A Therapeutic Strategy for Choroidal Neovascularization …

 

 

© The American Society of Gene & Cell Therapy

original article

A Therapeutic Strategy for Choroidal
Neovascularization Based on Recruitment
of Mesenchymal Stem Cells to the Sites of Lesions

Hui-Yuan Hou1, Hong-Liang Liang2, Yu-Sheng Wang1, Zhao-Xia Zhang1, Bai-Ren Wang3, Yuan-Yuan Shi1,
Xiao Dong1 and Yan Cai1

1Department of Ophthalmology, Eye Institute of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi’an, China; 2Department
of Cardiovascular Surgery, Institute of Cardiovascular Disease of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi’an, China;
3Institute of Neurosciences, Fourth Military Medical University, Xi’an, China

Choroidal neovascularization (CNV) is a common cause
of severe and irreversible visual loss; however, the treat-
ment of CNV has been hindered by its complex and
poorly understood pathogenesis. It has been postulated
that bone marrow (BM)–derived cells (BMCs) contrib-
ute to CNV, but little is known about the role of mesen-
chymal stem cells (MSCs) in CNV and their therapeutic
potential for CNV treatment. We found that BM-derived
MSCs transplanted by intravenous injection into laser-
induced CNV mouse models were specifically recruited
into CNV lesions, where they differentiated into mul-
tiple cell types and participated in the development of
neovascularization, without stagnation in other organs.
By taking advantage of this recruitment potential, engi-
neered MSCs were used to produce the antiangiogenic
pigment epithelial-derived factor (PEDF) at the CNV sites,
thereby inhibiting the growth of CNVs and stimulating
regressive features. Further studies indicated that the
effect may be mediated, at least partly, by retinal pig-
ment epithelial (RPE) cells, which function as important
regulators for CNV development. These results suggest
that MSCs contribute to CNV and could serve as delivery
vehicles of antiangiogenic agents for the treatment of a
range of CNV-associated diseases.

Received 8 February 2010; accepted 10 June 2010; published online
20 July 2010. doi:10.1038/mt.2010.144

INTRODUCTION
Pathological angiogenesis in the eye often leads to serious con-
sequences, including intractable high intraocular pressure, visual
impairment, and even irreversible blindness. One major mani-
festation of ocular angiogenesis is choroidal neovascularization
(CNV). CNV is characterized by the formation of new blood
vessels that arise from the choriocapillaris through Bruch’s mem-
branes into the subretinal space, causing exudation of fluid and
hemorrhaging. Furthermore, CNV is often accompanied by the

atrophy and senescence of retinal pigment epithelial (RPE) cells
and microfractures in Bruch’s membranes. Consequently, the
overlying neurosensory retina may detach, and the ensuing dam-
age to the retinal photoreceptors could lead to irreversible visual
loss.1 CNV is now known to be a common process in nearly 40
ophthalmic diseases affecting people of all ages, especially the
elderly.2 The most common condition associated with CNV is age-
related macular degeneration, which has emerged as the leading
cause of blindness among people aged ≥50 (ref. 3).

In light of the serious social and economic costs of CNV-
related diseases, several CNV treatment options, such as ion-
izing radiation, laser photocoagulation, surgical removal, and
photodynamic therapy, have been developed.4 Among them,
pharmaco therapy with antiangiogenic agents that target the
angiogenic vascular endothelial growth factor (VEGF) pathway
has shown relatively high efficacy. Most other therapies, how-
ever, are largely ineffective. Even in the case of pharmacother-
apy, regression of neovascularization is rarely permanent, and
the regrowth of new vessels, often within a few months, requires
multiple treatments. Moreover, frequent, invasive, intravitreal
injections of antiangiogenic agents may be associated with
serious side effects, such as endophthalmitis.3,5 Therefore, it is
a pressing issue to develop innovative therapeutic strategies
that are less invasive and safer, with enhanced specificity and
efficacy.

Mesenchymal stem cells (MSCs) have been shown to differen-
tiate into endothelial cells (ECs) and vascular smooth muscle cells
(VSMCs) and incorporate into the new blood vessel wall and form
vascular tubes.6 On the other hand, MSCs play distinct roles in
different angiogenic models. In contrast to angiogenic activities in
various organs other than the eyes,6–8 MSCs display antiangiogenic
effects in the cornea.9 Recently, MSCs, which were recruited into
tumors and function as potential precursors for tumor stroma,
have been used as delivery vehicles for anticancer agents via the
systemic circulation.10–15 Yet, little is known about the contribution
of MSCs to CNV, although accumulating evidence has indicated
that bone marrow (BM)–derived cells (BMCs), a heterogeneous

The first two authors contributed equally to this work and should be regarded as co-first authors.
Correspondence: Yu-Sheng Wang, Department of Ophthalmology, Fourth Military Medical University, No. 15, Changle West Road, Xi’an, China.
E-mail: wangys003@126.com

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Therapeutic Application of MSCs in CNV Treatment

© The American Society of Gene & Cell Therapy

cell population comprised multiple types of stem/progenitor cells,
participate in CNV formation.16–19 Accordingly, the purpose of
this study was to investigate whether MSCs contribute to CNV
formation and to explore the potential application of MSCs in
CNV treatment.

RESULTS
Isolation and characterization of MSCs
Using the well-established method described above, we enriched
plastic-adherent mouse BMCs expressing surface markers char-
acteristic of multipotent MSCs. Following their third passage, cell
cultures were devoid of hematopoietic cells and highly enriched
for MSCs, as judged by the lack of the hematopoietic markers
CD34 and CD45, and the expression of CD44, CD29, and CD105.
The multipotent nature of the MSCs was further confirmed by
their capacity to differentiate into the adipogenic and osteogenic
lineages in vitro. Thus, we concluded that the enriched stem
cells were bona fide MSCs, which were subsequently used in the
following experiments.

Specific recruitment of MSCs to CNV and their
differentiation in CNV
We first examined whether MSCs can be specifically recruited
to sites of CNV. We evaluated whether MSCs may remain in
other tissues outside the eyes. After laser photocoagulation
and transplantation of MSCs with a green fluorescent protein
(GFP) reporter, the number of MSCs in the peripheral blood
(PB) slightly increased on day 1 after treatment and decreased
afterwards. Some MSCs underwent a transient retention in the
BM. The number of GFP-labeled cells in the BM peaked on
day 1. Afterwards, however, MSCs decreased rapidly (Figure 1).
GFP+ cells were not found in lung, liver, spleen, or heart tissues.
Recruitment of MSCs to sites of CNV was confirmed by choroidal
flat mount. On day 1, a large number of green cells were found to
be dispersed around the laser photocoagulation sites (Figure 2,

1 day). On day 3, cell rings composed of green cells were found to
surround the laser spots, indicating that the MSCs were moving
directionally closer toward the CNV lesions (Figure 2, 3 days).
On day 7, GFP-labeled MSCs were found at the sites of CNV and
appeared to participate in vascular structure formation (Figure 2,
7 days).

Next, we conducted confocal microscopy to examine the loca-
tion and differentiation of MSCs in the eye. Most GFP-labeled
MSCs appeared to be integrated into CNV between the choroid
and the photoreceptor cell layer at the laser spots. We detected
MSCs that had differentiated into ECs, VSMCs, macrophages,
and RPE cells, which are the major cell types in CNV.20 Expression
of all five cell markers (CD31, α-smooth muscle actin (αSMA),
F4/80, vimentin, and keratin) were detected in the MSCs examined
(Figure 3).

Transduction efficiency and PEDF expression in MSCs
The transduction efficiency of adenoviral vectors in mouse
MSCs was analyzed. Reporter GFP expression in MSCs was
detected 24 hours after transduction using an inverted fluores-
cent microscope (Figure 4a,b). Flow cytometry analyses further
showed that 73.6 ± 5.3% of the total cell population was GFP+
(Figure 4c). Adenoviral-expressed pigment epithelial-derived
factor (PEDF) persisted for at least 8 days in MSCs in vitro, with
the maximum production observed during the first 24 hours
after transduction (Figure 4d). In contrast, human PEDF was
absent in the supernatant from MSCs transfected with control
vectors (AdNull) and untransduced MSCs throughout the entire
experimental period.

In vivo PEDF expression was examined by immunofluorescent
staining and enzyme-linked immunosorbent assays (ELISAs).
Human PEDF+ staining was found in MSCs and in the nearby
extracellular matrix in serial eye sections from mice transplanted
with adenoviral vectors expressing PEDF (AdPEDF)-transduced
MSCs (Figure 5a). ELISAs (Figure 5b) further showed that, in

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Figure 1 Quantitative analysis of GFP-expressing mesenchymal stem cells (MSCs) in PB and BM by flow cytometry after laser photocoagula-
tion and GFP-expressing MSC transplantation. (a) Representative data from three experiments are shown. (b) The quantity of GFP-expressing
MSCs in BM increased obviously in the first 24 hours of the experiment and decreased rapidly thereafter. The number of MSCs in PB slightly increased
on day 1 (error bars, SEM, n = 6). BM, bone marrow; GFP, green fluorescent protein; PB, peripheral blood.

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© The American Society of Gene & Cell Therapy

Therapeutic Application of MSCs in CNV Treatment

contrast to the pattern observed in vitro, the average production of
human PEDF in the eyes from the group transduced with AdPEDF
was constant throughout the experimental period and more than
fourfold higher than the quantity required to elicit antiangiogenic
effects and CNV inhibition.21 In contrast, human PEDF was absent
in the blood samples from all experimental mice and eye samples
from the AdNull, nontransduced, and control groups.

Agglutinin

GFP

Merge

Reduction in the severity of CNV in mice treated
with AdPEDF-transduced MSCs
Based on the confirmation that MSCs were recruited to sites of
CNV, we then examined whether MSCs could be utilized to inhibit
CNV growth. CNV lesion severity was measured by quantitative
analyses of both histopathology and flat mount. First, the effect
of MSCs themselves was examined. The nontransduced group
showed a slight increase in CNV severity compared to the control
group, although there was no statistically significant difference.
Therefore, we subsequently explored the effect of the antiangio-
genic factor PEDF on CNV. There was a significant reduction in
CNV thickness, diameter (Figure 6a), and surface area (Figure 6b)
in the group treated with AdPEDF-transduced MSCs. There was
no statistically significant difference between the control group, the
AdNull group, and nontransduced group, and we confirmed that it
was PEDF secreted by MSCs that affected CNV severity.

Using histopathological analyses, we noticed that 7 days
after the injection of AdPEDF-transduced MSCs, many CNVs
were encapsulated by pigmented cells, suggesting a regression
in neovascularization;22 however, on the other hand, this pheno-
menon was rarely observed in the other groups (Figure 6a). As a
result, we investigated the effect of AdPEDF-transduced MSCs on
RPE cells in vitro.

Enhanced proliferation and migration of RPE cells
by PEDF produced by MSCs
We examined whether AdPEDF-transduced MSCs affected pro-
liferation and migration of RPE cells in vitro. When cocultured
with AdPEDF-transduced MSCs, RPE cells displayed a markedly
increased migration rate (Figure 7a), suggesting that the AdPEDF-
transduced MSCs were chemotactic for RPE cells. Furthermore,
RPE cells that were cocultured with AdPEDF-transduced MSCs
were found to proliferate more rapidly relative to cells under other
coculture conditions (Figure 7b). Meanwhile, the migration and
proliferation of RPE cells were compared to RPE cells that were
cocultured with untransduced MSCs and AdNull-transduced
MSCs, but there was no statistically significant difference.

(cid:65)SMA

GFP

DAPI

1 d

3 d

7 d

Figure 2 Representative choroidal flat mount preparations after
laser photocoagulation and GFP-expressing mesenchymal stem
cell (MSC) transplantation. Blood vessels were stained by rhodamine-
conjugated agglutinin (red) in choroidal flat mounts. Panels show the
recruitment of GFP-expressing MSCs (green) to the choroidal neovascu-
larization (CNV) lesion (red) on days 1, 3, and 7, indicating a directional
movement of the MSCs into the CNV lesions. On day 7, GFP-expressing
MSCs in CNV participated in vascular structure formation (yellow). Bar =
50 μm. GFP, green fluorescent protein.

CD31

GFP

DAPI

F4/80

GFP

DAPI

Vimentin

GFP

DAPI

Keratin

GFP

DAPI

Figure 3 Immunofluorescence staining of eye sections showing differentiated GFP-expressing mesenchymal stem cells (MSCs) in choroidal
neovascularization (CNV) 1 week after CNV induction. The differentiation of MSCs in CNV was analyzed by cell maker staining. Representative
confocal images show that MSCs (green) express (a) the mature vascular endothelial cell marker CD31 (red), (b) the vascular smooth muscle cell
marker (cid:65)SMA (red), (c) the macrophage marker F4/80 (red), (d) the fibroblast marker vimentin (red), and (e) the epithelial cell marker keratin (red).
Blue: DAPI-stained nuclei. Bar = 20 μm. (cid:65)SMA, (cid:65)-smooth muscle actin; GFP, green fluorescent protein.

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Therapeutic Application of MSCs in CNV Treatment

© The American Society of Gene & Cell Therapy

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Figure 4 Analysis of adenoviral transduction efficiency 24 hours after
transduction and enzyme-linked immunosorbent assays of human
pigment epithelial-derived factor (PEDF) released by adenoviral
vectors expressing PEDF (AdPEDF)–transduced mesenchymal stem
cells (MSCs). Representative inverted microscopy images show reporter
green fluorescent protein (GFP) expression in MSCs (a, light microscope
image; b, fluorescent image). (c) A representative flow cytometry analy-
sis shows that 78.1% cells within the total cell population expressed GFP.
(d) Human PEDF production by AdPEDF-transduced MSCs was detected
in vitro during an 8-day experimental period. Maximum production
was observed during the first 24 hours after infection (error bars, SEM,
n = 9). Bar = 20 μm.

Therefore, we confirmed that the migration and proliferation of
RPE cells were stimulated by PEDF.

DISCUSSION
In this study, we demonstrated that BM-derived MSCs were
specially recruited into CNV lesions to participate in CNV

development. As an antiangiogenic therapy, engineered MSCs
were exogenously administered by intravenous injection, and they
locally produced a therapeutic dose of the antiangiogenic factor
PEDF to inhibit CNV growth in vivo. Furthermore, the MSC-
derived PEDF also enhanced RPE cells, which regulate CNV
development, proliferation, and migration.

Both vasculogenesis and angiogenesis play roles in neovas-
cularization in the eye and elsewhere. Previous studies have pro-
vided accumulating evidence for a role of BMCs in CNV. In these
studies, following lethal irradiation, labeled BMCs were trans-
planted into animal models, and laser photocoagulation was con-
ducted after BM reconstruction. Laser injury alone was sufficient
to induce recruitment of BMCs,20 which may have contributed up
to 50% of the total vasculature.17 Our study specifically examined
the role of MSCs, a major cell type of the BM. Compared to previ-
ous studies, we performed intravenous injection of labeled MSCs,
but we avoided BM re-establishment to allow direct and special
detection of MSCs.

We found that MSCs contributed to CNV formation. CNV is
a complex tissue composed of both vascular components (ECs,
VSMCs, and pericytes) and extravascular cells (inflammatory
cells, myofibroblasts, glial cells, and RPE cells).3,23 ECs, VSMCs,
macrophages, and RPE cells are the major cell types of CNV.20
In light of the multilineage potential of MSCs, and the demon-
strated differentiation of MSCs to vascular cells6 and connective
tissue cells,12 it is conceivable that MSCs can differentiate into
both vascular and extravascular cells in CNV. In this study, we
examined cell markers for five cell types, including ECs, VSMCs,
macrophages, fibroblasts, and RPE cells, and found that MSCs
differentiated into these cell types in CNV. Considering that GFP+
MSCs were transplanted without irradiation, BMCs from the
recipient, which were undetectable, must have also participated in
CNV formation. Accordingly, some cells of each cell type in CNV
were probably derived from the autologous MSCs. Therefore, we
do not presume that the differentiated cells detected in this study
represented the overall contribution of MSCs to CNV.

In contrast to the antiangiogenic effect of MSCs in corneal
wound healing following chemical injury,9 MSCs alone did not
inhibit CNV in this model. In alcohol-injured corneas, the role of
MSCs was attributed to their anti-inflammatory effects.9 Although
CNV involves some degree of inflammation, the inflammatory
component varies in intensity from minimal inflammation to
pronounced inflammation depending on the underlying disease
and dynamic stage of CNV development.2 Therefore, although
MSCs might exert antiangiogenic effects under certain conditions
in CNV or with an increased transplantation of MSCs, we turned
to explore a more efficient way of treating CNV, which is to use
MSCs as delivery vehicles of antiangiogenic factors.

The application of MSC vectors in CNV treatment is their
recruitment to the lesion. A putative CNV tropism is based pri-
marily on the innate physiological ability of MSCs to move to the
sites of inflammation and tissue repair,11 and CNV appears to be
a component of several key processes that can be referred to as
wound healing or tissue repair.24 Two main chemokines, VEGF
and stromal-derived factor-1, that navigate BMC trafficking and
homing in CNV, contain receptors on MSCs.6,25–28 In the current
study, we revealed the specific and rapid recruitment of MSCs

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Figure 5 Immunofluorescent staining and enzyme-linked immuno-
sorbent assays (ELISAs) of human PEDF expression in the eyes of
mice treated with adenoviral vectors expressing PEDF (AdPEDF)–
transduced MSCs. (a) In eye sections of the AdPEDF-expressing mice,
positive human PEDF staining (red) was found in AdPEDF-transduced
MSCs (green) and the extracellular matrix nearby. Blue: DAPI-stained
nuclei. (b) ELISAs show that the average production of human PEDF
in eyes of the AdPEDF-expressing mice was relatively steady for 1 week
(error bars, SEM, n = 6). Bar = 20 μm. GFP, green fluorescent protein;
PEDF, pigment epithelial-derived factor.

to the sites of laser injury without movement into other organs
(except for very short retention in the BM). Moreover, expression
of PEDF in MSCs was absent in the blood, which further sug-
gests the potential to avoid possible side effects. Previous report-
ers showed other advantages of MSCs as therapeutic candidates.
MSCs are amenable to genetic manipulation in vitro,10 and have
high metabolic activities and efficiently secrete therapeutic pro-
teins. MSCs exhibit low intrinsic mutation rates.29 Finally, MSCs
show little or low immunogenicity due to the lack of expression
of co-stimulatory molecules.11 Therefore, MSCs probably act as
cellular vectors and transgenic protein factories to safely and effi-
ciently deliver therapeutic agents in CNV. This administration
strategy may be especially suitable for ocular diseases because
the existence of the blood–ocular barrier confines agents in the
circulation. Moreover, delivery of proteins, which are not suitable
for intravitreous injection due to poor permeability through the
retina to CNV lesions, would be improved by using MSCs.

It is believed that CNV may be attributed to an imbalance in
the expression of angiogenic and antiangiogenic factors. Among
the endogenous inhibitors of neovascularization, PEDF is a strong
inhibitor of angiogenesis.1,30 Compared with other antiangiogenic
agents, PEDF may be ideally suited for CNV treatment. PEDF
targets aberrant neovascularization and has no known deleterious
effects on mature blood vessels.1,5 Furthermore, PEDF is the only
known therapeutic agent for CNV inhibition, which can cause
regression of already established ocular neovascularization.22

It has been reported that 6 ng of PEDF per eye was sufficient to
inhibit CNV.21 In the present study, sufficient PEDF was delivered
by transduced MSCs recruited to CNV. Consequently, the growth
of CNV was inhibited.

PEDF is known as an antiangiogenic factor because it can
induce apoptosis and inhibit migration of ECs,1,31 downregulate
VEGF expression, and inhibit VEGF–VEGF receptor 2 binding.32
In fact, the effects of PEDF are complex, and its target cells are
various.33–35 PEDF can also induce proliferation1 and migration,33
among other effects,1,34,35 which suggests that distinct signal
transduction pathways are affected. It is possible that the bio-
logical activities of PEDF rest with the differential binding and
dissociations of multiple receptors, which are affected by differ-
ent microenvironments.1,33 A lipase-linked cell membrane recep-
tor for PEDF has been found on RPE cells,36 although the exact
signal that would be transduced is still unknown. Under nor-
mal conditions, RPE is a major source of PEDF in the eye.1,34,35,37
Under pathological conditions, however, with the atrophy of RPE,
reduced PEDF production may permit the formation of CNV.1,30,38
Our study showed improved proliferation and migration of
RPE cells when cocultured with AdPEDF-transduced MSCs.
Therefore, we presume that a positive feedback loop may exist, in
which PEDF improves RPE proliferation, and PEDF expression
by RPE cells increases accordingly, causing enhanced antiangio-
genic effects. CNV inhibition by AdPEDF-transduced MSCs may
be partly mediated by RPE cells; however, the detailed mechanism
by which this occurs needs further investigation.

The

laser-induced Bruch’s membrane photocoagulation
model, in which the course of CNV is characterized by a tissue
repair response, is the most widely accepted and most frequently
utilized experimental CNV model by virtue of its recapitula-
tion of many important features of human age-related macu-
lar degeneration.3 However, there are multiple causative factors
for CNV in patients, such as pathological myopia, trauma, and
infectious diseases, which mandate further studies using distinct
experimental systems. In addition, although it remains unclear
whether BMCs that can develop into MSCs under in vitro con-
ditions are identical to circulating mesenchymal precursors in
PB, it is assumed that cultured BM adherent cells possess MSCs
potentials.10,39 The potential concerns over the use of MSCs as
delivery vehicles mainly stem from studies that have shown that
MSCs can be precursors of tumor stroma.10,11 Yet, the possibility
that MSCs are able to enhance or initiate tumor growth is unlikely
because of the intrinsic homeostasis of MSCs.29 MSCs may pos-
sess tumorigenic properties only after extensive passage, whereas
lower-passage MSCs do not form tumors in vivo.29 In this study,
third-passage MSCs were used. As a result, we could exclude the
possibility that MSCs were single-handedly tumorigenic.

In summary, we have demonstrated that MSCs play a role in
CNV and serve as a powerful cellular delivery system for antian-
giogenic therapeutic agents in CNV. Our findings may facilitate
further understanding of the mechanisms that underlie CNV and
represent new therapeutic strategy for CNV treatment.

MATERIALS AND METHODS
Isolation, culture, and characterization of MSCs. Isolation and enrich-
ment of mouse MSCs was carried out using standard protocols.40 Briefly,

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Therapeutic Application of MSCs in CNV Treatment

© The American Society of Gene & Cell Therapy

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P < 0.05 P < 0.05 Length Thickness C ontrol N ontransduction A d N ull A d P E D F AdNull AdPEDF P < 0.05 i ) l e x p ( a e r a V N C 400,000 300,000 200,000 100,000 0 A d N ull A d P E D F C ontrol N ontransduction Figure 6 Hematoxylin and eosin (H&E) staining, choroidal flat mount, and quantitative analysis of choroidal neovascularization (CNV) sever- ity 1 week after laser photocoagulation. (a) Representative H&E images show that CNVs from the AdPEDF group were encapsulated by pigmented cells (arrows), whereas CNVs in other groups were not. CNVs are indicated by dotted lines. The average thickness and diameter of CNVs from the AdPEDF group significantly decreased compared to other groups. No statistical difference was found among the other groups (error bars, SEM, n = 8). Bar = 20 μm. (b) The images of choroidal flat mounts show smaller surface areas of CNV (red) in the AdPEDF group. CNV area was signifi- cantly reduced in the AdPEDF group. No statistically significant difference was found among the other groups (error bars, SEM, n = 8). Bar = 50 μm. AdPEDF, adenoviral vectors expressing pigment epithelial-derived factor. BM cells from tibias and femurs of 4-week-old female C57BL/6 mice were cultured on plastic dishes in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal calf serum (Gibco, Grand Island, NY) and incubated at 37 °C with 5% CO2 in an incubator. After 3 days, nonadher- ent cells were removed by washing with phosphate-buffered saline, and the remaining monolayer cells were cultured in fresh medium until they reached confluence. After 6–7 days, cells were trypsinized (0.25% trypsin with 0.1% EDTA) and subcultured. The third-passage cells were used for experiments. To validate enrichment of MSCs, flow cytometry was carried out to characterize the surface antigens, and in vitro differentiation of cultured MSCs was performed as previously reported.41 Briefly, flow cytometry analysis was performed on a BD FACSAria Flow Cytometer (BD Biosciences, San Diego, CA). When the cells became confluent, MSCs were trypsinized and stained using fluorescein isothiocyanate– labeled antibodies against CD44, CD45, CD34, or phycoerythrin-labeled CD105, and CD29 (all from eBioscience, San Diego, CA). Negative control immunofluorescence experiments were performed in parallel with unrelated antibodies. Adipogenic differentiation was induced with dexamethasone, isobutylmethylxanthine, hydrocortisone, indomethacin, and insulin in low-glucose Dulbecco’s modified Eagle’s medium and 10% fetal calf serum. After 16 days, cells were fixed with 10% formalin and stained with Oil Red O for the detection of lipid vacuoles. Osteogenic differentiation was induced with dexamethasone, ascorbic acid, and β-glycerol phosphate in low-glucose Dulbecco’s modified Eagle’s medium and 10% fetal calf serum. Cultures were harvested at day 18. Calcium deposition was evaluated qualitatively by von Kossa staining of formalin- fixed cultures. All chemicals used for the induction of differentiation were obtained from Sigma-Aldrich (St Louis, MO). Cultures grown in MSC medium for the entire period served as negative controls. CNV induction in mice and MSC transplantation. Laser induction of CNV was performed as previously reported.42 Briefly, recipient mice were anesthetized, and their pupils were dilated. Laser photocoagulation (532 nm wavelength, 75 μm spot size, 0.1 second duration, and 90 mW intensity) was delivered with a slit lamp and a cornea contact lens. Six burns were performed in positions 1–1.5 disc diameters from the optic nerve. Only laser spots where rupture of Bruch’s membrane was confirmed with a vaporization bubble without hemorrhage were considered effective and included in the study. To investigate the role of MSCs in experimental CNV, MSCs derived from GFP+ transgenic C57BL/6 homozygous mice were cultured and transplanted into wild-type C57BL/6 mice. The third-passage GFP+ MSCs were trypsinized, and the concentration of cells was assessed by microscopy. 1842 www.moleculartherapy.org vol. 18 no. 10 oct. 2010 © The American Society of Gene & Cell Therapy Therapeutic Application of MSCs in CNV Treatment P < 0.05 C ontrol N ontransduction A d N ull A d P E D F P < 0.05 a l d o F b l d o F 2.0 1.5 1.0 0.5 0.0 4 3 2 1 0 C ontrol N ontransduction A d N ull A d P E D F Figure 7 Proliferation and migration assays of RPE cells cocultured with MSCs. (a) Migration of RPE cells was stimulated by the secretion of AdPEDF-transduced MSCs after coculture for 8 hours. (b) Three days after coculture, proliferation of RPE cells cocultured with AdPEDF-transduced MSCs was markedly enhanced. There was no statistically significant dif- ference between the control group, the AdNull group, and the non- transduction group in both assays (error bars, SEM, n = 10). AdPEDF, adenoviral vectors expressing pigment epithelial-derived factor. The cell suspensions were adjusted to 1.0 × 107 cells/ml with saline, and the GFP+ MSCs (4.0 × 106 cells/0.4 ml/mouse) were injected into adult C57BL/6 mice tail veins 0.5–1 hour after laser photocoagulation. Detection of MSC recruitment. To investigate the recruitment of MSCs, PB, BM, some highly vascularized organs, and CNV lesions were examined for GFP+ MSCs. On days 1, 3, and 7 after laser-induced CNV, PB samples from tail veins were collected into heparinized tubes. The mice were then killed and perfused transcardially with 20 ml warm 0.9% saline followed by 50 ml cold 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4). BM from femurs and tibiae were collected into heparinized tubes. Red blood cells in PB and BM were schizolysed, and the percentage of cells expressing GFP