Non-Invasive Stem Cell Therapy in a Rat Model for
Retinal Degeneration and Vascular Pathology
Shaomei Wang1*, Bin Lu1, Sergei Girman1, Jie Duan1, Trevor McFarland1, Qing-shuo Zhang2, Markus
Grompe2, Grazyna Adamus1, Binoy Appukuttan1, Raymond Lund1
1Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States of America, 2Oregon Stem Cell Center, Oregon Health & Science University,
Portland, Oregon, United States of America
Background: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular
permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy
has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer
generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic
administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular
pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP.
Methodology/Principal Findings: Animals received syngeneic MSCs (16106cells) by tail vein at an age before major
photoreceptor loss. Principal results: both rod and cone photoreceptors were preserved (5–6 cells thick) at the time when
control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with
controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of
pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular
leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was
upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors
within eyes of animals that received MSCs.
Conclusions/Significance: These results underscore the potential application of MSCs in treating retinal degeneration. The
advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for
autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be
readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal
degeneration and other ocular vascular diseases.
Citation: Wang S, Lu B, Girman S, Duan J, McFarland T, et al. (2010) Non-Invasive Stem Cell Therapy in a Rat Model for Retinal Degeneration and Vascular
Pathology. PLoS ONE 5(2): e9200. doi:10.1371/journal.pone.0009200
Editor: Christophe Egles, Tufts University, United States of America
Received September 25, 2009; Accepted January 21, 2010; Published February 15, 2010
Copyright: ? 2010 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants of Foundation Fighting Blindness, Research Prevent Blindness and Lincy Foundation. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Retinitis pigmentosa (RP) refers to a subset of inherited retinal
degenerations, for which over 180 disease associated loci have
been mapped and of these over 130 genes have been identified
that when mutated result in severe vision impairment. An
estimated 100,000 people in the U.S. have RP [1,2,3,4], with a
worldwide prevalence between 1 in 3000 to 1 in 7000. Retinitis
pigmentosa is not only genetically heterogeneous but can be
inherited in multiple forms, including autosomal dominant,
autosomal recessive, X-linked, nonsyndromic and digenic-diallelic.
A universal feature of all forms of RP is initial degeneration of
photoreceptors and with time the pathology involves the inner
retina, leading to a loss in its lamination, vascular leakage, invasion
of RPE cells into the retina and subsequent loss of ganglion cells
[5,6,7]. The majority of people with RP are usually legally blind by
age 40–50, with visual symptoms manifesting in the early teens.
Notably, there is no effective treatment available. Experimental
animal models that mimic the human RP condition allow
investigation and development of potential treatments. Viral
mediated delivery of a normal copy of the affected gene has lead
to partial reversal of the phenotypic changes in animal models and
has led to human clinical trials [8,9,10,11,12,13]. However,
specific genetic defects have been found in only a few of the known
retinal degenerative diseases, which thereby limit the potential
application of gene therapy to those few patients. A generic
blanket therapy for all retinal dystrophies may be a better global
strategy, and indeed therapies with calcium channel blockers,
vitamin supplementation and neuroprotective growth factors have
been tested although in some cases with limited success
[2,14,15,16]. Cell-based therapy, especially the development of
stem cell biology for application in treating neurodegenerative
diseases to the retina has been shown to be effective. Direct
injection of donor cells into vitreous does not have much merit, as
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donor cells tend to cover the back of the lens and block the passage
of light into the eye, and thus preventing functional tests to
determine efficacy of treatment. Intravitreal implantation of
encapsulated factor secreting cells has the disadvantage of non-
specific exposure of intraocular structures to potentially deleterious
levels of growth factor, and there is limitation of allowing repeat
implantation, which is required to sustain long-term efficacy. An
advantage of delivering donor cells via subretinal injection method
is that the therapeutic material is placed directly in the space
where the defective RPE cells or degenerating photoreceptors are
targeted, and in this fashion both photoreceptors and visual
function can be partially preserved [17,18,19]. However, donor
cells are usually distributed across at most about a quarter of the
total retinal area, the rest of the retina undergoes progressive
degeneration, especially the development of the secondary
vascular pathology, which compromises donor cell survival and
related beneficial effect. An attractive therapeutic intervention
would be one that affords generic neuro-and vascular-protective
effects via a non-invasive method and bestows protection to both
rod and cone photoreceptors. The pluripotent bone marrow-
derived mesenchymal stem cells (MSCs) are an ideal cell source for
therapy of inherited or degenerative disease, because of its
autologous characteristic, ease of isolation; secreting growth
factors know to be neuro-vascular protective, less contentious
relative to other stem cells [20,21,22,23]. We found that MSCs
preserved vision and limited vascular pathology when intrave-
nously injected into the Royal College Surgeon (RCS) rat, a well-
established animal model of RP.
A mutation in the gene for the receptor tyrosine kinase Mertk,
in the RCS rat, results in dysfunction of retinal pigment epithelium
(RPE) cells . Compromising the ability of the RPE to
phagocytize photoreceptor outer segments leads to a progressive
loss of both rods and cone cells overtime in the RCS rat [25,26].
Interestingly, mutation within the human orthologue of Mertk
results in RP, whereby patients exhibit progressive poor visual
acuity and visual field losses with age .
Neuroprotection of Cones and Rods
In the RCS rat by postnatal day (P) 90 only a single layer of
photoreceptors remains compared to the 10 layers observed at
P30. To investigate whether MSCs could provide a neuroprotec-
tive effect, we isolated and injected intravenously syngeneic MSCs
into RCS rats at P30 (n=12), at which time the retinal
degeneration is at an early stage. Eyes were collected and
processed at P90 to determine efficacy of MSC treatment in
comparison to controls (sham injection (carrying medium alone):
n=8, and untreated: n=8). Retinal sections were stained with
cresyl violet for examining general retinal lamination and with
photoreceptor cell-specific antibodies (rhodopsin, cone arrestin),
which showed the preservation of cone and rod photoreceptors
within the MSC treated animals. We found that photoreceptors
were substantially rescued across the retina (Figure 1A). Although
an uneven distribution of cell layer thickness was noted with more
prominent rescue in peripheral than central retina. There were 5–
6 layers of photoreceptors in the peripheral retina compared with
2–3 layers in the central retina (Figure 1: A1&A3 vs. A2). The
retina appeared orderly laminated. In contrast, there was a single
layer of photoreceptor remaining in both sham injected (Figure 1B)
and untreated retinas (Figure 1C). Morphologically both rod and
cone photoreceptors were rescued (Figure 1 D&E). Further
analysis revealed that cone density was 1863 cells/300 mm in
retina that had received MSCs (2062 cells/300 mm in wild type);
while in control retina (Figure 1F) it was impossible to conduct
meaningful counts due to severe degeneration.
Functional Preservation of Photoreceptors
In the RCS rat, visual function deteriorates as photoreceptors are
lost. Visual acuity in the RCS rat as tested by an optokinetic system
(under photopic condition) has been shown to decrease with age
from0.5cycle/degree (c/d)atP30to0.3 c/d atP90.Thistest is
non-invasive, rapid and allows for repeated measurements of spatial
frequency and contrast sensitivity thresholds of the optokinetic
response (OKR). Another test for functional photoreceptors is a
luminance threshold (LT)recording fromthe superiorcolliculus(SC)
under standard background luminance level. In the RCS rat, the LT
was elevated from1.2 log units at P30 to 3 log units atP90 (,0.4
log units in wild rat). Although LT recording is time-consuming, it
measures functional sensitivity across the visual field, which in turn
provides a topographic indication of the magnitude and area of
photoreceptor rescue across the whole retina. To examine whether
MSCs preserved visual function afterintravenous administration, we
conducted the aforementioned functional tests that correlated very
well with the morphological neuroprotective data. The OKR
analysis revealed that there was significant difference between MSC
treated and control eyes (P,0.001) (Figure 2A). An average of
0.4160.01 c/d was recorded at P90 in MSC treated animals
(n=12), whereas 0.3060.01 c/d in medium injected (n=8) and
0.2960.02 c/d in untreated (n=8) controls were observed.
Luminance threshold recordings revealed that MSC injected eyes
(n=6) produced thresholds less than 2.76 log units over 60% of the
total SC area; while in controls (n=6), no SC area produced
thresholds less than 2.76 log units. Thus, MSC treated eyes had
significantly lower threshold than untreated eyes (p,0.05), indicat-
ing a convincing degree of functional preservation (Figure 2B).
In the RCS rat, retinal vascular pathology develops as
photoreceptors degenerate. Leakage from vessels within the deep
capillary plexus is first detected at P60, using horseradish
peroxidase (HRP) perfusion method, and this seepage is initially
located around the optic nerve disc and eventually with increasing
age spreads to the whole retina . In the RCS rat vascular
complexes, determined as abnormal vessels associated with clusters
of retinal pigment epithelium cells (RPE), are clearly evident by
P90. The RPE cells appear to migrate along the abnormal vessels
and form pathological vascular complexes. To examine whether
MSCs confer a vascular-protective role, retinal vessels were stained
with the nicotinamide adenine dinucleotide phosphate-diaphorase
(NADPH-diaphorase) on whole mount preparation as described
previously . The NADPH-diaphorase staining reveals the
outline of retinal vessels and also allows identification of migrating
RPE cells attached to pathological vessels. We found that the
number of pathological vascular complexes was dramatically
reduced in MSC treated retina (n=8), compared with medium
(n=6) or untreated controls (n=6) (0–8 vs. 25–30 of vascular
complexes; p,0.001) (Figure 3A vs.D). In controls, the vascular
complexes were located immediately ventral to the optic nerve
head, with spreading to middle and eventually to the peripheral
parts of the retina. It is common to find many vascular complexes
at multiple sites along a major vessel (Figure 3B). High power
image showed the vessels were twisted and covered by pigment
granules (Figure 3C). We also noticed that the vascular complexes
in MSC treated retinas appeared to be smaller (Figure 3E&F) and
if present usually isolated instead of clustered as observed in the
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To further examine vascular integrity, animals were injected
with fluorescein isothiocyanate (FITC)-dextran via the tail vein
and the retinal whole mount was examined under fluorescence
microscopy. We found that typical leakage around the optic
nerve head was observed in control animals at P90 (n=6)
(Figure 3G&H); abnormal vascular profiles (dilated, torturous
with evidence of leakage) were seen in the mid to peripheral
retina (Figure 3I–K) and it was common to see multiple
abnormal vascular profiles on one vessel. However, vascular
leakage and abnormal vascular profiles were dramatically
reduced in animals that had received MSCs (n=6) (Figure 3L–
N). The abnormal vascular profiles were much smaller, isolated
and located around the optic nerve disc and rarely seen
anywhere else in the retina.
We hypothesized that the neuro-vascular protection afforded by
the introduction of MSCs was achieved by the increase in
production of neurotrophic growth factors within the retina. To
investigate this theory we performed semi-quantitative RT-PCR
from retinal tissue isolated from animals at P90. We found that
growth factors including ciliary neurotrophic factor (CNTF), basic
fibroblast growth factor (bFGF), and brain derived neurotrhophic
factor were upregulated in MSC treated eyes (n=3) compared
with control eyes (n=3) (Figure 4A&B). However, only CNTF and
BDNF were significantly increased over controls as determined by
densitometry analysis. To determine the cells responsible for the
increase in this growth factor production in the retina, antibodies
against CNTF, bFGF and BDNF were applied to retinal sections
Figure 1. Rod and cone protection. A. Retinal sections stained with cresyl violet indicate substantial preservation of photoreceptors across the
retina in MSC treated eyes at P90, while in control eyes (sham injected (B) and untreated (C)): only a single layer of photoreceptors remained. A1, A2
&A3: higher power images showing preservation of photoreceptors from the insets A1, A2 &A3 in A. D, E&F: confocal images showing rhodopsin
(green in D) and cone arrestin (red in E) positive staining at P90 in MSC treated retina, while in sham injected retina, cone arrestin staining was
dramatically reduced (F). All sections were counterstained with DAPI (blue) (scale bars equal 50 mm).
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of MSC-injected (Figure 4C) and control (Figure 4G). Strong
staining of CNTF was found in MSC-injected retina compared
with controls (Figure 4D vs.H). There were no obvious difference
observed for the other proteins between MSC treated and controls
(data not shown). The retinal sections were double stained with
glial fibrillary acidic protein (GFAP) (Figure 4E&I) for Mu ¨ller cells
and CNTF, which revealed their co-localization in MSC-injected
retina (Figure 4F), not in control (Figure 4J), suggesting that Mu ¨ller
glia cells upregulate expression of CNTF in response to the
presence of MSC within the eye.
Distribution of MSCs
To track the distribution of MSCs after systemic administration,
MSCs (Figure 5A) were harvested and labeled with cell-linker
PKH26 (Sigma) before injection (Figure 5B). Retinal whole mount
and sections were examined 2 weeks after MSC injection. To see
the relation between retinal vessels and MSCs, animals were
perfused with FITC-dextran to highlight retinal vasculature before
sacrifice. The PKH26 labeled MSCs were found in the eyes
(Figure 5C, whole mount) and in other tissues including lungs,
kidneys and liver (data not shown). In retinal sections, PKH
labeled cells were seen in the retinal ganglion cells layers, inner
and outer plexiform layers (Figure 5D–F).
This study demonstrated that bone marrow derived mesenchy-
mal stem cells, when administrated intravenously, preserved both
rod and cone photoreceptors, maintained visual function and
limited vascular pathology.
Ocular vascular pathology is the most common cause of
blindness and associated with several disorders. Age-related
macular degeneration affects over 10 million individuals over
age 65 in the United States alone. About 10215% of these
individuals suffer loss of vision as a direct effect of neovascular-
ization of choroidal vessels [31,32]. An estimated 100,000 people
in the U.S. have RP with initial progressive photoreceptor loss
followed by secondary vascular pathology (1). Over 40,000
patients with diabetes suffer from ocular compilations each year
. Many premature infants suffer from retinopathy of
prematurity . Vision loss has significant social and economic
impact. This study demonstrates that renewable adult stem cells
preserve vision and limit vascular pathology by systemic
administration. Both rod and cone photoreceptors were preserved
morphologically; visual functions tested by optomotor response
and luminance threshold were significantly better than controls;
vascular pathology including leakage and formation of vascular
complexes was also dramatically reduced. The advantages of
systemic administration of stem cells are that cells exert their effect
over the whole retina, and multiple administrations can be easily
performed if needed. The intravenous MSCs offer unique neuro-
vascular protection as an auto-cell therapy.
Previous studies suggested that MSCs secrete a variety of
cytokines and growth factors that have neuronal protective
activities [20,21,35]. Our results indicated that the level of bFGF,
BDNF and CNTF in MSC treated retina was higher than
controls. Interestingly, CNTF was strongly expressed in Mu ¨ller
cells, which are the main source of trophic factors within the retina
[36,37]. The CNTF is a member of IL-6 family of cytokines that
modulates survival of retinal neuronal cells. Up-regulation of
endogenous CNTF is believed to promote photoreceptor survival,
to protect them from mechanical injury. Direct intraocular
injection of CNTF has been shown to retard photoreceptor death
caused by inherited forms of retinal degeneration  or by light
induced retinal damage . A recent study by Hauk and
colleagues  showed that intravitreal injection of toll-like
receptor 2 agonist Pam3Cys (caused lens injury) can induce glial
activation and upregulations of GFAP and CNTF, which
significantly stimulated retinal ganglion cell axon regeneration
into the injured optic nerve. An encapsulated cell therapy device
that delivers CNTF has been used in clinical trials to rescue
photoreceptors and is currently showing promising outcome .
Our current study showed that substantial photoreceptor rescue
was observed across the whole retina and that both rods and cones
were preserved. Systemic administration of whole bone marrow
cells promoted photoreceptor survival in a mouse model of RP
. Sasahera and colleagues reported that the rescued photore-
ceptors were mainly cones in this RP model. In the current study
we showed that both rod and cone photoreceptors were rescued at
morphological and functional level. Further analysis indicated that
cone density was comparable to the wild type rat; while in the
Figure 2. Preservation of visual function. A. Visual acuity tested by
Optomotor response. Unrestrained animals were placed on a platform,
where they tracked the grating with reflexive head movements. The
acuity threshold was quantified by increasing the spatial frequency of
the grating. RCS rats received MSCs and medium injection via tail vein
at P30 and tested at P90. Visual acuity was significantly better in MSC
treated eyes compared with controls (P,0.001). A value of 0.43 c/d was
recorded, which was 78% of normal value (0.55 c/d in wild-type). B. The
luminance threshold was evaluated by recording single and multiunit
activity close to the surface of the superior colliculus (SC). It measures
functional sensitivity across the visual field, which in turn provides a
topographic indication of the magnitude and area of photoreceptor
rescue across the retina. MSC treated rats recorded around P90–100
revealed significantly lower threshold than controls (P,0.005), for
example, over 60% of the SC area had threshold at 2.76 log units in MSC
treated eyes, no detectable response in control eyes.
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RCS control retina, cone photoreceptors degenerated with a
disorganized profile. It would appear that stem cells exert their
effect over the whole retina when administered systemically. In
comparison, subretinal delivery of cells including bone marrow
derived cells [43,44,45,46] usually results in rod and cone rescue
close to the injection site and/or correlated with donor cell
distribution, and thus there is no noticeable protection at sites
distant from the grafted cells.
The vascular protection bestowed by MSCs is partly mediated
by increased expression of angiopoietin-1/Tie2 and vascular
Figure 3. Vascular protection. A–F: Retinal whole mount was stained with NADPH-diaphorase: A. typical vascular pathology in the eye at P90 in
untreated RCS rat: vascular complexes (abnormal vessels associated with RPE cells) were mainly located around the optic nerve disc (arrows) and
spread peripheral with age. B. vascular complexes in the middle to peripheral retina (arrows). C. high power image showing vascular complexes
(arrows) from B. D. RCS retina treated with MSCs at P90: the vascular complexes were dramatically reduced around the optic nerve disc. E. two
vascular complexes (arrows) in the middle field of the retina. F. high power image from E showing vascular complexes (arrow). G–L. animal was
perfused with FITC-dextran, whole mount was prepared: G. typical vascular leakage, mainly around the optic disc in untreated eye at P90. H–K. high
power images from G showing vascular leakage (arrows in H) and abnormal vessels (arrows in I–K). L. MSC treated retina, the vascular leakage around
the optic nerve disc was greatly reduced. M&N. high power images from L showing much reduced leakage (arrows in M) and small abnormal vessels
(arrow in N) (Scale bars equal 250 mm for A, D, G &L; 100 mm for F).
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endothelial growth factor/FLK1 in the animal model for stroke
. We performed RT-PCR and immunohistochemistry to test
the presence of angiopoietin-1/Tie2 and vascular endothelial
growth factor/FLK1 on retinal tissue isolated from MSC treated
and untreated RCS rats. We observed no difference in the
expression profiles between the control and treated animals (data
not shown). It is possible that the mechanism of MSC mediated
vascular protection in this degeneration model is acting through an
alternative pathway. Further study is under way to investigate this
phenomenon in the RCS model.
The mechanism by which MSCs home into degenerating eyes is
not fully understood. Studies have shown that stem cell migration
and organ-specific homing are regulated by chemokines and their
receptors. The expression of CXCR4 has been reported on
embryonic stem cells [48,49] and bone marrow derived stem cells
[50,51,52,53]. The specific CXCR4 ligand, stromal cell-derived
factor-1 (SDF-1) is expressed by several tissues and upregulated by
injury or ischemia. The SDF-1/CXCR4 axis plays an important
role in the recruitment of circulating progenitor cells to home to
sites of ischemic injury to facilitate repair . Our study showed
that MSCs were found in the retina two weeks after intravenous
injection. It is likely that retinal degeneration leads to upregula-
tions of certain chemokines, which promote MSCs to home into
the eye. It would be interesting to investigate whether CXCR4 and
its ligand are involved; how the MSCs are distributed and
differentiated in the eye with time; when is the best time to
administrate MSCs to achieve optimal efficacy.
The results of this study provide preliminary evidence in support
of potential clinical application, whereby a patient’s own bone
marrow cells can be used to treat retinal degeneration and ocular
vascular pathology, such as that observed in diabetic retinopathy.
Diabetic retinopathy is associated with increased capillary
permeability, which can lead to retinal edema and retinal
neovascularization. The MSCs can provide neuro-vascular
protection and may avoid many of the unwanted potential side
effects associated with the use of viral vectors in gene therapy. The
Figure 4. Upregulation of trophic factors. A. Semi-quantitative RT-PCR for CNTF, bFGF, BDNF and beta actin. Lane 1: RNA isolated from MSC
prior to injection; Lane 2–4: RNA isolated from retinas treated with MSC; Lane 5–7: RNA isolated from non-treated control retinas. B. Densitometry
analysis of CNTF, BDNF and bFGF in treated versus untreated samples. Beta actin was used to normalize the data for comparison. Level of CNTF and
BDNF in the treated retinas were significantly higher than non-treated controls (p,0.05), while the level of bFGF in MSC treated retina did not
increase significantly. C–J: confocal images of retinal sections double stained with antibodies to CNTF (green) and GFAP (red), counterstained with
DAPI (blue in C and G) from MSC treated and controls. Strong CNTF staining in MSC treated retina (D) compared with untreated control (H); E&I:
retinal sections stained with GFAP (red) showing upregulation of GFAP in Mu ¨ller glia in both MSC treated and untreated control; F&J: merged images
showing colocalization of CNTF and GFAP in MSC treated retina (F), which was not observed in untreated control (J) (Scale bar equals 50 mm).
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issue of rejection associated with non-autologous stem cells may
also be limited. The advantages of this non-invasive cell-based
therapy are: cells are easily isolated and can be expanded in large
quantity for autologous graft; hypoimmunogenic nature as
allogeneic donors; less controversial in nature than other stem
cells; can be readministered with minor discomfort and non
surgical procedures. Currently, there are over 80 clinical trials
using bone marrow derived cells to treat various human diseases.
Therefore, MSCs may prove to be the ideal cell source for auto-
cell therapy for retinal degeneration and other ocular vascular
Isolation and Culture of Rat MSCs
MSCs were obtained from the bone marrow of RCS rats (6–8
weeks old) according to the method previously described [55,56].
Briefly, bone marrow was flushed from femurs and tibias with
Dulbecco’s modified Eagle’s medium (DMEM)(Gibco, Invitro-
gen, USA) and centrifuged at 600 g for 10 minutes. Freshly
isolated cells were resuspended in DMEM supplemented with
10% fetal bovine serum (HyClone, UT, USA), 100 U/ml
penicillin G and 100 mg/ml streptomycin sulfate (Invitrogen,
USA) and then seeded into T75 flasks (Corning, MA, USA). After
8 days, nonadherent cells were removed and adherent cells were
detached with 0.05% trypsin/0.53 mM EDTA and replated.
After 3 days, cells were detached with 0.1% trypsin/0.02%
EDTA and plated at 2000 cells/cm2. MSCs were passaged upon
reaching 50% confluency and cells from passage 2–4 were used
for this study.
Distribution of MSCs
To track the distribution of MSCs after systemic administra-
tion, cell-linkerPKH26 (Sigma)
manufacturer’s protocol to label MSCs prior to injection.
Retinal whole mount and sections were examined 2 weeks after
MSC injection. To see the relation between retinal vessels and
MSCs, animals were perfused with FITC-dextran to highlight
retinal vasculature before sacrifice. Fluorescence microscopy was
used to visualize PKH26 labeled MSCs within eyes and other
Intravenous Administration of MSCs
MSC suspension containing 1 million cells/ml in balanced salt
solution (BSS) was administered via tail vein using 31G needle to
RCS rats at P30; as a control, age-matched RCS rats received BSS
alone. These studies were conducted with approval and under the
supervision of the Institutional Animal Care and Use Committee
at the Oregon Health & Science University.
Spatial Visual Acuity
Animals were tested for spatial visual acuity at P90 using an
Optomotry testing apparatus (CerebraMechanics, Lethbridge,
Can) . The optomotry set-up comprises of four computer
monitors arranged in a square that projected a virtual three-
dimensional (3-D) space of a rotating cylinder lined with a vertical
sine wave grating. Unrestrained animals were placed on a
platform in the center of the square, where they tracked the
grating with reflexive head movements. The spatial frequency of
the grating was clamped at the viewing position by re-centering
the ‘cylinder’ on the animal’s head. The acuity threshold was
quantified by increasing the spatial frequency of the grating using
a psychophysics staircase progression until the following response
was lost, thereby defining the acuity.
Luminance threshold (LT) was measured with the objective of
providing parallel data to the surface of the superior colliculus (SC)
using previously described procedures . Recordings were
made in rats using glass-coated tungsten electrodes (resistance:
0.5 MV; bandpass 500 Hz–5 KHz). The brightness of a 5u spot
was varied using neutral density filters (minimum steps of 0.1 log
unit) over a baseline level of visual acuity. The LT was evaluated
by recording single and multiunit activity close to the 5.2 log units
until a response double the background activity was obtained: this
was defined as the threshold level for that point on the visual field.
A total of 15–20 positions were recorded from each SC from P90–
P95. Data was expressed as a graph of percentage of the SC area
with a LT below defined levels and as raw results.
Statistical analyses were performed using GraphPad Prism
version 5 for Windows (California, USA). All variables were
Figure 5. Distribution of MSCs. A. phase contrast microphotograph of bone marrow derived mesenchymal stem cells at passage 2. B. MSCs were
preincubated with PKH26 before intravenous injection. C. PKH26 labeled MSCs in the retina two weeks after intravenous injection (arrows); blood
vessels were perfused with FITC-dextran (green). D–F. showing PKH26 labeled MSCs in the retinal section (D, arrows pointing PKH26 labeled MSCs;
double arrows indicating background staining in debris zone); sections counterstained with DAPI (E); F. merged image from D&E showing PKH26
labeled MSCs counterstained with DAPI (scale bar equals 100 mm).
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expressed as mean6 standard error of the mean. Differences
between groups were compared by either Student’s two tailed
unpaired t test or analysis of variance. Newman-Keuls procedure
was used for multiple comparision analysis. Differences were
considered to be significant at P,0.05.
Whole Mount Preparation
PBS first, followed by 2% paraformaldehyde. The dorsal pole of
each eye was marked before enucleation. Whole mount
preparations of the retinas were prepared; four radial cuts were
placed in the dorsal, ventral, temporal, and nasal poles permitting
the retina to be laid flat. The retinas were postfixed for 30 minutes
in the same fixative, washed and incubated in a solution
containing 0.02% NADPH-diaphorase and 0.04% nitroblue
tetrazolium (Sigma) in 3% Triton X-100 for 90 minutes at 37uC
on a shaker. Retinas were washed with PBS, mounted on slides,
dehydrated with alcohol and covered with DPX. Retinal whole
mount was examined under a light microscope.
Animals were injected via tail vein
with FITC-dextran and the dorsal pole of each eye was marked
before enucleation. Eyes were fixed in 2% paraformaldehyde for
30 minutes, and then retinal whole mount was prepared as above
and examined under a confocal microscope.
Animals were perfused with
RNA was isolated from cells and retinal tissue using the
RNAqueous-4PCR kit (Ambion, USA) following the manufactur-
er’s protocol including a DNAse I step. The RNA concentration
for each sample was determined by UV spectrophotometry and
quality was assessed by the ratio of 260/280. The iScript cDNA
synthesis kit (Bio-Rad Laboratories, USA) was used to generate
cDNA. Briefly, equal concentrations of RNA from each sample
was reverse transcribed in the presence of 1X reaction buffer
which included dNTPs, random hexamers, oligo(dT), MgCl2and
MMLV-derived reverse transcriptase. Samples were incubated for
5 minutes at 25uC, 30 minutes at 42uC and 5 minutes at 85uC.
Following reverse transcription, PCR was performed using
standard protocols for CNTF, bFGF, BDNF and b-actin. Briefly,
2 ml of each RT reaction were mixed with 23 ml of a PCR cocktail
containing 1X PCR Buffer, 1.5 mM MgCl2, 1 unit of Taq
polymerase, 10 mM dNTPS, 20 pmols of forward and reverse
primers. Reactions were incubated at 95uC for 5 minutes followed
by 30 cycles of 95uC for 30 seconds, 55uC for 30 seconds and 72uC
for 30 seconds. PCR reactions were loaded onto 1% agarose gels
containing ethidium bromide and visualized on a strategene UV
gel-doc system. Digital photos of the results were used for
CNTF forward 59-TGGGACAGTTGATTTAGGG-39 and
reverse primers 59-GCTACATCTGCTTATCTTTGG-39
bFGF forward 59-GAGAAGAGCGACCCACAC-39 and re-
verse primers 59-GCAGACATTGGAAGAAACAG-39
BDNF forward 59-CCTGGCTGACACTTTTGAG-39 and
reverse primers 59-ATTGGGTAGTTCGGCATTGCG-39
b-actin forward 59-GAGCGTGGCTACAGCTTCACCAC-
39 and reverse primers 59-TACTCCTGCTTGCTGATCCA-
Cresyl violet and immunohistochemistry.
functional tests, all animals were euthanized with an overdose of
sodium pentobarbital (Sigma) and perfused with phosphate
buffered saline (PBS). The eyes were then removed, immersed in
2% paraformaldehyde for one hour, infiltrated with sucrose,
embedded in OCT and cut in sequence 10 mm horizontal sections
apart on a cryostat. Every sixth section was placed on the same
slide as the first section and a total of four sections (50 mm apart)
were collected per slide. Approximately 80 slides were generated
per eye, thus one eye contained a set of 16 and each set contained
5 slides. One slide from each set was stained with cresyl violet for
assessing integrity of retinal lamination. The remaining slides were
used for immunohistochemistry staining using retinal specific
antibodies, following previously described protocols , and were
examined by regular light and confocal microscopy. The retinal
specific antibodies to rhodopsin (1:1000, abcam, USA), cone
arrestin (1:3000, Chemicon), CNTF (1:500, Santa Cruz, USA),
GFAP (1:1000, Sigma) and BDNF (1:1000, Millipore) were used
After all the
We would like to thank Mr. Benjamin Cottam for assisting with confocal
Conceived and designed the experiments: SW BL. Performed the
experiments: SW BL SG JD TM QsZ. Analyzed the data: SW BL SG
JD TM GA BA. Contributed reagents/materials/analysis tools: QsZ MG
GA RDL. Wrote the paper: SW BA. Support works performed in his
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