ARTHRITIS & RHEUMATISM

Vol. 64, No. 1, January 2012, pp 243253

DOI 10.1002/art.33313
2012, American College of Rheumatology
Reversal of Serologic, Immunologic, and Histologic Dysfunction
in Mice With Systemic Lupus Erythematosus by
Long-Term Serial Adipose TissueDerived
Mesenchymal Stem Cell Transplantation
Eun Wha Choi,
1
Il Seob Shin,
2
So Young Park,
1
Ji Hyun Park,
1
Jong Sung Kim,
1
Eun Ji Yoon,
2
Sung Keun Kang,
2
Jeong Chan Ra,
2
and Sung Hwa Hong
3
Objective. To investigate the efficacy of human
adipose tissuederived mesenchymal stem cell (AD-
MSC) transplantation in systemic lupus erythematosus
(SLE) and to determine the optimal transplantation
window for stem cells either before or after disease
onset.
Methods. (NZB NZW)F
1
mice with SLE were
administered human AD-MSCs (5 10
5
) intravenously
every 2 weeks from age 6 weeks until age 60 weeks, while
the control group received saline vehicle on the same
schedule. Another experiment was carried out with a
different initiation time point for serial transplantation
(age 6 weeks or age 32 weeks).
Results. Long-term serial administration (total of
28 times) of human AD-MSCs ameliorated SLE without
any adverse effects. Compared with the control group,
the human AD-MSCtreated group had a significantly
higher survival rate with improvement of histologic and
serologic abnormalities and immunologic function, and
also had a decreased incidence of proteinuria. Anti
double-stranded DNA antibodies and blood urea nitro-
gen levels decreased significantly with transplantation
of human AD-MSCs, and serum levels of granulocyte
macrophage colony-stimulating factor, interleukin-4
(IL-4), and IL-10 increased significantly. A significant
increase in the proportion of CD4FoxP3 cells and a
marked restoration of capacity for cytokine production
were observed in spleens from the human AD-MSC
treated group. In the second experiment, an early stage
treatment group showed better results (higher survival
rates and lower incidence of proteinuria) than an ad-
vanced stage treatment group.
Conclusion. Serial human AD-MSC transplanta-
tion had beneficial effects in the treatment of SLE,
without adverse effects. Transplantation of human AD-
MSCs before disease onset was preferable for ameliora-
tion of SLE and restoration of immune homeostasis.
Systemic lupus erythematosus (SLE) is a multi-
system autoimmune disease. Antigenantibody com-
plexes are produced and subsequently lodge in small
vessels, the basement membrane zone of the skin and
kidney, and various organ systems. The exact immuno-
pathogenesis of SLE is unknown. Many factors might be
involved, such as genetic factors, T cell defects, B cell
hyperactivity, hormonal alterations, and virally induced
antigenantibody complex formation. Some studies have
indicated that the etiopathogenesis of murine lupus can
be attributed to defects that reside at the level of stem
cells (17).
(NZB NZW)F
1
mice develop a spontaneous
autoimmune disease process with striking similarities to
Supported by the Basic Science Research Program through
the National Research Foundation of Korea, which is funded by the
Ministry of Education, Science, and Technology (basic research grants
2010-0016648 and 2009-0077165). Additional support was provided by
the Samsung Biomedical Research Institute (grant SBRI P-A9-005).
1
Eun Wha Choi, DVM, PhD, So Young Park, BS, Ji Hyun
Park, BS, Jong Sung Kim, DVM, MS: Samsung Biomedical Research
Institute, Seoul, Republic of Korea;
2
Il Seob Shin, DVM, PhD, Eun Ji
Yoon, BS, Sung Keun Kang, DVM, PhD, Jeong Chan Ra, DVM, PhD:
RNL Bio, Seoul, Republic of Korea;
3
Sung Hwa Hong, MD, PhD:
Samsung Biomedical Research Institute, Samsung Medical Center,
and Sungkyunkwan University School of Medicine, Seoul, Republic of
Korea.
Address correspondence to Eun Wha Choi, DVM, PhD,
Laboratory Animal Research Center, Samsung Biomedical Research
Institute, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of
Korea. E-mail: vet.cew@gmail.com.
Submitted for publication September 14, 2010; accepted in
revised form August 25, 2011.
243
human SLE. In female (NZB NZW)F
1
mice, antinu-
clear antibodies (including antidouble-stranded DNA
[anti-dsDNA] antibodies) are produced and the mice
develop a severe immune complexmediated glomeru-
lonephritis. Thus, all of these mice die of renal failure by
age 1012 months (8).
Immunosuppressive drugs such as cortico-
steroids, cyclophosphamide (CYC), azathioprine, and
methotrexate are currently used to treat SLE. These
agents may be effective in some cases of SLE, but they
are not uniformly efficacious and are associated with
substantial toxicities (9). Through a better understand-
ing of the immunopathogenesis of autoimmune disease,
many researchers seek to identify and test novel immu-
notherapeutic strategies. One such strategy is immuno-
ablation followed by hematopoietic stem cell transplan-
tation. In 2007, Smith-Berdan et al reported that
nonmyeloablative transplantation of allogeneic hemato-
poietic stem cells improved survival and decreased pro-
teinuria, circulating immune complexes, and autoanti-
bodies to nuclear antigens in (NZB NZW)F
1
mice
with SLE (10). However, this method requires chemo-
ablation combined with polyclonal antibodies to elimi-
nate lymphocytes and reduce the risk of fatal graft-
versus-host (GVH) reaction.
Recently, many in vitro studies have shown that
mesenchymal stem cells (MSCs) exert immunomod-
ulatory and immunosuppressive effects by inhibiting
naive, memory, and activated T cells, B cells, natural
killer cells, and dendritic cells. In 2009, Sun et al
reported that bone marrowderived MSC (BM-MSC)
transplantation reverses multiorgan dysfunction in one
mouse model of SLE (the MRL/lpr mouse) and in
humans (11). However, in another mouse model of SLE
(the [NZB NZW]F
1
mouse), short-term BM-MSC
transplantation did not affect the anti-dsDNA antibody
level, proteinuria, or survival at the advanced stage of
disease (12).
Adipose tissues are becoming an alternative
source of MSCs for therapeutic applications, because
large amounts of adipose tissuederived MSCs (AD-
MSCs) are easily obtainable with little discomfort
(13,14). In addition, many in vitro studies have shown
that human AD-MSCs have an immunosuppressive ca-
pacity similar to that of BM-MSCs (15).
We previously demonstrated in vivo that human
AD-MSCs have beneficial effects on autoimmune thy-
roiditis (16). To our knowledge, there are no data
regarding the efficacy of human AD-MSC transplanta-
tion for treatment of SLE. Therefore, the current study
was conducted to investigate the effects of long-term
serial human AD-MSC transplantation on SLE using
(NZB NZW)F
1
mice, and to determine the optimal
therapeutic window of stem cell transplantation either
before or after disease onset.
MATERIALS AND METHODS
Isolation and culture of human AD-MSCs. Human
adipose tissue samples were obtained by simple liposuction
from abdominal subcutaneous fat after donors had provided
informed consent. Human AD-MSCs were prepared under
Good Manufacturing Practice conditions (RNL Bio) as de-
scribed previously (16,17).
Experiment 1. Experiment 1 animals. This study was
reviewed and approved by the Institutional Animal Care and
Use Committee (IACUC) of Samsung Biomedical Research
Institute. Samsung Biomedical Research Institute is accredited
by the Association for the Assessment and Accreditation of
Laboratory Animal Care International and abides by the
guidelines of the Institute of Laboratory Animal Resources.
Female (NZB NZW)F
1
mice (n 52) were purchased from
Japan SLC.
Experiment 1 groups. For the in vivo study, the experi-
mental groups were divided into a control group (n 26) and
a human AD-MSCtreated group (n 26). Each mouse in the
human AD-MSCtreated group was administered 5 10
5
human AD-MSCs/150 l of saline intravenously (IV) every 2
weeks from age 6 weeks until age 60 weeks. The control mice
were infused with 150 l of saline on the same schedule. The
survival rate and incidence of proteinuria (percent of individ-
ual mice with urine protein 300 mg/dl) were calculated.
Survival was monitored through age 60 weeks. The total
injected number of human AD-MSCs in each surviving mouse
of the human AD-MSCtreated group at the end point was
1.4 10
7
(5 10
5
human AD-MSCs 28 times).
Determination of proteinuria. During the experiment,
the urine protein:creatinine ratio was measured every 2 weeks.
Fresh urine was collected by performing abdominal massage.
Urine protein was measured with the Coomassie brilliant blue
method (18,19). Urine creatinine was measured using a mod-
ified Jaffe reaction by diluting the urine 1:100 in deionized
water.
Determination of body weight, spleen weight, spleen
weight:body weight ratio, spleen index, and spleen cell number.
The spleens were harvested and weighed at the time of death
or at the end of the study (n 26 in the control group, n 25
in the human AD-MSCtreated group). Body weights were
also measured at that time. The spleen index was calculated as
follows (20):
Spleen weight, human AD-MSCtreated total weight,
human AD-MSCtreated
Spleen weight, controls total weight, controls
.
Different time points (depending on when the mice died) were
left unaccounted for in the analysis. Spleen cells from the
surviving mice (age 62 weeks) in the control group (n 3) and
the human AD-MSCtreated group (n 6) were isolated and
counted.
244 CHOI ET AL
Determination of blood urea nitrogen (BUN), serum
creatinine, and anti-dsDNA antibodies. Blood samples were
collected from the mice under isoflurane anesthesia every 2
months, and the obtained sera were stored at 70C until
assayed. BUN and serum creatinine levels were determined
with a DRI-CHEM 3000 Colorimetric analyzer (Fujifilm).
Anti-dsDNA antibody concentrations were measured using a
mouse anti-dsDNA enzyme-linked immunosorbent assay
(ELISA) kit (Shibayagi).
ELISA for levels of multiple cytokines in spleen cell
culture supernatants and sera. Spleens were removed from 3
mice per group at age 40 weeks. Spleen cells from each mouse
were isolated and cultured in 96-well flat-bottomed plates at
2.5 10
5
/well to a final volume of 200 l with or without 2.5
g/ml concanavalin A (Con A). The plates were incubated at
37C in a humidified atmosphere. After 48 hours, the cell
culture supernatant in each well was collected and stored at
70C until used. The samples were assayed using a multiplex
cytokine ELISA kit for granulocytemacrophage colony-
stimulating factor (GM-CSF), tumor necrosis factor ,
interferon- (IFN), interleukin-1 (IL-1), IL-1, IL-2, IL-4,
IL-6, IL-10, IL-15, IL-17, monocyte chemotactic protein 1, and
RANTES (Millipore). Serum samples (n 1115 per group)
were also assayed using the same multiplex cytokine ELISA
kit. The IFN level was determined using a commercial mouse
IFN ELISA kit (VeriKine Mouse IFN- ELISA kit no.
42120; Pestka Biomedical Laboratories).
Determination of the proportion of Treg cells by flow
cytometry. Spleen cells were analyzed for Treg cell markers
using antibodies to CD4, CD25, and FoxP3 (fluorescein iso-
thiocyanate [FITC]conjugated rat anti-mouse CD4,
allophycocyanin-conjugated rat anti-mouse CD25, and
phycoerythrin-conjugated rat anti-mouse FoxP3; BD Biosci-
ences).
Hematoxylin and eosin (H&E), periodic acidSchiff
(PAS) reagent, and Massons trichrome staining. Kidneys were
removed from 3 mice per group at age 16 weeks or 40 weeks.
For light microscopy, tissues were fixed in 10% neutral buff-
ered formalin, embedded in paraffin wax, sectioned at 5 m,
and stained with H&E using a routine histologic technique.
PAS staining and Massons trichrome staining were used for
detection of proteinuria and fibrosis, respectively.
Immunofluorescence analysis of kidneys. Fresh kidneys
were embedded in OCT compound and frozen in
2-methylbutane slush. Sections (4 m thick) were fixed in
ice-cold acetone for 5 minutes and washed 2 times in ice-cold
phosphate buffered saline (PBS). Nonspecific binding was
blocked with 1% bovine serum albumin in PBSTween 20 for
30 minutes. The slides were drained and wiped with tissue
paper. Slides were incubated at room temperature with FITC-
conjugated goat anti-mouse IgG (1:200, AP 308F; Millipore)
or FITC-conjugated goat anti-mouse C3 (1:100; Cappel) for 1
hour and then washed 3 times in PBS. The slides were then
counterstained with mounting medium containing DAPI (Vec-
tor) and examined with a confocal laser scanning microscope
(Radiance 2100 confocal microscope; Bio-Rad).
Confocal microscopy examination of CM-DiIlabeled
human AD-MSCs. Three mice from the human AD-MSC
treated group were administered human AD-MSCs that had
been fluorescence-labeled using conjugated red fluorochrome
Cell Tracker CM-DiI (Invitrogen) for identification in histo-
pathologic sections. However, the evaluation time points for in
vivo distribution of human AD-MSCs differed among the 3
mice since the organs were harvested immediately upon death.
The presence of CM-DiIlabeled cells was examined in various
tissues, such as spleen, kidney, liver, lung, and heart (by
counterstaining with mounting medium containing DAPI),
with a confocal laser scanning microscope.
Experiment 2. Experiment 2 animals. This study was
reviewed and approved by the IACUC of Samsung Biomedical
Research Institute. Female (NZB NZW)F
1
mice (n 13)
were purchased from The Jackson Laboratory.
Experiment 2 groups. Experiment 2 was conducted to
evaluate the therapeutic potential of human AD-MSCs using a
different window of opportunity. To determine the efficacy of
serially administered human AD-MSCs initiated at a different
time point, 30 experimental mice with early stage disease were
used (n 15 each) for the control 1 group and the early stage
treatment group. Each mouse in the control 1 group was given
150 l of saline IV once every 2 weeks from age 6 weeks, while
each mouse in the early stage treatment group was treated with
5 10
5
human AD-MSCs resuspended in 150 l of saline on
the same schedule as that in experiment 1.
The remaining 43 animals were maintained without
any treatment until age 32 weeks. After a urine test, the mice
were divided into the control 2 group (n 14), a group of mice
with advanced stage disease treated with human AD-MSCs at
8 months (n 15), and a CYC-treated group (a positive
treatment control group; n 14). From age 32 weeks, each
mouse in the control 2 group was given 150 l of saline IV once
every 2 weeks until age 42 weeks, each mouse in the group with
advanced stage disease treated with human AD-MSCs was
given 2 10
6
human AD-MSCs IV once every 2 weeks until
age 42 weeks, and each mouse in the CYC-treated group was
injected intraperitoneally with 50 mg/kg CYC 3 times per week
for 10 weeks. The survival rate and incidence of proteinuria
(percent of individual mice with urine protein 300 mg/dl)
were calculated. Survival of mice was monitored until age 54
weeks. The control 1 and control 2 groups combined (n 29),
the early stage treatment group (n 15), the advanced stage
treatment group (n 15), and the CYC-treated group (n
14) were compared. The incidence of proteinuria and survival
rate were similar in all of the groups (in each group, 1 mouse
exhibited severe proteinuria [300 mg/dl] and 1 mouse
died).
Statistical analysis. All results are expressed as the
mean SEM. Data were compared between groups using
Students t-test or the Mann-Whitney U test. Proteinuria and
survival rate data were analyzed using Kaplan-Meier curves
and the log rank test. P values less than 0.05 were considered
significant. All statistical analyses were conducted using SPSS
software, version 17.0.
RESULTS
Experiment 1 findings. Survival rate, incidence
of proteinuria, and degree of proteinuria. The survival rate
was significantly higher in the human AD-MSCtreated
group than in the control group (P 0.029 by log rank
LONG-TERM SERIAL AD-MSC TRANSPLANTATION FOR SLE 245
test) (Figure 1A), and the proteinuria rate did not differ
significantly between the groups by log rank test (P
0.109), but was significantly lower in the human AD-
MSCtreated group by Breslow test (P 0.04). When
the sample size was larger, a statistically significant
difference between groups could be observed by log rank
test. The onset of proteinuria was delayed 8 weeks by
human AD-MSC treatment (Figure 1B), and the onset
of death was delayed 5 weeks (Figure 1A). The degree of
proteinuria and the urine protein:creatinine ratio were
lower in the human AD-MSCtreated group. The urine
protein:creatinine ratio in the human AD-MSCtreated
group was maintained at 1 up to age 34 weeks.
Clinicopathologically, a urine protein:creatinine ratio of
1 indicates the presence of glomerular lesions.
Body weight, spleen weight, spleen weight:body
weight ratio, spleen index, and spleen cell number. To
investigate whether host immune responses occur after
human AD-MSC transplantation, we measured body
weight and spleen weight and calculated the spleen
weight:body weight ratio. During the experiment, body
weights were not significantly different between groups
(data not shown). The degree of splenomegaly was
expressed as the spleen index. Spleen weight and
the spleen weight:body weight ratio were also not signif-
icantly different between groups. The mean SEM
spleen weight of the control group was 0.21 0.05 grams
(n 26), and that of the human AD-MSCtreated
group was 0.15 0.02 grams (n 25). The mean
SEM spleen weight:body weight ratio in the control
Figure 1. Survival, progression of severe proteinuria, and serology in (NZB NZW)F
1
mice after transplantation of human adipose tissuederived
mesenchymal stem cells (human AD-MSCs; hATMSC). (NZB NZW)F
1
mice were divided into 2 groups. Each mouse in the control group was
administered 150 l of saline intravenously, and each mouse in the human AD-MSCtreated group was administered 5 10
5
human AD-MSCs/
150 l of saline intravenously every 2 weeks from age 6 weeks. A, Survival was monitored through age 60 weeks. The survival rate was significantly
higher in the human AD-MSCtreated group than in the control group (P 0.029). B, Urinary protein was measured using Coomassie brilliant blue
reagent every 2 weeks. Fresh urine was collected by performing abdominal massage. The frequency of proteinuria (percent of individual mice with
urine protein 300 mg/dl) was calculated. C, Blood urea nitrogen (BUN) and serum creatinine levels were determined every 2 months, and BUN
levels at 12 months were significantly lower in the human AD-MSCtreated group than in the control group. D, Antidouble-stranded DNA antibody
(anti-dsDNA Ab) concentrations were measured by enzyme-linked immunosorbent assay. Data from A and B were analyzed using Kaplan-Meier
curves and a log rank test. Data from C and D were analyzed using the Mann-Whitney U test. Values in C and D are the mean SEM. In A and
C, P 0.05 versus control mice. In D, P 0.05 versus human AD-MSCtreated mice.
246 CHOI ET AL
group was 0.00633 0.00102, and that in the human
AD-MSCtreated group was 0.00488 0.00054; thus,
the spleen index was 0.77.
The number of spleen cells from the mice at age
62 weeks was 11.11 10
7
0.44 10
7
in the control
group (n 3) and 6.49 10
7
0.85 10
7
in the human
AD-MSCtreated group (n 6). Thus, in this animal
model of SLE, spleen cell numbers were significantly
reduced by human AD-MSC transplantation (P 0.008
versus the control group).
BUN, serum creatinine, and anti-dsDNA antibody
levels. To investigate kidney function and the level of
autoantibodies, we measured BUN, serum creatinine,
and anti-dsDNA antibodies. Anti-dsDNA antibodies
and BUN at 12 months were significantly lower in the
human AD-MSCtreated group than in the control
group (P 0.046 and P 0.047, respectively, by
Mann-Whitney U test) (Figures 1C and D).
Cytokine levels in spleen cell culture supernatants.
We examined the capacity for cytokine production in
spleen cells and compared the levels between the
groups. GM-CSF, IL-2, and IL-4 levels in the culture
supernatants of spleen cells at 10 months were signifi-
cantly higher in the human AD-MSCtreated group
than in the control group (P 0.046, P 0.009, and P
0.001, respectively) (Table 1). GM-CSF and IFN levels
in the culture supernatants of spleen cells stimulated
with Con A were significantly higher in the human
AD-MSCtreated group than in the control group (P
0.003 and P 0.007, respectively) (Table 1).
Cytokine levels in sera. The levels of cytokines in
the serum were measured at different time points by
ELISA to examine exactly which cytokines changed
significantly after human AD-MSC transplantation. As
shown in Table 1, serum levels of GM-CSF, IL-4, and
IL-10 in the human AD-MSCtreated mice at age 16
weeks (4 months), serum levels of IL-4 and IL-10 in
the human AD-MSCtreated mice at age 32 weeks
(8 months), and serum levels of GM-CSF in the human
AD-MSCtreated mice at age 48 weeks (12 months)
were significantly higher than those in the control
mice at the same time point. Data were also analyzed
by repeated-measures analysis of variance (General
linear model; SPSS). The time elapsed (ages 4 months,
8 months, and 12 months) had a significant influence
on levels of GM-CSF (P for time 0.028 by multi-
variate test) and IL-4 (P for time 0.011 by multivariate
test), and there was also an effect of interaction be-
tween time elapsed and group (administration of
human AD-MSCs) on levels of IL-4 (P for time-by-
group interaction 0.011 by multivariate test). There
was a difference between the groups, which was due to
different mean levels of GM-CSF, IL-4, and IL-10 (P for
Table 1. Cytokine levels in spleen cell culture supernatants and sera from (NZB NZW)F
1
mice after transplantation of human AD-MSCs*
Control mice
Human AD-MSC
treated mice P
Culture supernatants, pg/ml
GM-CSF 3.10 3.10 (3) 42.35 13.34 (3) 0.046
IL-2 0.81 0.81 (3) 63.24 13.05 (3) 0.009
IL-4 0.00 0.00 (3) 16.78 2.14 (3) 0.001
GM-CSF after Con A stimulation 30.98 19.18 (3) 226.72 24.83 (3) 0.003
IFN after Con A stimulation 478.74 245.46 (3) 1,805.59 77.00 (3) 0.007
IL-4 after Con A stimulation 11.18 10.48 (3) 26.69 2.98 (3) 0.228
Sera, pg/ml
GM-CSF at 4 months 0 0 (15) 17.9 7.3 (15) 0.017
GM-CSF at 8 months 43.7 25.2 (11) 215.4 72.2 (15) 0.172
GM-CSF at 12 months 0 0 (11) 28.7 11.2 (13) 0.025
IL-4 at 4 months 1.2 0.2 (15) 91.6 31.9 (15) 0.001
IL-4 at 8 months 2.1 1.1 (12) 69.1 27.8 (15) 0.002
IL-4 at 12 months 1.2 0.2 (11) 11.4 4.5 (13) 0.087
IL-10 at 4 months 13.9 13.9 (15) 2,881.7 1,184.3 (15) 0.001
IL-10 at 8 months 120.2 92.2 (12) 953.4 569.4 (15) 0.018
IL-10 at 12 months 44.7 20.0 (11) 207.5 92.3 (13) 0.085
* Values are the mean SEM (number of samples). AD-MSCs adipose tissuederived mesenchymal stem cells; GM-CSF granulocyte
macrophage colony-stimulating factor; IL-2 interleukin-2; IFN interferon-; Con A concanavalin A.
By t-test for culture supernatants and by Mann-Whitney U test for sera.
Determined in mice at age 10 months.
LONG-TERM SERIAL AD-MSC TRANSPLANTATION FOR SLE 247
between-subject effects, by group: 0.017 for GM-CSF,
0.001 for IL-4, 0.009 for IL-10).
IFN levels in sera. Since IFN may prove to
be the chief cytokine involved in SLE (21), we mea-
sured IFN levels in sera obtained from 2 groups of
mice at ages 4, 8, and 12 months. With the commer-
cial ELISA kit used (detection range 12.5400 pg/ml),
serum IFN levels were too low to detect except in
1 sample obtained from a control mouse at age 12
months (20.1 pg/ml).
Figure 2. Hematoxylin and eosin (H&E), periodic acidSchiff (PAS)
reagent, and Massons trichrome staining of kidneys obtained from mice
at age 16 weeks (before disease onset) and at 40 weeks (after disease
onset). A, H&E staining. Before disease onset, kidneys from the control
group and the human adipose tissuederived mesenchymal stem cell
(human AD-MSC; hATMSC)treated group were normal in appearance.
After disease onset, kidneys from the human AD-MSCtreated mice
without proteinuria appeared normal, whereas extensive mesangial matrix
deposition and crescent formation was observed in kidneys from the
control mice with severe proteinuria. B, PAS staining. After disease onset,
pink material was seen in the kidney tubules of control mice, which
indicated proteinuria. C, Massons trichrome staining. After disease onset,
a blue color was seen in the kidneys of control mice, which indicated
fibrosis. Original magnification 400.
Figure 3. Immunofluorescence of kidneys. After incubation at room
temperature with fluorescein isothiocyanate (FITC)conjugated goat
anti-mouse IgG (1:200) or FITC-conjugated goat anti-mouse C3
(1:100) for 1 hour and then washing in phosphate buffered saline,
slides were mounted, stained with DAPI, and examined by confocal
laser scanning microscopy. A, IgG deposition in kidney. B, C3 depo-
sition in kidney. Note the striking reduction of IgG and C3 deposits in
the group treated with human adipose tissuederived mesenchymal
stem cells (human AD-MSCs; hATMSC). At age 40 weeks, the
intensity of the fluorescence of IgG or C3 deposits was very severe
(from 3 to 4) in kidneys from control mice but was milder (1) in
kidneys from human AD-MSCtreated mice. Original magnification
400 in A; 200 in B. () negative control (before the onset of
disease).
248 CHOI ET AL
Proportion of Treg cells. Because the serum level
of IL-10 was significantly higher in the human AD-
MSCtreated group than in the control group, we com-
pared the proportion of Treg cells between the groups.
The mean proportion of CD4CD25FoxP3 cells in
the spleen was higher in the human AD-MSCtreated
group than in the control group. However, the difference
was not statistically significant (mean SEM 0.92
0.13 versus 0.43 0.18; P 0.095). The proportion of
CD4FoxP3 cells in the spleen was significantly
greater in the human AD-MSCtreated group than in
the control group (11.0 0.4 versus 8.7 0.5; P
0.016).
Histologic staining results. At age 16 weeks, be-
fore the onset of disease, H&E staining showed that
Figure 4. Confocal microscopy examination of CM-DiIlabeled hu-
man adipose tissuederived mesenchymal stem cells (human AD-
MSCs; hATMSC). Three mice in the human AD-MSCtreated group
were administered human AD-MSCs that were fluorescence-labeled
using conjugated red fluorochrome Cell Tracker CM-DiI, for histo-
pathologic examination. After counterstaining with mounting medium
containing DAPI, the presence of CM-DiIlabeled cells was examined
by confocal microscopy. Human AD-MSCs labeled with the CM-DiI
red fluorescent tracker dye were mostly found in the spleen, and many
human AD-MSCs labeled with the dye were present in the kidney and
liver. There was little evidence of the dye in the lung and heart.
Original magnification 400.
Figure 5. Comparison of the effects of human adipose tissuederived
mesenchymal stem cells (human AD-MSCs) between the early stage
and advanced stage disease groups. Each mouse in the early stage
treatment group (hATMSC) was treated with 5 10
5
human AD-
MSCs/150 l of saline intravenously every 2 weeks from age 6 weeks
until age 54 weeks. Each mouse in the advanced stage treatment group
(hATMSC 8M) was treated with 2 10
6
human AD-MSCs/150 l of
saline intravenously every 2 weeks from age 32 weeks until age 42
weeks. Mice in the control group (n 29) were administered 150 l of
saline every 2 weeks from age 6 weeks until age 54 weeks (n 15) or
every 2 weeks from age 32 weeks until age 42 weeks (n 14). A fourth
group of mice was administered 50 mg/kg of cyclophosphamide
intraperitoneally 3 times per week from age 32 weeks for 10 weeks
(CTZ). Survival (A) and the progression of severe proteinuria (B) were
monitored. Data were analyzed using Kaplan-Meier curve and log
rank test. P 0.05 versus control mice from age 24 weeks to age
54 weeks.
LONG-TERM SERIAL AD-MSC TRANSPLANTATION FOR SLE 249
kidneys from the control and human AD-MSCtreated
groups were normal in appearance. After the onset of
disease, kidneys appeared normal in the human AD-
MSCtreated mice without proteinuria but showed
extensive mesangial matrix deposition and crescent for-
mation in the control mice with severe protein-
uria (Figure 2A). After disease onset, PAS staining
showed pink material in the kidney tubules of the
control mice, which indicated proteinuria (Figure 2B),
and Massons trichrome staining showed a blue color
in the kidneys of the control group, which indicated
fibrosis (Figure 2C).
Kidney immunofluorescence findings. The fluores-
cence intensity of IgG or C3 deposits in the kidneys
harvested from age 37 weeks to age 40 weeks was
evaluated (n 7 per each group). The mean SEM
mean fluorescence intensity of deposits of IgG (Figure
3A) or C3 (Figure 3B) was significantly higher in the
control group than in the human AD-MSCtreated
group (for IgG, 3.9 0.1 versus 1.9 0.5; P 0.006).
CM-DiIlabeled human AD-MSCs identified by
confocal microscopy. Human AD-MSCs labeled with
CM-DiI red fluorescent tracker dye were mostly
present in the spleen; however, many were evident in
the kidney and liver as well (Figure 4). There was little
evidence of fluorescence-labeled cells in the lung and
heart.
Experiment 2 findings. At age 54 weeks, 20.7%,
73.3%, 33.3%, and 78.6% of the control 1 and control 2
groups combined (the control group), the early stage
group, the advanced stage group, and the CYC-treated
group, respectively, had survived (Figure 5A). The sur-
vival rate was significantly higher and the incidence of
proteinuria was significantly lower in the early stage
human AD-MSC treatment and CYC treatment groups
than in the control group. However, the survival rate
and proteinuria incidence of the control group and the
advanced stage group (the group with advanced stage
disease treated with human AD-MSCs at 8 months) did
not differ significantly.
At age 48 weeks, 89.7%, 33.3%, 66.7%, and
14.3% of the control, early stage, advanced stage,
and CYC-treated groups, respectively, had severe
proteinuria (300 mg/dl) (Figure 5B). Although the
early stage group had a lower percentage of protein-
uria than did the advanced stage group at age 48 weeks,
rapid occurrence subsequently developed, and the early
stage group eventually reached the same incidence of
proteinuria (73.3%) as the advanced stage group at
54 weeks.
DISCUSSION
GVH disease is the most frequent complication
associated with the transplantation of allogeneic hema-
topoietic grafts (22). Spleen enlargement results from
proliferation of both the CD4 and CD8 T cell
populations. A spleen index of 1.3 is considered to
indicate a positive GVH reaction (20). The causes of
splenomegaly are diverseinflammation, hyperplasia,
congestion, and infection. Among these, inflammatory
splenomegaly develops in association with various infec-
tions or inflammatory processes and results from an
increase in the defense activities of the organ. The
demand for increased antigen clearance from the blood
may lead to increased numbers of reticuloendothelial
cells in the spleen and stimulate accelerated antibody
production with resultant lymphoid hyperplasia. In this
study, the spleen index was 0.77 and no clinical signs of
host immune response were observed. Splenomegaly is
one of the symptoms of SLE as well as a clinical sign of
adverse immune responses. Indeed, a comparison of the
spleen index and spleen cell count data between the
groups showed that transplantation of human AD-MSCs
did not worsen splenomegaly. However, IgG and IgM
antibodies against transplanted human AD-MSCs devel-
oped, which was probably the result of the humoral
response.
The survival rate was significantly higher in the
human AD-MSCtreated group, and the onset of pro-
teinuria was delayed by 8 weeks compared to the control
group. Considering that the mean lifespan of (NZB
NZW)F
1
mice is 3539 weeks, an 8-week delay (one-
fifth of their lifespan) in the onset of proteinuria is
meaningful and would be a clinically worthwhile im-
provement. Anti-dsDNA antibodies and BUN levels
were significantly lower in the human AD-MSCtreated
group at 12 months.
In this study, human AD-MSC transplantation
significantly increased serum levels of IL-4 and IL-10
and the proportion of CD4FoxP3 cells in the spleen.
Thus, CD4FoxP3 cells were thought to be involved
in the therapeutic mechanism of human AD-MSCs.
Ectopic FoxP3 expression in CD4 non-Treg cells is
sufficient to confer suppressor function both in vitro and
in vivo (2326), and FoxP3 is thus a very important
target for the development of an immunosuppressive
agent for both autoimmune and allergic diseases (27).
Recently, Choi et al reported that cell-permeable FoxP3
protein alleviates autoimmune disease associated with
inflammatory bowel disease and allergic airway inflam-
mation (27).
250 CHOI ET AL
Findings of preliminary studies by our group
indicated that the function of Treg cells (sorted by
CD4CD25 cells) was slightly reduced in diseased
(NZB NZW)F
1
mice compared to the normal mouse
strain (BALB/c), based on the suppression index of T
cell proliferation and GM-CSF and IL-10 levels in
supernatants from cultures (wells from 10
5
T effector
cells, 10
5
antigen-presenting cells, 10
5
Treg cells) (Choi
EW, et al: unpublished observations). Similar to our
results, Abe et al (28) reported that (NZB NZW)F
1
mice have few defects in the function of Treg cells, and
the failure of Treg cells to control disease (autologous T
cells) might be predominantly caused by extrinsic factors
such as cytokine milieu and costimulatory signals pro-
vided by antigen-presenting cells.
SLE exhibits many features of immunomodula-
tion, including both hyperactivity and deficiency of
the immune system (11). Ikehara et al reported that
older (NZB NZW)F
1
mice produced less IL-2 than
did young (NZB NZW)F
1
mice and that the older
mice did not generate cytotoxic T lymphocytes. IL-2
production increased significantly in (NZB NZW)F
1
mice that underwent bone marrow transplantation
and was comparable with levels in normal mice (1). This
is consistent with our finding of a marked restoration
of cytokine production in the spleen cells of the human
AD-MSCtreated group. Aging is associated with a
progressive decline in the T cellmediated immune
response, and whole CD4CD25 T cells from aged
mice showed a diminished proliferative response (29).
SLE patients die not only from renal failure but also
from opportunistic infections attributable to immuno-
suppression. Treatment with immunosuppressive agents
such as cyclosporin A, azathioprine, CYC, and steroid
hormones exerts suppressive effects on T cells and on
other immunologic functions. However, our results showed
that human AD-MSC treatment not only restored CD4
FoxP3 cells, which have an immunosuppressive func-
tion, but also markedly restored cytokine production and
modulated both the hyperactivity and deficiency of the
immune system. The results indicate that human AD-
MSC transplantation can restore immune homeostasis.
Human AD-MSC transplantation delayed the progres-
sion of proteinuria and death but could not correct
severe proteinuria.
Necropsy was performed on all of the animals
after death, and no tumors were observed on gross
examination. Serially sectioned slides from the spleen
and kidney were microscopically examined, and again
no tumors were observed. Further, no tumors were
found in the 3 animals from each group in which
microscopic examination of the liver, lung, and heart was
performed. In a comparison between the early stage and
advanced stage treatment groups, the early stage treat-
ment group showed better results (higher survival rate
and less proteinuria) than the advanced stage treatment
group.
At age 54 weeks, the early stage human AD-
MSCtreated group from experiment 2 had a survival
rate of 73.3%, while the human AD-MSCtreated group
from experiment 1 had a survival rate of 42.1%. This
discrepancy seems to have been due to the different
vendors (experiment 1 mice were from Japan SLC and
experiment 2 mice were from The Jackson Laboratory).
At age 40 weeks (up to age 4248 weeks), the
kidney histologic findings and urine protein levels were
significantly better in the human AD-MSCtreated
group than in the control group. However, with time, the
human AD-MSCtreated group also showed occurrence
of proteinuria and aggravation of disease.
In the study by Schena et al (12), (NZB
NZW)F
1
mice were treated with allogeneic C57BL/6J
mousederived BM-MSCs via tail vein injection at ages
27, 28, and 29 weeks. Proteinuria was evaluated at ages
2532 weeks. Histopathologic assessment of kidneys was
conducted at age 33 weeks. In vivo treatment with
BM-MSCs reduced glomerular immune complex depo-
sition, lymphocyte infiltration, and glomerular prolifer-
ation but did not affect the levels of anti-dsDNA anti-
bodies or proteinuria. This discrepancy with our results
could be related to the different sources of the MSCs
used in the treatments (allogeneic [murine source] ver-
sus xenogeneic [human source]) as well as to the differ-
ent treatment schedule. Our study had a long-term
monitoring period (from age 4 weeks to age 62 weeks).
MSCs inhibit antigen-dependent proliferation and dif-
ferentiation of follicular and marginal zone B cells to
plasma cells in vitro (12), and Hoyer et al demonstrated
that in (NZB NZW)F
1
mice, the number of splenic
antibody-secreting cells increases from age 1 month to
age 5 months and becomes stable thereafter (30). Thus,
serum autoantibodies in the (NZB NZW)F
1
mice
used in the study by Schena et al could have been
maintained by a pool of long-lived plasma cells gener-
ated in young mice and not affected by lately added
MSCs. However, the (NZB NZW)F
1
mice used in our
study were treated from age 6 weeks; thus, it seems that
MSCs can affect the number of splenic antibody-
secreting cells.
We have shown for the first time in a mouse
model of SLE that serial human AD-MSC transplanta-
tion at the early stage of disease has beneficial effects in
LONG-TERM SERIAL AD-MSC TRANSPLANTATION FOR SLE 251
the treatment of SLE. Additional studies of different
human AD-MSC treatment protocols are required to
determine optimal doses, frequencies, and timing for the
treatment of SLE.
ACKNOWLEDGMENTS
The authors thank the staff of the Laboratory Animal
Research Center, Samsung Biomedical Research Institute for
their technical support and assistance. The authors are grateful
to Kyung-Su Park and Mi-La Cho (Division of Rheumatology,
Department of Internal Medicine, The Catholic University of
Korea School of Medicine), Seoul, Republic of Korea) for
critical advice and to Su Won Chun (Animal Science, Univer-
sity of California, Davis) for editorial assistance.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Choi had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Choi, Hong.
Acquisition of data. Choi, Shin, S. Y. Park, J. H. Park, Kim, Yoon,
Kang, Ra.
Analysis and interpretation of data. Choi, Yoon.
ROLE OF THE STUDY SPONSOR
Samsung Biomedical Research Institute encourages academic
research by providing grants after review of study design. Samsung
Biomedical Research Institute had no control over the writing or
content of the manuscript or the decision to submit the manuscript for
publication.
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DOI 10.1002/art.33389
Clinical Images: Corneal melt in a woman with longstanding rheumatoid arthritis
The patient, an 83-year-old woman with a 35-year history of severe rheumatoid arthritis (RA), presented with blurred vision, pain,
and redness of the right eye, which had been present for 2 weeks. She had deforming arthritis (left) with low current disease activity
and positive anticyclic citrullinated peptide and antinuclear antibodies (ANAs). She was currently being treated with methotrexate
10 mg/week. She had received infliximab treatment from 2001 until the end of 2010, when the drug was discontinued due to a
respiratory tract infection. Ocular examination revealed ulcerative keratitis, which rapidly led to corneal perforation with iris
prolapse and loss of vision. The visual activity was 20/30 in the left eye, while with the right eye the patient could only count fingers
held 30 cm from her face. Slit lamp examination of the right eye showed a peripheral corneal melt (right) (white arrow) and
moderate conjunctival injection adjacent to the ulcer. A Wessely ring, which is thought to be an immunoprecipitation line in the
cornea caused by immune complexes and indicating an immune reaction to an infectious or noninfectious antigen, was present
(right) (black arrow). The adjacent sclera was not inflamed. Corneal melting was rapidly progressive, and the patient was treated
with antibiotics. She refused further surgical or immunosuppressive treatment. Corneal disease in RA may involve significant
inflammation in association with anterior scleritis, although it can occur in eyes with little scleral involvement. Peripheral ulcerative
keratitis occurs in 5% of RA patients, with a predominance in women. It is considered a manifestation of systemic vasculitis, which
may occur in patients with longstanding RA and positive ANA. Ulcerative keratitis results from collagen breakdown initiated by
significant inflammation in the corneal extracellular matrix in RA. It requires aggressive treatment. Antibiotics may be indicated for
a perforation that could lead to endophthalmitis. Surgical intervention and immunosuppressive agents such as pulse steroids or
cyclophosphamide have been used for the treatment of ulcerative keratitis; biologic agents (infliximab, adalimumab, rituximab) have
also been shown to be effective for ulcerative keratitis, in addition to controlling joint disease.
Chrisoula Iliou, MD
Nikolaos Anthis, MD
Niki Tsifetaki, MD
George Kitsos, MD
Paraskevi V. Voulgari, MD
University of Ioannina
Ioannina, Greece
LONG-TERM SERIAL AD-MSC TRANSPLANTATION FOR SLE 253