Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes.

Abstract

A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo.
NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined.
NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.
These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

Full text

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Congenic Mesenchymal Stem Cell Therapy Reverses
Hyperglycemia in Experimental Type 1 Diabetes
Mollie Jurewicz,1Sunmi Yang,1Andrea Augello,1Jonathan G. Godwin,1Robert F. Moore,1
Jamil Azzi,1Paolo Fiorina,1Mark Atkinson,2Mohamed H. Sayegh,1and Reza Abdi1
OBJECTIVE—A number of clinical trials are underway to test
whether mesenchymal stem cells (MSCs) are effective in treating
various diseases, including type 1 diabetes. Although this cell
therapy holds great promise, the optimal source of MSCs has yet
to be determined with respect to major histocompatibility com-
plex matching. Here, we examine this question by testing the
ability of congenic MSCs, obtained from the NOR mouse strain,
to reverse recent-onset type 1 diabetes in NOD mice, as well as
determine the immunomodulatory effects of NOR MSCs in vivo.
RESEARCH DESIGN AND METHODS—NOR MSCs were
evaluated with regard to their in vitro immunomodulatory func-
tion in the context of autoreactive T-cell proliferation and
dendritic cell (DC) generation. The in vivo effect of NOR MSC
therapy on reversal of recent-onset hyperglycemia and on immu-
nogenic cell subsets in NOD mice was also examined.
RESULTS—NOR MSCs were shown to suppress diabetogenic
T-cell proliferation via PD-L1 and to suppress generation of
myeloid/inflammatory DCs predominantly through an IL-6-depen-
dent mechanism. NOR MSC treatment of experimental type 1
diabetes resulted in long-term reversal of hyperglycemia, and
therapy was shown to alter diabetogenic cytokine profile, to
diminish T-cell effector frequency in the pancreatic lymph nodes,
to alter antigen-presenting cell frequencies, and to augment the
frequency of the plasmacytoid subset of DCs.
CONCLUSIONS—These studies demonstrate the inimitable
benefit of congenic MSC therapy in reversing experimental type
1 diabetes. These data should benefit future clinical trials using
MSCs as treatment for type 1 diabetes. Diabetes 59:3139–3147,
2010
M
appreciation for the morbidity associated with immuno-
suppression. MSCs have been demonstrated to exhibit
profound immunomodulatory effects in vitro and in vivo,
and these immunomodulatory capabilities have been
shown to be exerted through both direct contact and
production of soluble markers (1–4). Moreover, upregula-
esenchymal stem cell (MSC) therapy has in
recent years emerged as a promising treat-
ment modality for diseases with immune eti-
ology,particularlygiventheincreasing
tion of B7-H1/PD-L1 by IFN-? has been shown to play a
central role in the immunosuppressive properties of MSCs
via direct contact with activated T-cells (5,6). In vitro
studies have also demonstrated the ability of MSCs to
regulate the function of T-cell effector pathways through
promotion of regulatory dendritic cell (DC) generation,
due to MSC-modulated alteration of DC cytokine profiles
as evidenced by increased production of regulatory cyto-
kines such as IL-10 and reduction of inflammatory cyto-
kines including IFN-?, IL-12, and TNF-?, thereby inducing
a more anti-inflammatory or tolerant DC phenotype (7,8).
These immunomodulatory effects as well as an extensive
capacity for in vitro expansion of MSCs have prompted the
launch of numerous clinical trials (1). MSC therapy has
yielded promising results in the treatment of graft versus
host disease (GVHD) as well as in the resolution of
Crohn’s disease-associated fistulas, in stabilization of re-
fractory progressive multiple sclerosis, and in reversal of
multiorgan dysfunction in patients with systemic lupus
erythematosus (9–12). However, although the therapeutic
value of MSCs for attenuating the autoimmune disorder
type 1 diabetes has logical potential, MSC treatment of this
particular disease remains largely unexplored. Trials using
MSC therapy in patients with type 1 diabetes are under-
way, yet these efforts have been initiated in the near
absence of preclinical data. In this regard, we and others
have recently demonstrated delayed onset of experimental
type 1 diabetes as well as reversal of recent-onset diabetes
in response to allogeneic MSC therapy, whereas in our
study administration of autologous diabetic MSCs showed
no beneficial effect (13,14). Our previous work also indi-
cated that congenic NOR MSCs imparted the greatest
benefit in preventing type 1 diabetes. The NOR/LtJ strain,
while resistant to insulitis due to the protective Idd alleles
(15),sharesthediabetogenicH2g7complexwiththeNOD/LtJ
strain. NOR mice are 85% homologous to spontaneously
diabetic NOD mice and are thus somewhat analogous to
nondiabetic siblings of type 1 diabetic patients. Here, we
sought to further examine the therapeutic efficacy of NOR
MSCs on reversal of recent-onset diabetes and to elucidate
the mechanisms by which NOR MSCs may act to ameliorate
diabetes pathogenesis.
RESEARCH DESIGN AND METHODS
Mice. NOR/LtJ, NOD/LtJ, and NOD.Cg-Tg(TcraBDC2.5)1DoiTg(TcrbBDC2.5)2Doi/
DoiJ(BDC2.5)werepurchasedfromtheJacksonLaboratories(BarHarbor,ME).All
procedures used in animal experiments were in accordance with the stan-
dards set forth in the Guidelines for the Care and Use of Laboratory Animals
at Harvard University.
MSC culture and differentiation. To generate NOR MSCs, bone marrow
mononuclear cells were isolated from the femurs and tibiae of NOR/LtJ mice.
Cells were seeded in tissue culture flasks at a concentration of 1 ? 106/cm2as
previously described (13) and were trypsinized at 80% confluence and
consolidated 2:1 until passage 4 (P4); from P4 to P6, cells were used for
injection, characterization, or in vitro assays. 7.5 ? 105MSCs/well were
From the1Transplantation Research Center, Children’s Hospital and Brigham
and Women’s Hospital, Harvard Medical School, Boston, Massachusetts;
and the2Departments of Pathology and Pediatrics, University of Florida
College of Medicine, Gainesville, Florida.
Corresponding author: Reza Abdi, rabdi@rics.bwh.harvard.edu.
Received 16 April 2010 and accepted 1 September 2010. Published ahead of
print at http://diabetes.diabetesjournals.org on 14 September 2010. DOI:
10.2337/db10-0542.
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ORIGINAL ARTICLE
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cultured for 48 h in 6-well plates with 0.05, 0.5, or 5 ng/ml recombinant murine
IFN-? (Peprotech, Rocky Hill, NJ). MSC differentiation to mesodermal tissues
was performed as previously described (13).
Flow cytometric analysis. MSCs were analyzed for surface expression of a
battery of markers at P4. Anti-mouse antibodies purchased from BD Bio-
sciences (San Jose, CA) included CD45, Ly-6A/E/Sca-1, CD44, CD90.2, and
CD73. Antibodies purchased from eBioscience (San Diego, CA) were CD105,
CD29, CD106/VCAM-1, PD-1, B7-H1/PD-L1, and B7-H2/PD-L2. For ex vivo
studies, the spleen and pancreatic lymph nodes (PLNs) were harvested and
subjected to analysis for CD4 effectors, CD8 effectors, and Tregs as previously
described (13). Splenocytes or DC cultures were also stained with CD11c,
CD11b, F4/80, CD45R/B220, CD40, CD80, CD86, and Ly-6c (BD Biosciences).
The biotinylated lineage panel was purchased from Miltenyi Biotec (Auburn,
CA), and cells were secondarily stained with streptavidin (BD Biosciences).
Immunohistochemistry. Adherent NOR MSCs were fixed on slides and
stained with hematoxylin–eosin for morphological evaluation, as previously
described (13). Immunohistochemistry was also performed as previously
described (13).
T-cell receptor-stimulated proliferation. NOD CD4?T-cells were isolated
by magnetic bead separation using CD4 microbeads (Miltenyi Biotec). 1 ? 105
CD4?T-cells were stimulated with 1 ?g/ml anti-mouse CD3 and anti-mouse
CD28 (BD Biosciences) alone or in combination with 1 ? 104, 2 ? 104, or 4 ?
104control or IFN-?-challenged (0.05, 0.5, or 5 ng/ml for 48 h) NOR MSCs or
NOR splenocytes in 96-well plates for 48 h, followed by pulsing with 1 ?Ci
tritiated thymidine (Perkin Elmer, Waltham, MA) for 16 h. Tritium uptake was
assessed using a MicroBeta FilterMate-96 Harvester and a 1,450 MicroBeta
TriLux (both from Perkin Elmer).
Autoreactive T-cell proliferation. BDC2.5 CD4?cells were extracted from
isolated splenocytes using magnetic bead separation (Miltenyi Biotec, Auburn,
CA), and the BDC2.5 autoreactive T-cell assay was performed as previously
described (13). In experiments with siRNA knockdown, PD-L1 or nontargeting
pool siRNA were added to MSCs using the Accell platform (Dharmacon,
Lafayette, CO) for 3 days prior to culturing with BDC2.5 T-cells, NOD DCs, and
BDC2.5 islet peptide, followed by pulsing with 1 ?Ci tritiated thymidine as
above.
ELISPOT. The autoreactive T-cell assay was performed as above using NOR
MSCs in an ELISPOT assay as previously described (16). ELISPOT kits (BD
Biosciences) to assess IFN-? and IL-6 production were used according to the
manufacturer’s instructions.
Luminex and ELISA. To assess cytokine production of murine serum and
culture supernatant samples, a 21-plex cytokine kit (Millipore, St. Charles,
MO) was used according to the manufacturer’s instructions and as previously
described (16). To assess production of IFN-?, M-CSF, and Flt3L, murine
ELISA kits (R&D Systems, Minneapolis, MN) were used according to the
manufacturer’s instructions.
DC culture. Bone marrow-derived NOD DCs were generated as previously
described (16). In DC and MSC coculture, 1 ? 105NOR MSCs were plated at
day 0 of NOD DC culture. For blocking studies, 10 ?g/ml anti-IL-6 (eBio-
science) was added at day 0 and replaced on days 3 and 6 at the same time as
media additions or changes.
Giemsa staining. Cytospins of DC cultures at day 8 were stained with Giemsa
stain (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instruc-
tions, and images were obtained using a Nikon TE300 system.
Reversal studies. Female NOD mice were monitored beginning at 10 weeks
of age, and on day 2 of hyperglycemia (?240 mg/dl), a sustained-release
insulin pellet (LinBit, LinShin Canada, Inc., Ontario, Canada) was placed
subcutaneously into the dorsum. The initial MSC injection (1 ? 106cells i.v.)
was injected within 24 h of pellet placement, and 1 ? 106NOR MSCs were
injected intravenously twice per week thereafter for 4 weeks. Normoglycemia
was maintained as needed during the last two weeks of treatment by 250 ng/dl
insulin (Lantus, Sanofi-Aventis, Bridgewater, NJ). Mice were monitored daily
by measuring blood glucose until the time of sacrifice, and measurements
were performed by tail bleeding according to National Institutes of Health
guidelines.
RESULTS
NOR MSC phenotype is consistent with mesenchymal
lineage. NOR MSCs were evaluated by immunohisto-
chemical and flow cytometric analysis for their expression
of classical MSC markers as well as costimulatory mole-
cules. Cultured cells were shown to be positive for the
MSC markers CD29, CD105, and CD44 but were negative
for the hematopoietic lineage-restricted marker CD34 after
immunohistochemical staining (Fig. 1A). Surface staining
revealed substantial expression of CD29, CD44, and
CD105, with moderate expression of CD73 and Sca-1, and
MSCs were found to be negative for the leukocyte antigen
CD45 (Fig. 1B). Our NOR MSCs were additionally found to
be capable of differentiating into cells of mesodermal
H&E CD34
CD29
CD44
CD105
A
B

Control medium Differentiation medium
Osteogenesis Chondrogenesis
CD29 CD44 CD45 CD73
CD106 Sca-1 CD90.2 CD105
100
101
102
CD29
103
104
0
20
40
60
80
100
% of Max
80.6±4.8%
100
101
102
CD44
103
104
0
20
40
60
80
100
% of Max
54.2±5.8%
100
101
102
103
104
AA CD45
0
20
40
60
80
100
% of Max
1.53±1.2%
100
101
102
CD73
103
104
0
20
40
60
80
100
% of Max
23.5±2.4%
100
101
102
103
104
CD90.2
0
20
40
60
80
100
% of Max
3.7±0.31%
100
101
102
CD105
103
104
0
20
40
60
80
100
% of Max
76.1±5.5%
100
101
102
CD166
103
104
0
20
40
60
80
100
% of Max
19.13±7.68%
100
101
102
Sca-1
103
104
0
20
40
60
80
100
% of Max
29.3±3.0%
C
FIG. 1. Characterization of NOR MSCs. A: Immunohistochemical staining of NOR MSC cultures demonstrates fibroblast cell morphology by
hematoxylin–eosin staining, substantial expression of the MSC markers CD44 and CD105, moderate expression of CD29, and lack of expression
for the hematopoietic stem cell marker CD34. B: Flow cytometric analysis of NOR MSC P4 cultures (n ? 5, data shown as mean ? SEM) shows
abundant expression of the classical MSC markers CD29, CD44, and CD105, while MSCs were negative for the hematopoietic lineage-restricted
markers CD45 and CD90.2. Sca-1, CD73, and VCAM (CD106) were expressed at moderate levels. C: NOR MSCs were shown to undergo
osteogenesis and chondrogenesis after exposure to differentiation factors. (A high-quality digital representation of this figure is available in the
online issue.)
MSC THERAPY FOR T1D
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Page 3

lineage (Fig. 1C). These data confirm that our cultured
NOR MSCs are phenotypically and lineally mesenchymal
as well as functionally multipotent.
NOR MSCs suppress autoantigen-specific and non-
specific T-cell proliferation. To further characterize
NOR MSCs, we performed cytokine studies of NOR MSC
cultures, which demonstrated the presence of various
cytokines, with IL-6 most notable in its production (n ? 4,
Fig. 2A). As MSCs are defined by their immunomodulatory
ability as well as their surface marker profile and multipo-
tent potential, we added increasing numbers of NOR MSCs
previously challenged with increasing concentrations of
IFN-? to an anti-CD3-/anti-CD28-stimulated proliferative
assay using NOD CD4?T-cells to assess the capacity of
NOR MSCs to suppress T-cell proliferation. NOR MSCs
potently inhibited T-cell receptor (TCR)-stimulated prolif-
eration in a dose-dependent manner (Fig. 2B, n ? 5, P ?
0.027 for indicated conditions), and pretreatment with
IFN-? enhanced suppression of proliferation dose-depen-
dently. To examine whether NOR MSCs are able to spe-
cifically suppress autoreactive T-cells, MSCs were added
to an autoreactive T-cell assay, in which isolated BDC2.5
CD4?T-cells, or H2g7-restricted diabetogenic T-cells, are
cocultured with NOD DCs and BDC2.5 islet peptide. As
shown in Fig. 2C, NOR MSCs significantly suppressed
autoreactive T-cell proliferation, as assessed by carboxy-
fluorescein succinimidyl ester (CFSE) dilution and calcu-
lation of proliferation index (n ? 5, P ? 0.047). Addition of
BA
D
E
F
HI
Control +NOR MSC
CFSE
CD4
PD-1
0 0.05 0.5 5
0
10
20
30
40
50
60
70
80
90
IFN-γ concentration (ng/ml) IFN-γ concentration (ng/ml) IFN-γconcentration (ng/ml)
PD-L1
0 0.050.55
PD-L2
0
0.050.5 5
0124
0
10000
20000
30000
40000
50000
60000
70000
Control
0.05 ng/ml IFN-γ
0.5 ng/ml IFN-γ
5 ng/ml IFN-γ
*
*
*
Number of MSC added (x104)
Number of cells added (x104)
0
20
40
60
80
100
500
1500
2500
3500
IFN-γ
IFN-α
TNF-α
IL-1β
IL-6
IL-9
IL-10
IL-13
IL-15
FIt3L
M-CSF
IL-12(p70)
pg/ml
% positive cells
0
10
20
30
40
50
60
70
80
90
% positive cells
0
10
20
30
40
50
60
70
80
90
0
10
20
30
40
50
60
70
80
90
% positive cells
% PD-L1 expression
3H-thymidine incorporation
3H-thymidine incorporation
3H-thymidine incorporation
PD-L1 copies/copies GAPDH
Frequency of cytokine-producing cells
Proliferation index
0124
0
100000
200000
300000
400000
500000
NOR MSC
NOR splenocytes
*
*
*
C

G
BaselineNOR MSC
NOR MSC post co-culture
*
0
2500
5000
7500
Control
+NT siRNA-MSC
+PD-L1 siRNA-MSC
*
0.000
0.001
0.002
0.003
0.004
0.005
0.006
NT siRNA-MSC
PD-L1 siRNA-MSC
*
IFN-IL-6
0
1000
2000
3000
Control
+2x104 NOR MSC
*
*
100
101
102
CFSE
103
104
100
101
102
103
104
CD4
100
101
102
CFSE
103
104
100
101
102
103
104
CD4
Ctrl +NOR MSC
0
1
2
3
4
5
6
*
FIG. 2. NOR MSC suppression of diabetogenic autoreactive T-cells via PD-L1. A: Cytokine studies of NOR MSC cultures revealed considerable
levels of IL-6, with M-CSF and Flt3L produced at lesser but substantial levels in comparison to other growth factors (n ? 4). B: NOR MSCs
suppressed TCR-stimulated proliferation of NOD CD4?cells in a dose-dependent manner, in which increasing numbers of IFN-?-stimulated NOR
MSCs were added to 1 ? 105NOD CD4?cells in the presence of 1 ?g/ml anti-CD3 and anti-CD28 (n ? 5, P < 0.027 for 1 ? 104MSCs, P < 0.0001
for 2 ? 104and 4 ? 104MSCs), and IFN-? challenge enhanced the suppressive effect of NOR MSCs. (C) 2 ? 104NOR MSCs were shown to
significantly reduce autoreactive T-cell proliferation (n ? 5, P ? 0.047), as evaluated by CFSE dilution and calculation of proliferation index
when added to a BDC2.5 autoreactive assay containing BDC2.5 CD4?T-cells, NOD DCs, and 100 ng/ml BDC2.5 islet peptide. D: IFN-? production
was similarly suppressed in the presence of 2 ? 104NOR MSCs by ELISPOT in the BDC2.5 autoreactive assay (n ? 5, P ? 0.0024), while IL-6 levels
were enhanced (P ? 0.0005). E: Addition of 1, 2, or 4 ? 104NOR splenocytes had no suppressive effect on anti-CD3/-CD28 T-cell proliferation as
compared with addition of identical numbers of NOR MSCs (n ? 4, P < 0.0001 for NOR MSCs, not significant for NOR splenocytes). F: NOR MSCs
stimulated with 0.05, 0.5, or 5 ng/ml recombinant murine IFN-? show dose-dependent upregulation of PD-L1 expression by flow cytometric
analysis (n ? 3, P < 0.008 for 0.5 and 5 ng/ml), a minor increase in PD-1 (p ? not significant), and no increase in expression of PD-L2. G: NOR
MSCs after coculture with BDC2.5 CD4?T-cells, NOD DCs, and BDC2.5 peptide exhibited marked upregulation of PD-L1 expression at 72 h by
flow cytometric analysis (n ? 5, P < 0.0001). H: siRNA knockdown of PD-L1 in MSCs abrogated the suppressive effect observed on autoreactive
T-cell proliferation when 2 ? 104MSCs were added (n ? 6, P ? 0.0034 for control versus nontargeting siRNA, not significant for control versus
PD-L1 siRNA for representative experiment shown), and (I) gene expression analysis of MSCs demonstrated efficient suppression of PD-L1
transcripts in response to siRNA treatment (n ? 4, P ? 0.016). Experiments were performed between 3 and 6 times, and data are displayed with
means and SEM.
M. JUREWICZ AND ASSOCIATES
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NOR MSCs was also able to suppress production of IFN-?
by BDC2.5 T-cells, which has been described previously as
a characteristic of proinflammatory autoreactive T-cells
(17), and the production of IL-6 was shown to be enhanced
in the presence of NOR MSCs (Fig. 2D, n ? 6, P ? 0.0024
for IFN-?, P ? 0.0005 for IL-6). These data demonstrate
that not only are NOR MSCs capable of suppressing T-cell
proliferation stimulated through the TCR via anti-CD3/-
CD28 stimulation but that, in the specific context of
diabetogenic T-cells, NOR MSCs have a potent immuno-
modulatory effect on autoreactive T-cell proliferation and
production of IFN-?. NOR splenocytes were also tested for
their suppressive ability in all of the assays above to assess
the effects of congenicity, and no effects on anti-CD3/
-CD28-stimulated T-cell proliferation, autoreactive T-cell
proliferation, or autoreactive T-cell IFN-? production were
observed (Fig. 2E [n ? 4, P ? 0.0001 for NOR MSCs, not
significant for NOR splenocytes], and data not shown,
respectively).
Suppression of autoreactive T-cell proliferation by
NOR MSCs is mediated by PD-L1. Recent studies have
highlighted the central role of the negative costimulatory
PD-1 pathway ligand PD-L1 in suppressing the prolifera-
tion of autoreactive T-cells and in consequently halting the
progression of type 1 diabetes in NOD mice (18–21). Given
these data, we hypothesized that inhibition by PD-L1
serves as a mechanism by which NOR MSCs exert their
immunomodulatory effects on diabetogenic T-cells. We
thus examined the expression of PD-L1 on resting NOR
MSCs and after activation with IFN-? to evaluate expres-
sion of components of the PD-1 pathway in the context of
inflammation. Although NOR MSCs expressed PD-L1 at
insubstantial levels at baseline, stimulation with increas-
ing doses of recombinant murine IFN-? resulted in prodi-
gious upregulation of PD-L1 expression in a dose-
dependent manner, while PD-1 and PD-L2 expression
underwent modest changes after IFN-? challenge (Fig. 2F,
n ? 3, P ? 0.0002 for PD-L1 expression of baseline
compared with 5 ng/ml IFN-?). We also investigated
whether addition of unstimulated NOR MSCs to the
BDC2.5 autoreactive assay would result in upregulation of
PD-L1, as substantial IFN-? was detected in the autoreac-
tive T-cell experiments (Fig. 2D); indeed, coculture of
NOR MSCs with autoreactive T-cell assay components
resulted in considerable NOR MSC expression of PD-L1
(Fig. 2G, n ? 5, P ? 0.0001), indicating that an autoreactive
inflammatory milieu induces marked upregulation of
PD-L1 on MSCs. To determine the functionality of PD-L1
expression in the suppression of autoreactive T-cell pro-
liferation by NOR MSCs, we treated NOR MSCs with
PD-L1 siRNA prior to adding MSCs to our autoreactive
T-cell assay. Indeed, treatment of NOR MSCs with PD-L1
siRNA abrogated the suppressive effect of MSCs on
BDC2.5 T-cell proliferation (Fig. 2H, n ? 6; P ? 0.0034 for
control versus nontargeting siRNA, not significant for
control versus PD-L1 siRNA for representative experiment
shown). Addition of greater numbers of PD-L1 siRNA-
treated NOR MSCs (4 and 8 ? 104; n ? 4) to the
autoreactive T-cell assay did not result in restoration of
the suppressive effect of NOR MSCs (data not shown),
perhaps due to the fact that MSCs have been shown to
exert their immunomodulatory effects through cell con-
tact. To confirm that our siRNA treatment resulted in
efficient knockdown of PD-L1 expression, siRNA-treated
MSCs were examined for PD-L1 copy number by real-time
PCR. As shown in Fig. 2G, PD-L1 transcripts were signif-
icantly decreased by PD-L1 siRNA treatment of MSCs (n ?
4, P ? 0.016), demonstrating effective suppression of
PD-L1. These data indicate that PD-L1 plays a significant
role in the specific context of MSC-mediated suppression
of diabetogenic T-cells.
NOR MSCs inhibit in vitro DC differentiation via
IL-6. Aberrant DC development and imbalance in anti-
gen-presenting cell (APC) subsets have been reported to
be responsible for the lack of tolerance mechanisms in
NOD mice (22,23). To examine the effect of NOR MSCs
on DC generation, we performed in vitro studies of NOR
MSCs and NOD DCs using an established method of DC
culture (16,24). Because of the substantial production of
IL-6 in our NOR MSC cultures (Fig. 2A) as well as the
fact that IL-6 has been demonstrated to both suppress
and alter DC differentiation (2,7,25,26), we performed
IL-6 blocking studies in conjunction with coculture of
MSCs and DCs. The presence of MSCs strikingly re-
duced CD11c and CD11b expression of DCs, so that
the predominant population induced by MSCs was
CD11clowCD11blow(Fig. 3A, CD11c?CD11b?cells ?
40.7 ? 2.6% and 22.1 ? 2.4% for control and NOR
MSC-treated, respectively, n ? 5, P ? 0.0007). Addition
of anti-IL-6 somewhat abrogated the change in pheno-
type observed in DC coculture with NOR MSCs (Fig. 3A,
n ? 4, not significant in comparison to control [?/?]
and in comparison to MSCs alone [?/?]). Of note,
costimulatory molecule expression in the CD11c?pop-
ulation was not found to be significantly different in the
presence or absence of MSCs (data not shown). The
CD11b?population was also evaluated with respect
to Ly-6c expression, as both CD11b?Ly-6chighand
CD11b?Ly-6cintcells have been demonstrated to be
inflammatory monocytes recruited to sites of inflamma-
tion (22,27). Coculture with NOR MSCs resulted in
downregulation of both
CD11b?Ly-6cintpopulations (Fig. 3A, n ? 4, P ? 0.0053
and P ? 0.02, respectively), and this difference was
again abrogated by blockade of IL-6. We then assessed
the number of lineage-negative cells as a function of
progenitor frequency or lack of differentiation, and NOR
MSC treatment was shown to increase lineage-negative
cells as well as increase the expression of Sca-1 within
the lineage-negative population (Fig. 3B, n ? 4, P ?
0.004 and P ? 0.0085, respectively). Treatment with
anti-IL-6 was somewhat efficacious in abrogating the
suppression of differentiation observed in response to
MSC coculture, suggesting that other factors may be
involved in the effect of MSCs on DC differentiation. We
therefore examined the supernatants of DC and MSC
cocultures at day 8 for cytokine production. As shown in
Fig. 3C, coculture with MSCs significantly enhanced IL-6
levels (n ? 4, P ? 0.0074), and addition of IL-6 blocking
antibody efficiently suppressed IL-6 production. More-
over, Flt3L and M-CSF production was increased in
response to MSCs (n ? 4, P ? 0.03 and P ? 0.04,
respectively), and IL-6 blockade had no effect on in-
creased levels of these cytokines (P ? 0.013 and P ?
0.018, respectively, in comparison to DCs alone, Fig.
3C). Conversely, production of TNF-?, a growth factor
involved in the maturation of DCs as well as a cytokine
secreted by mature DCs (16,28), was reduced in the
presence of MSCs (Fig. 3C, n ? 4, P ? 0.0056), and
blocking of IL-6 resulted in abrogation of this effect. To
examine the morphology of DCs in response to MSCs,
we performed Giemsa staining of day 8 DC cultures and
the CD11b?Ly-6chigh
and
MSC THERAPY FOR T1D
3142 DIABETES, VOL. 59, DECEMBER 2010diabetes.diabetesjournals.org

Page 5

found that the nuclear-to-cytoplasmic ratio appeared to
decrease after coculture with MSCs, a feature com-
monly associated with earlier stages of differentiation
(29), and IL-6 blockade appeared to partially reverse
this effect (Fig. 3D). Examination of side scatter of DCs
by flow cytometric analysis revealed that coculture with
MSCs resulted in a dramatic reduction in the degree of
granularity (data not shown), again demonstrating a
lack of differentiation in response to MSCs (30). Addi-
tion of MSCs to plasmacytoid DC (pDC) cultures re-
sulted in enhanced pDC frequency, and this effect was
fully reversed by IL-6 blockade (supplementary Fig. 1
in the online appendix available at http://diabetes.
diabetesjournals.org/cgi/content/full/db10-0542/DC1),
as IL-6 has been previously demonstrated to be impor-
tant for pDC generation (31). Taken together, these data
demonstrate a marked effect of NOR MSCs on DC
phenotype, differentiation, and cytokine production,
which is in large part mediated by IL-6.
NOR MSCs efficiently reverse recent-onset experi-
mental autoimmune type 1 diabetes. Given our previous
data in which congenic NOR MSCs were found to be most
CD11b
CD11c
+MSC
-anti-IL-6 +anti-IL-6
Ly-6c
-anti-IL-6 +anti-IL-6
Lineage
-MSC
-anti-IL-6 +anti-IL-6
104
+MSC
-MSC
Sca-1
-anti-IL-6 +anti-IL-6
A
B
C
D
IL-6
*
0
100
200
300
- + - +
- - + +
MSC
anti-IL-6
Flt3L
*
0
10
20
30
40
50
60
70
*
- + - +
- - + +
MSC
anti-IL-6
M-CSF
*
0
100
200
300
- + - +
- - + +
MSC
anti-IL-6
*
TNF-α
0
10
20
30
40
50
60
70
80
- + - +
- - + +
MSC
anti-IL-6
*
-MSC
+MSC
-anti-IL-6 +anti-IL-6
CD11b
% CD11b+CD11c+ Cells
% lineage-negative cells
IL-6 production (pg/ml)
Flt3L production (pg/ml)
M-CSF production (pg/ml)
TNF-α production (pg/ml)
% lineage-negative Sca-1+ cells
% CD11b+Ly-6chigh Cells
% CD11b+Ly-6cint Cells
+CD11c+
0
10
20
30
40
50
60
70
- + - + MSC
- - + + anti-IL-6
*
Ly-6chigh
0
2
4
6
8
10
- + - + MSC
- - + + anti-IL-6
*
Ly-6cint
0
10
20
30
- + - + MSC
- - + + anti-IL-6
*
Lineage-negative
0
10
20
30
- + - + MSC
- - + + anti-IL-6
*
Sca-1+
0
10
20
30
40
50
60
70
- + - + MSC
- - + + anti-IL-6
*
FSC-H
CD11c
100
101
102
CD11b
103
104
100
101
102
103
104
CD11c
100
101
102
CD11b
103
104
100
101
102
103
104
CD11c
100
101
102
CD11b
103
104
100
101
102
103
104
CD11c
100
101
102
CD11b
103
104
100
101
102
103
104
CD11c
100
101
102
Ly-6c
103
104
100
101
102
103
104
CD11b
100
101
102
Ly-6c
103
104
100
101
102
103
104
CD11b
100
101
102
Ly-6c
103
104
100
101
102
103
104
CD11b
100
101
102
Ly-6c
103
104
100
101
102
103
104
CD11b
100
101
102
Sca-1
103
104
0
10
20
30
# Cells
100
101
102
Sca-1
103
104
0
5
10
15
20
# Cells
100
101
102
Sca-1
103
104
0
5
10
15
# Cells
100
101
102
Sca-1
103
104
0
2
4
6
# Cells
02004006008001000
FSC-H
100
101
102
103
104
Lineage
02004006008001000
FSC-H
100
101
102
103
Lineage
02004006008001000
FSC-H
100
101
102
103
104
Lineage
02004006008001000
FSC-H
100
101
102
103
104
Lineage
FIG. 3. MSC suppression of DC differentiation. A: Using an established model of DC generation from NOD bone marrow mononuclear cells,
coculture with NOR MSCs was shown to markedly reduce the CD11c?CD11b?population, so that the predominant cell phenotype was
CD11clowCD11blow(CD11c?CD11b?cells ? 40.7 ? 2.6% and 22.1 ? 2.4% for control and NOR MSC-treated, respectively, n ? 5, P ? 0.0007),
whereas treatment with anti-IL-6 in large part abrogated this effect (CD11c?CD11b?cells ? 30.9 ? 4.7%, not significant in comparison to control
[?/?] and MSCs alone [?/?]). Analysis of expression of Ly-6c in the CD11b?fraction demonstrated that coculture with NOR MSCs resulted in
a decrease in both the CD11b?Ly-6chighand CD11b?Ly-6cintpopulations (n ? 4, P ? 0.0053 and P ? 0.02, respectively), which was fully abrogated
by blockade of IL-6 (p ? not significant). B: The population of lineage-negative cells was evaluated in DC culture as a function of progenitor
frequency; coculture with MSCs increased the percentage of Lin?cells (n ? 4, Lin?cells ? 8.94 ? 0.87% and 13.73 ? 1.08% for control and NOR
MSC-treated, respectively, P ? 0.004), which was in part rescued by addition of anti-IL-6 (p ? not significant). Similarly, Sca-1 expression within
the lineage-negative population was markedly increased in the presence of MSCs (n ? 4, Lin?Sca-1?cells ? 6.98 ? 1.27% and 30.53 ? 6% for
control and NOR MSC-treated, respectively, P ? 0.0085). Although treatment with anti-IL-6 resulted in loss of significance of this effect, IL-6
blockade appeared to be incompletely effective in reducing Sca-1 expression in response to MSCs. C: Cytokine analysis of cocultures of DCs and
NOR MSCs demonstrated marked IL-6 production in the presence of MSCs (n ? 4, P ? 0.0074) as well as efficient blockade of IL-6 in response
to treatment with anti-IL-6. Both Flt3L and M-CSF levels were substantially increased in response to MSC coculture (P ? 0.03 and P ? 0.04,
respectively), and IL-6 blockade had no effect on these growth factors (P ? 0.013 and P ? 0.018, respectively, in comparison to DCs alone).
Conversely, TNF-? production was reduced in the presence of MSCs (P ? 0.0056), and anti-IL-6 treatment resulted in abrogation of this effect
(p ? not significant). D: Giemsa staining of DC culture cytospins demonstrated a lower nuclear/cytoplasmic ratio in response to coculture with
MSCs, and IL-6 blockade appeared to in large part abrogate this effect. Experiments were performed between 3 and 5 times, and data are
displayed with means and SEM. (A high-quality color representation of this figure is available in the online issue.)
M. JUREWICZ AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 59, DECEMBER 20103143

Page 6

effective in preventing onset of diabetes in the NOD mouse
model in comparison to autologous or allogeneic MSC treat-
ment (13), we sought to determine the efficacy of NOR MSCs
in reversing recent-onset hyperglycemia in NOD mice. Our
previous work demonstrated that reversal of hyperglycemia
in response to BALB/c MSC therapy, although effective, was
only temporary, perhaps due to eventual rejection of the
allogeneic cells (13). Using a treatment protocol identical to
that of our previous study, we observed reversal of recent-
onset hyperglycemia in eight of nine NOD mice treated with
NORMSCsatthe5-weekpointofpost-treatmentobservation
(Fig. 4A). Of note, only one mouse in the NOR MSC-treated
group exhibited blood glucose levels ? 600 mg/dl (* in Fig.
4A) at ?5 weeks into treatment. Whereas two NOD mice
treated with NOR MSCs succumbed to unexplained deaths
with no evidence of remission break, the other mouse was
killed. Although several weeks of observation suffices for
most reversal studies, the remaining five mice were observed
for an extended period to ensure that their reversal was
maintained (i.e., with no return to hyperglycemia). At 12
weeks after treatment, their average blood glucose measure-
ment was 222 ? 13.4 mg/dl. All NOD mice (both NOR
MSC-treated and controls) were provided a slow-release
insulin pellet to allow for a limited period of metabolic
recovery, yet in contrast to NOR MSC-treated mice, all
control NOD mice (n ? 9) redisplayed hyperglycemia almost
immediately after dissolution of the insulin pellet. Weekly
mean blood glucose measurements of treated mice were
significantly reduced compared with controls from 2 to 12
weeks; notably, eight of nine hyperglycemic untreated mice
died within 6 weeks after the onset of diabetes (Fig. 4B, P ?
0.001 for all time points, one surviving control is shown
beyond week 6.5). No treatment bias was present, as glucose
measurements at the initiation of treatment did not differ
between groups (Fig. 4B, P ? not significant). These data
demonstrate efficient and long-term reversal of recent-onset
hyperglycemia in response to NOR MSC therapy.
NOR MSC treatment augments regulatory cytokine
levels and induces regulatory DCs. To elucidate the
mechanisms by which reversal of recent-onset hypergly-
cemia occurred in response to NOR MSC therapy, we
performed serum cytokine studies after the completion of
MSC administration at days 0, 7, 14, and 21. As shown in
Fig. 5A, NOR MSC treatment resulted in increased circu-
lating levels of IL-6, IL-7, IL-10, and IL-12(p40) (n ? 3–5
samples, P ? 0.00065 where indicated). Of note, IL-12(p70)
levels were decreased in serum of NOR MSC-treated
mice, but the difference did not reach statistical signifi-
cance (data not shown). Flow cytometric analysis of the
spleen and PLNs of treated and control mice at 2 weeks
after the initiation of treatment demonstrated a reduction
in the populations of CD4?CD44highCD62Llowand CD8?
CD44highCD62Lloweffector T-cells (Fig. 5B, n ? 5, P ?
0.041 and P ? 0.0022 for CD4 and CD8 effectors,
respectively), while no effect on Tregs was detected.
In studies in which NOD mice were treated for 2
weeks with NOR MSCs and in which the proliferative
capacity of isolated CD4 and CD8 T-cells in response to
concanavalin A and anti-CD3/-CD28 stimulation was
examined, no differential results were observed in pro-
liferation of CD4 or CD8 T-cells isolated from spleno-
cytes of NOR MSC-treated or control mice (data not
shown). In light of our in vitro DC data, we ana-
lyzed the splenic APC populations in control and
NOR MSC-treated mice. CD11c single-positive and
CD11chighCD11b?cells were found to be reduced in
frequency in response to treatment with NOR MSCs
(Fig. 5C, n ? 5, P ? 0.004 and P ? 0.015, respectively).
Fewer macrophages, identified as F4/80?CD11c?, were
also found in NOR MSC-treated mice (Fig. 5C, n ? 5,
P ? 0.015). Conversely, the CD11clowCD11b?popula-
tion was increased after NOR MSC therapy (Fig. 5C, n ?
5, P ? 0.029). The CD11b?population also showed
reduced expression of Ly-6c after treatment with NOR
MSCs (Fig. 5C, n ? 3–5, P ? 0.042). Further analysis of the
CD11c single-positive DC population revealed a dramatic
increase in B220 expression in this subset in response to
NOR MSC treatment (Fig. 5C, n ? 5, P ? 0.0094), and pDCs
ofthisphenotypehavebeenshowntopromotetoleranceand
to delay the onset of diabetes (32).
A
B
12345678910 111213
0
100
200
300
400
500
600
700
Blood glucose
Blood glucose (mg/dL)
*
Control
NOR MSC-treated
Weeks
02345678910 1112
0
100
200
300
400
500
600
700
Control
NOR MSC-treated
*
*
*
*
*
*
*
*
*
*
*
Weeks
FIG. 4. NOR MSC therapy induces long-term reversal of recent-onset hyperglycemia. A: NOD mice were monitored beginning at 10 weeks of age,
and on day 2 of hyperglycemia (blood glucose > 240 mg/dl), an insulin pellet was inserted subcutaneously for maintenance of normal glycemia
during treatment. Mice were randomized to control or NOR MSC-treated groups; for NOR MSC treatment, 1 ? 106cells were injected
intravenously twice per week for 4 weeks, and blood glucose measurements were taken daily. Eight of nine NOD mice treated with NOR MSCs
exhibited reversal of diabetes. Five of six treated mice followed for 12 weeks maintained their reversal, whereas controls reverted to
hyperglycemia (>600 mg/dl) soon after dissolution of the insulin pellet. B: Means of cumulative blood glucose measurements demonstrate no
difference in level of hyperglycemia at days ?1 and 0 (p ? not significant), while weekly mean measurements beginning at week 2 after the
initiation of treatment show significant decreases in blood glucose in response to NOR MSC therapy (P < 0.001 for all from week 2 to week 12).
Data are displayed with means and SEM.
MSC THERAPY FOR T1D
3144DIABETES, VOL. 59, DECEMBER 2010diabetes.diabetesjournals.org

Page 7

DISCUSSION
The incidence of type 1 diabetes is steadily rising at a global
level (33–35), and the most common form of intervention
seeking to reverse the disease in recently diagnosed patients
has been that of immunosuppression through use of agents
such as anti-CD3. However, as immunosuppressive regimens
are commonly associated with acute morbidity, novel treat-
ments to reduce the burden of immunosuppression are in
dire need of development. MSC therapy is one such treat-
ment modality that, because of the considerable immuno-
modulatory effects of these cells, has shown promising
results in treating autoimmune diseases and has the potential
to serve as a component of combination therapy to reduce
immunosuppressive regimen morbidity (9–12). Whereas
MSCs are capable of differentiating into a number of mesen-
chymal cell lineages, hematopoietic stem cells are multipo-
tent stem cells that give rise to all cells in the blood and that
have been shown to have immunomodulatory roles as well;
indeed, hematopoietic stem cell transplantation in patients
with newly diagnosed type 1 diabetes has resulted in im-
proved ?-cell function (36). Clinical trials examining the
effects of MSC therapy have also been initiated for a multi-
tude of disorders, including type 1 diabetes. However, type 1
diabetes trials have been initiated with a paucity of preclin-
ical data, which are necessary to determine the type and
course of MSC therapy as well as to elucidate the mecha-
nisms by which MSCs exert their immunomodulatory effects.
We and others have previously demonstrated the benefit of
MSC therapy in the specific setting of type 1 diabetes
(13,14,37,38). Importantly, our previous work demonstrated
no therapeutic benefit of autologous MSCs in vivo using a
NOD mouse model (13). Conversely, allogeneic BALB/c MSC
treatment was efficient in the treatment of type 1 diabetes,
but reversal was short-lived, perhaps due to the eventual
rejection of the allogeneic cells. Our previous work also
indicated that the most significant preventative effect on
experimental type 1 diabetes occurred with congenic NOR
MSC treatment. Given these data, we sought to examine the
effect of NOR MSCs on reversal of hyperglycemia as well as
to elucidate the mechanisms responsible for NOR MSC
immunomodulation.
In this report, we first demonstrate that NOR MSCs are
functionally and lineally mesenchymal as well as confirm
their immunomodulatory function in suppressing nonspe-
cific TCR-stimulated proliferation. We next examined their
immunosuppressive ability in the specific context of auto-
reactive T-cell proliferation through use of the BDC2.5
autoreactive T-cell assay; NOR MSCs were shown to
potently suppress diabetogenic T-cell proliferation and
IFN-? production. Although NOR MSCs expressed PD-L1
A
B
CD11clowCD11b-CD45R/B220+
Control NOR MSC-td
104
F4/80
CD11c
IL-6
*
pg/ml
pg/ml
pg/ml
% expression
% expression
pg/ml
Day 0 Day 7 Day 14 Day 21
Day 0 Day 7 Day 14 Day 21Day 0 Day 7 Day 14 Day 21
Day 0 Day 7 Day 14 Day 21
0
25
50
75
100
125
*
IL-7
0
25
50
75
100
125
*
*
Control
NOR MSC-treated
CD11b
Ly-6c+CD11b+
Control NOR MSC-td
104
IL-10
0
50
100
150
200
250
*
*
IL-12(p40)
0
100
200
300
*
*
0
10
20
30
40
50
Control
NOR MSC-treated
Ly-6c+
CD11b+
pDC
*
*
0
10
20
Control
NOR MSC-treated
CD4 eff CD8 eff Tregs
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
CD11c+CD11chigh CD11clowF4/80+
CD11b+
CD11b+
*
*
*
*
100
101
102
CD11b
103
104
100
101
102
103
104
CD11c
100
101
102
CD11b
103
104
100
101
102
103
CD11c
100
101
102
103
104
CD45R/B220
100
101
102
103
104

100
101
102
103
104
CD45R/B220
100
101
102
103
104

100
101
102
F4/80
103
104
100
101
102
103
CD11c
100
101
102
F4/80
103
104
100
101
102
103
104
CD11c
CD11c
100
101
102
Ly-6c
103
104
0
50
100
150
200
# Cells
100
101
102
Ly-6c
103
104
0
100
200
300
400
500
# Cells
C
FIG. 5. NOR MSC therapy alters DC phenotype, cytokine profile, and effector cell frequency in vivo. A: Serum cytokine studies of NOR
MSC-treated and control mice were performed at days 0, 7, 14, and 21 after completion of our treatment protocol at 4 weeks (n ? 3–5 samples).
NOR MSC therapy increased circulating levels of IL-6 at days 7 and 14 (P < 0.0065), increased IL-7 levels at days 0 and 7 (P < 0.00021), increased
levels of IL-10 at days 0 and 7 (P < 0.0052), and increased levels of IL-12(p40) at days 0 and 7 (P < 0.00091). B: CD4 and CD8 effector cell
frequency (identified as CD44highCD62Llow) was reduced in the PLN of NOR MSC-treated mice, while no difference was detected in Treg frequency
(n ? 5, P ? 0.041 and P ? 0.0022 for CD4 and CD8 effectors, respectively). C: CD11c single-positive cells and CD11chighCD11b?cells were reduced
in response to NOR MSC therapy (n ? 5, P ? 0.004 and P ? 0.015, respectively) while the CD11clowCD11b?population was increased (n ? 5,
1.12 ? 0.1% and 2.06 ? 0.34% for control and NOR MSC-treated, respectively, P ? 0.029). Analysis of the CD11c single-positive population
showed a marked increase in the plasmacytoid DC subset in NOR MSC-treated mice (gated on CD11c?CD11b?, followed by analysis of B220
expression, n ? 5, 21.9 ? 3.18% and 37.5 ? 3.31% for control and NOR MSC-treated, respectively, P ? 0.0094). The frequencies of CD11b?Ly-6c?
monocytes (n ? 3–5, P ? 0.042) and F4/80?macrophages (n ? 5, P ? 0.015) were also found to be reduced in response to NOR MSCs. All data
are displayed with means and SEM.
M. JUREWICZ AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 59, DECEMBER 20103145

Page 8

at low levels at baseline, treatment with recombinant
IFN-? resulted in abundant PD-L1 expression. Similarly,
previous reports have demonstrated IFN-? to be important
for MSC-mediated immunosuppression or PD-L1-mediated
MSC immunoregulation (6,39,40). In our model, IFN-?
production by autoreactive T-cells may upregulate PD-L1
expression on MSCs and thereby augment their immuno-
modulatory capability. Indeed, we show that addition of
NOR MSCs to the BDC2.5 autoreactive assay results in
prodigious upregulation of PD-L1 expression on NOR
MSCs; we postulate that a similar upregulation occurs
after in vivo administration of MSCs to diabetic mice. With
regard to functionality, the immunosuppressive effect of
NOR MSCs was shown to be mediated in part via PD-L1
through siRNA knockdown of MSC PD-L1 expression and
consequent abrogation of the suppressive effect on auto-
reactive T-cell proliferation. Given these results, further
exploration of the effects of administering PD-L1-positive
MSCs and the consequent potential for reducing the MSC
number injected to diabetic NOD mice is certainly of
merit. We also investigated the role of MSCs in regulating
the phenotype of DCs. Primary diabetic insult is identified
by peri-insulitis of the pancreas after DC and macrophage
infiltration, and MSCs have been shown to both suppress
and alter DC differentiation in other models (2,7). Herein,
we show that the presence of MSCs reduces CD11c and
CD11b expression, decreases inflammatory Ly-6c expres-
sion in the CD11b?population, and suppresses differenti-
ation as shown by increased frequency of lineage-negative
and Sca-1?cells. TNF-? production, a growth factor
involved in DC maturation and produced by mature DCs,
was suppressed in MSC cocultures, and DC morphology
was similarly altered in the presence of MSCs. Moreover,
we demonstrate that these effects are in large part medi-
ated by IL-6 through the use of a blocking IL-6 antibody
and the consequent reversal of this effect, although other
factors such as Flt3L and M-CSF may be involved due to
incomplete abrogation of all phenotypic alterations.
Our reversal studies in the NOD mouse show a marked
incidence in reversal of recent-onset hyperglycemia in eight
of nine mice in response to NOR MSC treatment, with
prolonged reversal of 83% of mice treated with NOR MSCs
(i.e., in five of six long-term survivors), whereas control mice
reverted to hyperglycemia soon after cessation of insulin
replacement. Serum cytokine studies of reversal mice
showed changes in the cytokine profile with respect to IL-6,
IL-7, IL-10, and IL-12(p40). IL-6 is a pleiotropic cytokine
purported to have both anti- and proinflammatory roles; in
type 1 diabetes, reports of its effects are conflicting, but it has
been shown to protect ?-cells from apoptosis and impaired
function as well as delay the onset of overt diabetes in
NOD mice (41–43). Our in vitro data also demonstrate the
central role of IL-6 in NOR MSC suppression of DC genera-
tion. IL-7, although important for effector memory T-cell
survival, has also recently been demonstrated to be neces-
sary for common lymphoid progenitor development, from
which plasmacytoid DCs arise (44). IL-10 is an established
immunoregulatory cytokine, whereas the homodimer IL-
12(p40) inhibits action of the bioactive heterodimer IL-
12(p70) (45,46). Taken together, changes in cytokines appear
to skew the inflammatory diabetogenic environment toward
amoreregulatoryprofileinresponsetoNORMSCtreatment.
Consistent with our in vitro data, we demonstrate altered DC
phenotype in response to NOR MSC treatment, manifested
by diverting the CD11c?subset toward a more regulatory
cell as well as possibly preventing myeloid APC differentia-
tion, as demonstrated by reduced expression of CD11c and
F4/80 after MSC injection in vivo. Interestingly, the
CD11c?CD11b?subset has been shown to prime autoreac-
tive T-cells, resulting in physiological ?-cell death (47), and
our treatment, particularly given our in vitro data, appears to
retard development of this cell subset. Moreover, plasmacy-
toid DC frequency was found to be markedly increased in
response to NOR MSC treatment, and DCs of this lineage
have been shown to prevent acceleration of insulitis in NOD
mice,aswellastosuppressmyeloidDCactivationofeffector
cells (48,49), which is consistent with the decreased fre-
quency of CD4 and CD8 effectors that we observed in
response to NOR MSC treatment. As it has also been shown
that type 1 diabetic patients have a reduced pDC compart-
ment (50), increased frequency of pDC after NOR MSC
treatment may thus contribute to the reversal of hyperglyce-
mia that we observed.
Taken together, NOR MSC treatment resulted in efficient
reversal of hyperglycemia, suppressed autoreactive T-cell
proliferation via PD-L1, and increased production of reg-
ulatory cytokines and frequency of plasmacytoid DCs.
This work is the first to demonstrate the distinct benefit of
congenic MSCs in reversing hyperglycemia and ameliorat-
ing diabetes pathogenesis. Further exploration to optimize
and to confirm the safety and efficacy of MSC therapy is
the subject of our future studies. These data should serve
to shape future type 1 diabetes clinical trials with regard to
optimal MSC source and therapeutic regimen.
ACKNOWLEDGMENTS
This work is supported by Juvenile Diabetes Research
Foundation (JDRF) Grant 4-2007-1065, JDRF R&D Grant
1-2007-713. R.A. is the recipient of a JDRF Regular Grant.
No potential conflicts of interest relevant to this article
were reported.
M.J. researched data, contributed to discussion, and
wrote the manuscript. S.Y., R.M., and J.A. researched data.
A.A. and J.G. researched data and contributed to discus-
sion. P.F. contributed to discussion. M.A. reviewed and
edited the manuscript. M.H.S. contributed to discussion
and reviewed and edited the manuscript. R.A. contributed
to discussion and wrote the manuscript.
We thank Drs. Carlo Capelli and Stefano La Rosa for
their contributions to immunohistochemistry studies.
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M. JUREWICZ AND ASSOCIATES
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