
r2=0.03450
P value=0.3637
0 6 12
Time (months)
0
P<0.05
N=11
N=6
N=8
Early
Mid
Late
8000
6000
4000
2000
No. of integrated photoreceptors
2 months 12 months
Integration
Gliosis
2 months 12 months
OLM
3 6 9 12
r2=0.2549
P value=0.0101
0
Time (months)
ii, Gnat1
-/-
15,000
5,000
0
Early
Mid
Late
10,000
20,000
P<0.001
N=9
N=11
P<0.01
N=11
No. of integrated photoreceptors
3 weeks 12 weeks
Integration
3 weeks 12 weeks
OLM
Gliosis
iii, Crb1
rd8/rd8
15,000
10,000
5,000
0
20,000
Early
N=9
N=7
N=7
N=9
Mid
Late
Late
P<0.01
P<0.05
No. of integrated photoreceptors
6 months 2 months 6 months
OLM
Integration
Gliosis
0 2 4 6 8 10
r2=0.2402
P value=0.004
Time (months)
iv, Prph2
+/Δ307
2 months
0
Early
Mid
Late
N= 13
N=7
N= 12
15,000
10,000
5,000
No. of integrated photoreceptors
GFAP
Integration
5 10 15
CSPG
0
r2=0.0234
P value=0.4036
Time (weeks)
Gliosis
v, Prph2
rd2/rd2
1500
1000
500
0
Early
Mid
Late
N=8
N=6
N=13
P<0.001
P<0.001
No. of integrated photoreceptors
4 weeks 10 weeks 4 weeks 10 weeks
Integration
Gliosis
OLM
2 4 6 8 10
r2=0.2942
P value=0.0003
vi, Rho
-/-
4 weeks 4 weeks 12 weeks 12 weeks
A B C D
Fig. 3. Disease progression significantly but differentially affects photoreceptor transplantation efficacy according to disease type. (A) Black: impact of disease
progression upon transplantation outcome, compared with wild-type. n = number of eyes examined. ANOVA with Tukey’s correction. Blue: linear regression
denotes integration trend. Note that changes in Crb1
rd8/rd8
retinae were bimodal (shown as dashed line). (B) Gliosis in early and late degeneration, as assessed
by CSPG (green) and GFAP (red) expression. (C) OLM integrity in early and late degeneration, as assessed by ZO-1 (red) expression. Disturbances in OLM integrity
indicated by white arrows. (Scale bar, 50 μm.) (D) Trend correlations (nonquantitative) for integration (black), OLM integrity (red), and gliosis (green).
Barber et al. PNAS | January 2, 2013 | vol. 110 | no. 1 | 357
NEUROSCIENCEThese data demonstrate that despite sharing the same final
common pathway of photoreceptor loss, different models and
stages of retinal degeneration present very different microenvironments through which donor cells must migrate, leading to
strikingly different outcomes in photoreceptor transplantation
efficacy. Specifically, the extent of glial scarring and changes to
OLM integrity appear important in determining transplantation
outcome in different types of retinal degeneration.
Manipulation of the Microenvironment in the Degenerating Rho
−/−
Retina Increases Integration and Permits Restoration of Visual Function.
The observed correlations between glial scarring, OLM integrity,
and transplant efficacy indicated it may be possible to improve
integration by administering a tailored modulation of the microenvironment for a given disease type and treatment timepoint.
To determine whether glial scarring and OLM integrity are indeed responsible for impeding integration in the degenerating
recipient retina, we manipulated these factors pharmacologically
and assessed their impact on transplanted rod-photoreceptor integration. We chose the Rho
−/−
mouse, the model with the poorest
transplantation outcome, which also presented with an intact
OLM together with a strong glial response.
We used siRNAs targeted against ZO-1 to provide a reversible
disruption of the OLM, a strategy we have previously shown to
increase photoreceptor integration (10). Rho
−/−
and wild-type
mice received either siRNA targeting ZO-1, a proven nontargeting control siRNA, or no injection 48 h before transplantation of Nrl.GFP
+ve
–rod-precursors to the same region. The
number of integrated rods was markedly increased in ZO-1
siRNA-treated Rho
−/−
retinae compared with eyes receiving
nontargeting siRNA or no pretreatment (4.2-fold and 2.3-fold
increases, respectively) (Fig. 4A). Although the overall number of
cells integrating into the wild-type mouse was higher than in the
Rho
−/−
, a similar pattern was observed (4.4-fold and 2.2-fold
increases, respectively). We next used chondroitinase ABC
(ChABC) to enzymatically digest CSPGs (11, 14, 19) and combined this with transplantation of Nrl.GFP
+ve
–rod-precursors in
Rho
−/−
and wild-type recipients. In Rho
−/−
mice, ChABC treatment lead to a highly significant increase in integration compared
with controls (eightfold increase) (Fig. 4 A, i). In wild-type
recipients, where there is no gliosis and CSPG expression is diffuse, the effect although present was less marked (2.5-fold increase) (Fig. 4 A, ii). We next examined the impact of combining
the two manipulations. Wild-type and Rho
−/−
recipients received
ZO-1 siRNA 48 h before coadministration of Nrl.GFP
+ve
–rodprecursors and ChABC. Combined treatment led to significant
increases in transplanted cell integration in both wild-type and
Rho
−/−
retinae (Fig. 4A, and Fig. S4). Thus, both the OLM and
CSPG deposition impede the integration of transplanted photoreceptors into the degenerating retina but integration can be
improved by targeted disruption of these barriers.
Finally, we sought to determine if the increase in integration
achieved using combined treatment was sufficient to restore visual function, as assessed by optokinetic head-tracking behavior
in Rho
−/−
mice (Fig. 4B, and SI Materials and Methods) (5, 20).
Seven of the Rho
−/−
mice receiving combined treatment did so
only in one eye and either no injection or cells only in the contralateral eye, before being tested 3–4 wk posttransplantation. No
consistent head-tracking behavior was observed upon presentation
of scotopic stimuli to control eyes. In contrast, head-tracking was
seen following stimulation of six of seven eyes receiving the
combined treatment. Histological assessment revealed a positive
correlation between improvement in contrast sensitivity and the
number of integrated rods (Fig. 4B), as shown previously when
testing optomotor head-tracking responses in transplanted Gnat1
−/−
mice (5). Of note, greater numbers of integrated cells were required in Rho
−/−
recipients to generate contrast sensitivities equivalent to those recorded in Gnat1
−/−
recipients.
Discussion
Photoreceptor transplantation has the potential to restore vision
following retinal degeneration (2, 5). While it could potentially
be applied to a wide range of retinal degenerations, there have
been no systematic assessments of efficacy across a spectrum of
retinal dystrophies. Here we show that it is possible to achieve
robust integration even in severely degenerate retinae and in
a variety of murine models with very different aetiologies. In
contrast to what might be expected, neither the rate nor extent
of degeneration affected photoreceptor integration, indicating
that photoreceptor replacement therapy may be an effective
therapeutic strategy for severe retinal degeneration. Indeed,
a more complex pattern was observed where integration increased, decreased, or remained constant with disease progression, and opposing trends were observed even in models with
similar rates of degeneration. We show that the aetiology specific
to each gene defect impacts both on the number and the morphology of the integrated rods within a given disease model. We
also demonstrate that two characteristics of retinal degeneration,
glial scarring and changes in OLM integrity, significantly affect
transplantation outcome. Broadly, integration decreases in those
models in which OLM integrity is maintained, but in which
gliosis increases with disease progression (Gnat1
−/−
; Rho
−/−
).
Integration remains constant in models in which the OLM is
disrupted, but gliosis increases (PDE6β
rd1/rd1
). Finally, integration increases in the model in which the OLM undergoes
remodeling and gliosis decreases with disease progression
(Prph2
+/Δ307
). Importantly, it is possible to manipulate these
barriers and increase transplanted photoreceptor integration to
levels sufficient to restore visual function.
There are notable differences in the pattern of gliosis in the
different models; GFAP
+ve
fibers were seen extending throughout
Cells only
Non-targeting
ChABC
ZO-1 siRNA
Combined
ii, Wildtype
No. of integrated photoreceptors
30,000
20,000
10,000
0
40,000
i, Rho
-/-
P < 0.001
P < 0.001 P < 0.001
P < 0.001
Contrast sensitivity
1.0
R2
= 0.882
No. of integrated photoreceptors
1.1
1.2
1.3
1.4
0 10,000 20,000 30,000
Cells only
Non-targeting
ChABC
ZO-1 siRNA
Combined
A
B
Fig. 4. Manipulation of OLM and gliosis significantly increases integration
and permits restoration of visual function. (A) impact of OLM disruption (using
ZO-1 siRNA) and CSPG degradation (using ChABC), singularly and combined,
on transplantation outcome in Rho
−/−
and wild-type recipients. n ≥ 7 per condition; ANOVA with Tukey’s correction. (B) Contrast sensitivity against number
of integrated rod-photoreceptors in subset (n = 7) of Rho
−/−
recipients that
underwent optomotor testing 3–4 wk after receiving combined treatment.
358 | www.pnas.org/cgi/doi/10.1073/pnas.1212677110 Barber et al.the ONL in those models in which integration efficiency declined
with degeneration (Gnat1
−/−
; Rho
−/−
), but the most poorly performing model (Rho
−/−
) also displayed significant CSPG deposition. Conversely, in the Prph2
rd2/rd2
, in which integration
efficiency remained similar at different stages of degeneration,
CSPG deposition decreased over time. In the Prph2
+/Δ307
model,
we observed a striking decrease in GFAP expression in the ONL,
although such regional changes were not reflected in the global
changes in GFAP expression. Thus, the specific characteristics of
the glial response may be as important as its absolute magnitude.
Integration efficiency is typically higher in models of degeneration in which OLM integrity is compromised than in those in
which it remains intact. Of all of the models studied, the Crb1
rd8/rd8
mouse, which has a profoundly disrupted OLM (10, 17), had the
highest levels of integration. Of note, we observed significant differences in OLM adherens junction composition in the different
models. In wild-type mice, these junctions form between photoreceptors and Müller glia. However, in the Prph2
+/Δ307
, Prph2
rd2/rd2
,
and PDE6β
rd1/rd1
models, many formed directly between Müller
glia, indicating significant OLM remodeling. This was supported
by the presence of photoreceptors mislocalized within the IPM. In
these models, integration efficiency increased or remained constant, presumably because of, at least in part, the continued
changes in OLM integrity. Surprisingly, despite significant endogenous rod cell death in the Rho
−/−
mouse, the adherens
junctions retain the typical photoreceptor-Müller glia association.
Although important, it is unlikely that OLM integrity and glial
scar formation solely govern transplantation outcome within the
degenerate retina; many more factors are likely to be involved.
Here, our assessment of glial scarring focuses primarily on the
up-regulation of GFAP and deposition of CSPG, and the application of ChABC leads to the breakdown of only some
CSPGs. However, gliosis has many attributes, including Müller
cell hypertrophy and proliferation and microglia accumulation.
The biological roles of all these changes are unclear and their
impact on cell transplantation has not been addressed. Others
have shown that CSPG degradation, either by ChABC (11, 14, 19)
or by endogenous enzymes (21), enhances the integration of
transplanted cells. However, there are conflicting reports of the
role of GFAP: one study reported an increase in transplanted cell
integration in the GFAP
−/−
/Vim
−/−
mouse (13), suggesting that
GFAP might be inhibitory to migration; others have reported
enhanced integration around sites of GFAP up-regulation (22).
Further work is needed to ascertain the exact role of intermediate
filament assembly in transplantation outcome.
Recently, we have shown that it is possible to restore vision in
the Gnat1
−/−
mouse by rod-photoreceptor transplantation (5). In
this model, the retinal cytoarchitecture is well preserved and the
integrating cells displayed morphologies almost indistinguishable
from wild-type rods. However, as we have shown here, transplanted photoreceptor morphologies are profoundly affected by
the recipient cytoarchitecture. Although the numbers of cells
integrating within the PDE6β
rd1/rd1
and Gnat1
−/−
models were
similar, in PDE6β
rd1/rd1
mice integrated photoreceptors often
had multiple processes, with few synaptic-like structures or segments. Similarly, photoreceptors integrated within the Rho
−/−
mouse developed only short outer segments (present study and
refs. 2 and 14). The failure to elaborate outer segments does not
necessarily prevent a photoreceptor from functioning: outer
segments fail to form in the Prph2
rd2/rd2
mouse, yet these animals
retain some residual function for several weeks postbirth (23).
However, it is likely that a greater number of integrated cells will
be required to restore visual function in these recipients than in
recipients with more normal outer-segment morphology. Accordingly, although we observed restoration of optokinetic headtracking in some Rho
-/−
mice following transplantation combined
with OLM disruption and CSPG degradation, more integrated
cells were required to generate contrast thresholds equivalent to
those we reported recently for Gnat1
−/−
recipients (5). This
finding highlights the need to find additional ways to achieve
high levels of integration in advanced degeneration.
Materials and Methods
Full methods are available in SI Materials and Methods. Single subretinal
transplantations of 200,000 live P6-8 Nrl.GFP
+ve
–rod-photoreceptors were
given to wild-type (C57BL/6J) mice and models of inherited retinal degeneration at early-, mid-, and late-stages of degeneration (Results). Cell integration was assessed 3–4 wk posttransplantation. Gliosis was manipulated
using ChABC (19) and OLM integrity was manipulated using siRNAs directed
against ZO-1 (10). Optomotor responses were recorded as previously described
(5). See Table S3 for immunohistochemistry.
ACKNOWLEDGMENTS. We thank A. Eddaoudi for technical assistance. This
work was supported by grants from the British Retinitis Pigmentosa Society
(GR566), Wellcome Trust (082217; 086128), Royal Society (RG080398), and
Medical Research Council UK (G03000341; G0901550 mr/j004553/1). R.A.P. is
a Royal Society University Research Fellow; J.C.S. is supported by Great
Ormond Street Hospital Children’s Charity; and R.R.A. is partially funded by
the Department of Health’s National Institute for Health Research Biomedical Research Centre, Moorfields Eye Hospital, and the Miller’s Trust.
1. Bainbridge JW, et al. (2008) Effect of gene therapy on visual function in Leber’s
congenital amaurosis. N Engl J Med 358(21):2231–2239.
2. MacLaren RE, et al. (2006) Retinal repair by transplantation of photoreceptor precursors. Nature 444(7116):203–207.
3. Lakowski J, et al. (2010) Cone and rod photoreceptor transplantation in models of the
childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive
donor cells. Hum Mol Genet 19(23):4545–4559.
4. Akimoto M, et al. (2006) Targeting of GFP to newborn rods by Nrl promoter and
temporal expression profiling of flow-sorted photoreceptors. Proc Natl Acad Sci USA
103(10):3890–3895.
5. Pearson RA, et al. (2012) Restoration of vision after transplantation of photoreceptors. Nature 485(7396):99–103.
6. Eiraku M, et al. (2011) Self-organizing optic-cup morphogenesis in three-dimensional
culture. Nature 472(7341):51–56.
7. Osakada F, Ikeda H, Sasai Y, Takahashi M (2009) Stepwise differentiation of pluripotent stem cells into retinal cells. Nat Protoc 4(6):811–824.
8. West EL, et al. (2012) Defining the integration capacity of embryonic stem cell-derived
photoreceptor precursors. Stem Cells 30(7):1424–1435.
9. Lamba DA, Gust J, Reh TA (2009) Transplantation of human embryonic stem cell-derived
photoreceptors restores some visual function in Crx-deficient mice. Cell Stem Cell 4(1):73–79.
10. Pearson RA, et al. (2010) Targeted disruption of outer limiting membrane junctional
proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina. Cell Transplant 19(4):487–503.
11. Singhal S, et al. (2008) Chondroitin sulfate proteoglycans and microglia prevent migration and integration of grafted Müller stem cells into degenerating retina. Stem
Cells 26(4):1074–1082.
12. Fawcett J (2009) Molecular control of brain plasticity and repair. Prog Brain Res 175:
501–509.
13. Kinouchi R, et al. (2003) Robust neural integration from retinal transplants in mice
deficient in GFAP and vimentin. Nat Neurosci 6(8):863–868.
14. Ma J, Kabiel M, Tucker BA, Ge J, Young MJ (2011) Combining chondroitinase ABC and
growth factors promotes the integration of murine retinal progenitor cells transplanted into Rho(-/-) mice. Mol Vis 17:1759–1770.
15. West EL, et al. (2008) Pharmacological disruption of the outer limiting membrane
leads to increased retinal integration of transplanted photoreceptor precursors. Exp
Eye Res 86(4):601–611.
16. Campbell M, et al. (2006) Aberrant retinal tight junction and adherens junction
protein expression in an animal model of autosomal dominant Retinitis pigmentosa:
The Rho(-/-) mouse. Exp Eye Res 83(3):484–492.
17. Mehalow AK, et al. (2003) CRB1 is essential for external limiting membrane integrity
and photoreceptor morphogenesis in the mammalian retina. Hum Mol Genet 12(17):
2179–2189.
18. Yao J, et al. (2011) XIAP therapy increases survival of transplanted rod precursors in
a degenerating host retina. Invest Ophthalmol Vis Sci 52(3):1567–1572.
19. Suzuki T, et al. (2007) Chondroitinase ABC treatment enhances synaptogenesis between transplant and host neurons in model of retinal degeneration. Cell Transplant
16(5):493–503.
20. Umino Y, Solessio E, Barlow RB (2008) Speed, spatial, and temporal tuning of rod and
cone vision in mouse. J Neurosci 28(1):189–198.
21. Zhang Y, Klassen HJ, Tucker BA, Perez MT, Young MJ (2007) CNS progenitor cells promote a permissive environment for neurite outgrowth via a matrix metalloproteinase-2-
dependent mechanism. J Neurosci 27(17):4499–4506.
22. Nishida A, et al. (2000) Incorporation and differentiation of hippocampus-derived
neural stem cells transplanted in injured adult rat retina. Invest Ophthalmol Vis Sci
41(13):4268–4274.
23. Reuter JH, Sanyal S (1984) Development and degeneration of retina in rds mutant
mice: The electroretinogram. Neurosci Lett 48(2):231–237 |
