Investigating the route of administration and efficacy of adipose tissue-derived mesenchymal stem cells and conditioned medium in type 1 diabetic mice

Abstract

Type 1 diabetes (T1D) is a chronic autoimmune disease destroying the insulin-producing beta cells. Recently, stem cell therapy has been tested to treat T1D. In the present study, we aim to investigate the effects of intraperitoneal and intravenous infusion of multipotent mesenchymal stem/stromal cells (MSCs) and MSC-conditioned medium (MSC-CM) in an experimental model of diabetes, induced by multiple injections of Streptozotocin (STZ). The adipose tissue-derived MSC and MSC-CM were isolated from C57Bl/6 male mice and characterized. Later, MSC and MSC-CM were injected intraperitoneally or intravenously into mice. The blood glucose, urinary glucose, and body weight were measured, and the percentages of CD4+ CD25+ FOXP3+ T cells as well as the levels of IFN-γ, TGF-β, IL-4, IL-17, and IL-10 were evaluated. Our results showed that both intraperitoneal and intravenous infusions of MSC and MSC-CM could decrease the blood glucose, recover pancreatic islets, and increase the levels of insulin-producing cells. Furthermore, the percentage of CD4+ CD25+ FOXP3+ T cells was increased after intraperitoneal injection of MSC or MSC-CM and intravenous injection of MSCs. After intraperitoneal injection of the MSC and MSC-CM, the levels of inflammatory cytokines reduced, while the levels of anti-inflammatory cytokines increased. Together current data showed that although both intraperitoneal and intravenous administration had beneficial effects on T1D animal model, but intraperitoneal injection of AD-MSC and AD-MSC-CM was more effective than systemic administration.

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Vol.:(0123456789)
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Inflammopharmacology
https://doi.org/10.1007/s10787-019-00661-x
ORIGINAL ARTICLE
Investigating theroute ofadministration andecacy ofadipose
tissue‑derived mesenchymal stem cells andconditioned medium
intype 1 diabetic mice
SeyedMahmoudHashemi1,2,3 · ZuhairMohammadHassan4· NikooHossein‑Khannazer1·
AliAkbarPourfathollah4· SaraSoudi4
Received: 3 June 2019 / Accepted: 29 October 2019
© Springer Nature Switzerland AG 2019
Abstract
Type 1 diabetes (T1D) is a chronic autoimmune disease destroying the insulin-producing beta cells. Recently, stem cell
therapy has been tested to treat T1D. In the present study, we aim to investigate the effects of intraperitoneal and intravenous
infusion of multipotent mesenchymal stem/stromal cells (MSCs) and MSC-conditioned medium (MSC-CM) in an experi-
mental model of diabetes, induced by multiple injections of Streptozotocin (STZ). The adipose tissue-derived MSC and
MSC-CM were isolated from C57Bl/6 male mice and characterized. Later, MSC and MSC-CM were injected intraperito-
neally or intravenously into mice. The blood glucose, urinary glucose, and body weight were measured, and the percentages
of CD4+ CD25+ FOXP3+ T cells as well as the levels of IFN-γ, TGF-β, IL-4, IL-17, and IL-10 were evaluated. Our results
showed that both intraperitoneal and intravenous infusions of MSC and MSC-CM could decrease the blood glucose, recover
pancreatic islets, and increase the levels of insulin-producing cells. Furthermore, the percentage of CD4+ CD25+ FOXP3+ T
cells was increased after intraperitoneal injection of MSC or MSC-CM and intravenous injection of MSCs. After intraperito-
neal injection of the MSC and MSC-CM, the levels of inflammatory cytokines reduced, while the levels of anti-inflammatory
cytokines increased. Together current data showed that although both intraperitoneal and intravenous administration had
beneficial effects on T1D animal model, but intraperitoneal injection of AD-MSC and AD-MSC-CM was more effective
than systemic administration.
Keywords Adipose tissue-derived stem cell· Conditioned medium· Intraperitoneal injection· Intravenous injection·
Immunomodulatory effect
Introduction
Type 1 diabetes (T1D) is a chronic, metabolic, organ-spe-
cific autoimmune disease characterized by irreversible and
progressive destruction of insulin-producing β cells (Figli-
uzzi etal. 2009; Aguayo-mazzucato and Bonner-weir 2010;
Neshati etal. 2010). T1D is one of the leading causes of
death in many countries and its prevalence is growing glob-
ally (Chen etal. 2004; Vija etal. 2009; Jurewicz etal. 2010;
Li etal. 2012). The autoimmune attack in T1D is driven by
T cell and can cause life-threatening implications including
absolute insulin deficiency, inadequate blood circulation,
hyperglycemia, dysfunction of kidney, stroke, infection, and
early death, although the anti-islet autoantibodies are known
as the main hallmarks of T1D progression. However, T cells,
B cells, dendritic cells (DCs), natural killer cells (NKs), and
inflammatory cytokines released by macrophages along
Inflammopharmacology
* Seyed Mahmoud Hashemi
smmhashemi@sbmu.ac.ir
* Zuhair Mohammad Hassan
hasan_zm@modares.ac.ir
1 Department ofImmunology, School ofMedicine, Shahid
Beheshti University ofMedical Sciences, Tehran, Iran
2 Urogenital Stem Cell Research Center, Shahid Beheshti
University ofMedical Sciences, Tehran, Iran
3 Department ofTissue Engineering andApplied Cell
Sciences, School ofAdvanced Technologies inMedicine,
Shahid Beheshti University ofMedical Sciences, Tehran,
Iran
4 Department ofImmunology, Faculty ofMedical Sciences,
Tarbiat Modares University, Tehran, Iran

S.M.Hashemi et al.
1 3
with activated T cells have significant contribution in the
pathogenesis of T1D (Brayman 2016). Conventional treat-
ment for T1D patients is the life-long external administra-
tion of insulin. However, insulin injection is not a cure and
is associated with hypoglycemic episodes (Figliuzzi etal.
2009; Zhao and Mazzone 2010; Brayman 2016). On the
other hand, pancreas or islet transplantation represents an
effective therapy for T1D patients but has certain restrictions
including immune rejection, immune response against islets,
donor availability, and need for life-long administration of
immunosuppressive drugs (Liu and Han 2008; Neshati etal.
2010; Jiang 2011; Brayman 2016). Thus, recent investiga-
tions focused on new treatment strategies based on control-
ling autoimmunity and preventing cell destruction (Zhao
and Mazzone 2010). Multipotent mesenchymal stem/
stromal cells (MSCs) have great immunomodulatory and
anti-inflammatory properties with low immunogenicity.
MSCs modulate innate and adaptive immune responses via
cell–cell contact or secretion of soluble factors(Madec etal.
2009; Jurewicz etal. 2010). It has been shown that MSCs are
capable of inhibiting CD4+ and CD8+ T cells proliferation
and DCs maturation, and suppressing the NK cell prolif-
eration and B cell function (Abdi etal. 2008; Institutes and
Wood 2012). Moreover, MSCs able to promote CD8+ Treg
survival and induce peripheral tolerance (Gao etal. 2016).
The immunomodulatory function of MSCs is often medi-
ated by production of regulatory mediators such as TGF-β,
IL10, hepatic growth factor (HGF), prostaglandin E2 and
IDO (indoleamine 2,3-dioxygenase) (Chen etal. 2004; Vija
etal. 2009; Sciences etal. 2011). Therefore, MSCs trans-
plantation has been introduced as a promising therapeutic
strategy for many human disorders, including T1D (Xu etal.
2008; Fiorina etal. 2011; Wang etal. 2013).
Besides the direct contribution of MSCs in tissue regener-
ation, recent studies have shown that the MSCs’ secreted fac-
tors, also known as conditioned medium (CM), have benefi-
cial therapeutic effects in many diseases such as skin wound
healing, acute lung injury, and myocardial infarction (Walter
etal. 2010; Timmers etal. 2011; Ionescu etal. 2012). Pre-
vious studies showed that the MSC-conditioned medium
(MSC-CM) has immunomodulatory properties (Yousefi
etal. 2016; Hossein-khannazer etal. 2019a). The MSC-CM
with different sources could inhibit immune cells migra-
tion into the injured tissue and induced M2-polarization of
macrophages (Parekkadan etal. 2007; Ionescu etal. 2012).
Although MSC and MSC-CM have shown great immu-
nomodulatory effects, but many aspects of these therapies
remained to be investigated. A critical factor in MSC and
MSC-CM therapies is the method of delivery. Intravenous
(IV) injection is the most examined and satisfactory route of
MSC or MSC-CM administration. However, systemic injec-
tion has some limitations; for example, over 80% of MSCs
were found in the lungs just a few minutes after IV infusion.
The accumulated cells in the lung vessels may cause emboli
and not able to reach the target tissues (Leibacher and Hen-
schler 2016; Wang etal. 2016). Although the intraperitoneal
(IP) injection of MSC has been tested in previous studies,
but the benefits and limitations of this methods have not
been well investigated (Ghionzoli etal. 2010). Previous
study showed that IP injection is much safer than IV admin-
istration and permits proper migration and homing of the
MSCs into the different tissues (Pierro etal. 2009; Wagner
etal. 2013). In the present study, we aim to investigate the
immunomodulatory effects of IV and IP administration of
adipose tissue-derived MSC (AD-MSC) and the AD-MSC-
conditioned media in a T1D mouse model.
Materials andmethods
Isolation ofadipose tissue‑derived mesenchymal
stem cell
Subcutaneous abdominal adipose tissue was harvested from
8-week-old C57Bl/6 male mice under sterile conditions. The
tissue samples were cut into 1mm3 fragments, then washed
with phosphate-buffered saline (PBS, Gibco, Germany), and
digested with 0.075% type I collagenase (Gibco, Germany)
for 20min at 37°C to obtain a homogeneous cell suspen-
sion. The cell suspension was then centrifuged at 500g for
5min to separate the adipocytes. The cells were cultured
in Dulbecco’s Modified Eagle’s Medium (DMEM, Biosera,
England) containing 10% fetal bovine serum (FBS; Gibco,
Germany), 1% penicillin/streptomycin (Biosera, England),
and 2mMl-glutamine (Invitrogen) at 37°C and 5% CO2.
After 48h, the medium was changed with fresh medium to
eliminate the non-adherent cells. The cells received fresh
medium twice a week until they reached 80% confluency,
then cells were detached using trypsin/EDTA for further
expansion.
AD‑MSC‑conditioned media preparation
For this purpose, 1 × 106 AD-MSC at passage 3 was seeded
in T75 tissue culture flask. Confluent cells were fed with
serum-free medium and cultured for 48h. The MSCs super-
natant was collected, filtered through a 0.22mm membrane
and then stored at − 80°C.
AD‑MSCs surface marker analysis
Cells at passage 2 were used for immunophenotyping. AD-
MSCs were detached and re-suspended in the staining buffer
(PBS containing 2% FBS). Cells were incubated with 100µl
of each antibody for 30min in the dark, then washed twice
using the washing buffer and analyzed using FACS Caliber

Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
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flow cytometer (BD Biosciences, San Diego, CA, USA). The
antibodies used for cell surface marker expression analyses
were mouse Sca-1, CD90, CD44, CD73, CD34, CD45 and
CD11b (all from eBioscience).
MSC dierentiation intoadipocyte andosteoblast
lineages
The AD-MSCs were examined for their ability to differen-
tiate into adipocyte. To induce adipogenic differentiation,
2 × 104 cell/cm2 cells were cultured in 24 well plates. The
confluent cells were incubated with DMEM supplemented
with 100mM indomethacin (Sigma), 250nM dexametha-
sone, 5µM insulin (Sigma), and 0.5mM 3-isobutyl-1-meth-
ylxanthine (Sigma). The differentiation tests were performed
in 3weeks and the medium was changed every 3days. For
detection of intercellular oil droplets, cells were stained with
Oil Red O and analyzed with microscopy.
For osteogenic induction, 2 × 104 cell/cm2 cells were
seeded in 24 well plates. An osteogenic induction medium
containing 50µg/ml ascorbic acid biphosphate (Sigma),
10mM beta-glycerolphosphate (Merck), and 100nM dexa-
methasone (Sigma) was added to the confluent cultures. The
osteogenic medium was replaced every 3days and the cells
were treated for 3weeks. Finally, cells were stained with
Alizarin Red to detect the mineralized matrix.
GFP+ AD‑MSCs tracking
In current study, and to detect the localization of stem cells
after transplantation, the GFP-positive MSCs were used.
GFP+ mice were obtained from Tehran University of Medi-
cal Sciences, Tehran. All mice were housed and handled
in accordance with the ethical principles of National Insti-
tutes of Health Guide for the Care and Use of Laboratory
Animals. GFP+ mice were euthanized and AD-MSCs were
isolated from adipose tissue as mentioned above.
Diabetes induction
Male C57BL/6 mice were purchased from the Pasteur Insti-
tute, Tehran, Iran. All animals were housed in standard
conditions under approved guidelines for care and use of
animals provided by Tarbiat Modarres University Ethics
Committee (ethics approval 525026). Male mice, 6–8weeks
old, were used to multiple low-dose injections of streptozo-
tocin (STZ), a β-cell-cytotoxic agent (Rahavi etal. 2015).
The STZ was dissolved in sodium citrate buffer (0.1M).
The pH was adjusted to 4.5 and the solution was prepared
fresh before injection. The mice were fasted for 6h before
induction, and then 50mg/ml of STZ solution was injected
through peritoneal. The animals were received the STZ
injection for five sequential days. Animals were fed with
10% sucrose-sweetenedwater. The mice were screened for
the diabetic state 3weeks after the first injection. The blood
glucose concentration was measured by a glucose meter and
glucose levels > 250mg/dl were considered as diabetic.
Administration ofAD‑MSC indiabetic mice
After induction of diabetes, mice were divided into three
groups for MSC administration. One group received 5 × 105
fresh MSCs at passage 2 through the tail vein (MSCs-IV).
The second group received 1 × 106 MSCs via IP injection
(MSCs-IP). The control animals were injected with DMEM
(control-IV and control-IP)(Fig.1).
Injection ofMSC‑CM intodiabetic mice
For the MSC-CM injection study, two groups were
employed: the first group received 0.5ml AD-MSC-CM by
IV injection (CM-IV), and the second group was injected
with 0.5ml AD-MSC-CM via IP injection (CM-IP). The
control animals were injected with DMEM and the injec-
tions were performed twice per week (control-IV and
control-IP)(Fig.1).
Blood glucose measurement
Blood samples were collected from the tip of the tail of ani-
mals in test and control groups. The blood glucose concen-
tration was measured every week using glucometer Accu-
Chek Go (Roche Diagnostic, Germany).
Intraperitoneal glucose tolerance measurement
To assess the peritoneal glucose tolerance, mice were fasted
for 6h and then 2mg/kg glucose was administered peritone-
ally. The tail blood glucose was determined before glucose
injection and at 15, 30, 90 and 120min after injection.
Histology
The mice were killed using chloroform inhalation at week 6.
For histology evaluation, the pancreas was removed imme-
diately, fixed in 10% buffered formalin, and then paraffin
embedded. The pancreas sections(5µm) were stained with
hematoxylin and eosin (H&E). The number of pancreas
islets and immune cell infiltration was determined using
ImageJ.
Immunohistochemistry
The pancreas and lymph node were subjected to immunohis-
tochemistry (IHC) analysis. In brief, the sections underwent
deparaffinization with xylene for 20min and rehydrated with

S.M.Hashemi et al.
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alcohol gradient. To block endogenous peroxidase activity,
slides were incubated with hydrogen peroxide solution (3%
H2O2) or 5min, then antigen retrieval solution was added
and samples were incubated for 25min. Sections were incu-
bated with primary antibodies: chicken polyclonal antibody
to GFP (1:1000; Abcam) and mouse monoclonal anti-insulin
(1:200; Abcam) for 1h at room temperature. Then, second-
ary antibodies including HRP-conjugated anti-chicken IgY
(1:500; Abcam) and HRP-conjugated rat anti-mouse IgG
(1:1000; Bioscience) were applied to the sections for 1.5h
at room temperature. Slides were exposed to diaminoben-
zidine (DAB)for 5min and finally counterstained with
hematoxylin.
Splenocyte proliferation assay
The spleen was removed from the killed animals aseptically
and splenocytes were extracted using a 5ml syringe with
a 23-gauge needle. The cells were washed two times with
lysis buffer (ammonium chloride solution) and then washed
in RPMI-1640 media. The number of viable cells was meas-
ured using 0.2% trypan blue dye. Then, 5 × 105 cells per well
were seeded in a round-bottom 96-well plate and cultured
in RPMI-1640 containing 10% FBS. After 48h, the prolif-
eration rate of splenocytes was measured in the presence of
the specific stimulator (pancreatic lysates) and nonspecific
stimulator (PHA) for the test and control groups.
Detection ofCD4+ CD25+ FOXP3+ T Cells
inthespleen
The levels of CD4+ CD25+ FOXP3+ T cells in the spleen
tissues after MSC or CM injections were evaluated. In brief,
the spleen was removed and DMEM containing 10% FBS
was injected to the spleen tissue and splenocytes extracted.
Then, cells were centrifuged at 300 × g for 5min and resus-
pended in lysis buffer. Finally, cells were passed through a
nylon wool column and stained with FITC-conjugated anti-
mouse CD4, PE-conjugated anti-mouse CD25 and APC-
conjugated anti-mouse FOXP3 antibodies. Rat IgG was used
as isotype control (Abcam). Finally, cells were analyzed
using FACS Calibur flow cytometer (BD Biosciences, San
Diego, CA, USA).
Cytokine measurement inserum andspleen cells
After AD-MSC or CM injections, the levels of IFN-γ, IL-10,
IL-17, TGF-β and IL-4 were measured quantitatively in
serum and splenocyte cell culture using the ELISA (Bender
MedSystems ELISA kits), followingthe manufacturer’s
instructions.
Statistical analysis
The statistical analysis was performed using Graphpad
Prism software (version 5). For multiple comparisons,
one-way ANOVA was used. The data are expressed as
mean ± standard deviation. P < 0.05 was considered statis-
tically significant.
Result
Characterization, adipogenic andosteogenic
dierentiation ofAD‑MSC
Flow cytometry analysis of AD-MSCs showed that they
were positive for MSCs markers CD90, CD44, CD73 and
Sca-1 and negative for CD11b, CD34 and CD45 (Fig.2).
The adipogenic and osteogenic differentiation potential of
AD-MSCs was confirmed using Oil Red O and Alizarin red
staining, respectively, after 21days (Fig.2). The control
groups failed to differentiate to adipogenic or osteogenic
lineages after 21days (Fig.2).
Blood glucose measurement
Hyperglycemia occurred in all the STZ-induced mice. Dur-
ing the study, the T1D mice (both IP and IV) showed ele-
vated glucose levels ranging from 300 to 550mg/dl. Inves-
tigating the level of blood glucose showed the glucose levels
stayed around 300mg/dl in the second and third weeks after
MSC infusion in both MSC-IP and CM-IP groups. The glu-
cose levels continued to decrease until 6weeks after MSC
administration. In the MSCs-IV group, the blood glucose
levels fell during the first 3weeks. This reduction was
maintained for 4weeks, after which the blood glucose con-
centration increased slightly. The blood glucose levels in
the MSCs-IV, MSCs-IP, and CM-IP groups were signifi-
cantly lower (P < 0.05) than the T1D groups (IP and IV) and
CM-IV group after 4, 5, and 6weeks of injection (Fig.3a).
Glucose tolerance measurement
After 6h of fasting, 2mg/g glucose was intraperitoneally
injected into the mice. The levels of blood glucose were
assessed at 15, 30, 90, and 120min after injection (Fig.3b).
The data showed that the blood glucose levels in MSCs-IV,
MSCs-IP, and CM-IP groups at 30, 90, and 120min after
injection were significantly lower than CM-IV group in T1D
animals (P < 0.05) (Fig.3b).
Moreover, the body weight measurement showed the
marked reduction in the body weight of T1D groups (T1D-
IV and T1D-IP). The MSCs-IP and CM-IP groups at 4, 5,
and 6weeks following injection showed significantly higher

Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
1 3
body weight compared with the T1D groups (P < 0.05). The
weight gain after injection of CM in both groups (CM-IP and
CM-IV) groups was higher than MSC injection (P < 0.05);
moreover, animals in CM-IV group showed higher weight
gain rather than CM-IP (Fig.3c).
Histology
To evaluate the effects of AD-MSC administration on recov-
ery of pancreatic β-cells, at 6weeks after AD-MSCs or CM
injections, the pancreatic sections (5μm) were stained with
H&E and then the size and number of islets were evaluated.
The MSC-IP, CM-IP, and MSCs-IV groups showed signifi-
cant increase in the size and numbers of islets in comparison
with T1D groups (IV and IP) (Fig.4).
Immunohistochemistry
To determine the number of insulin-producing cells, the
pancreas tissue sections were stained using mouse mono-
clonal anti-insulin antibody (Fig.5a). The tissues from the
control groups contained smaller islets along with a reduced
number of islets. The sections from MSC-IP and CM-IP
groups showed significant increases in the number of insulin
positive cells (P < 0.05) (Fig.5b). The number of insulin
positive islets in MSC-IP, MSC-IV, and CM-IP groups was
significantly higher than the control groups and CM-IV in
STZ-diabetic mice (Fig.5b).
Splenocyte proliferation assay
The splenocyte proliferation rate was determined after 48h
in the presence of PHA (nonspecific stimulator) and pan-
creatic lysates (specific stimulator) in the test and control
groups. The results indicated that the proliferation of spleno-
cytes was significantly (P < 0.05) higher in the T1D groups
(IP and IV) after stimulation with pancreas islet lysate
(Fig.6). The splenocytes proliferation was significantly
lower in the normal and MSC-IP groups after stimulation
with antigen (P < 0.05, Fig.6). Furthermore, the prolifer-
ation of splenocytes in the CM-IV, MSC-IV, and CM-IP
groups was lower than the T1D groups but the data were
not statistically significant. After nonspecific stimulation
with PHA, the splenocytes proliferation in MSC-IP group
showed a significant suppression as compared with other
study groups (Fig.6). MSC-IP group was more effective in
inhibiting proliferation of splenocytes.
Fig. 1 Study design of the therapeutic effects of AD-MSC and MSC-CM in STZ-induced diabetes

S.M.Hashemi et al.
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Detection ofCD4+ CD25+ FOXP3+ T cells
After MSC and MSC-CM injections, the levels of splenic
CD4+ CD25+ FOXP3+ T cells were evaluated using flow
cytometry (Fig.7a, b). The percentage of the splenic
Treg Cell subset has increased significantly in the MSC-
IP groups as compared with T1D IP and T1D IV groups
(P < 0.05; Fig.7). There was a significant increase in
splenic CD4+ CD25+ FOXP3+ T cells in the CM-IP
groups as compared with T1D IP and T1D IV groups.
There was a slight increase in CD4+ CD25+ FOXP3+ T
cells in CM-IV group (Fig.7c). The data showed that the
amount of CD4+ CD25+ FOXP3+ T cells in CD4+ T cells
increased significantly (P < 0.05) in MSC-IP, CM-IP and
Fig. 2 Characterization of isolated AD-MSCs from C57BL/6 mice
at passage 2. a Expression of positive and negative markers of AD-
MSCs. b Adipogenic differentiation of AD-MSCs assessed after
21days by oil red O staining. AD-MSCs differentiated into oil red
O-positive adipocytes. c Alizarin red staining showed calcium min-
eralization in AD-MSCs 21days after culture in osteogenic medium

Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
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MSC-IV groups as compared with T1D groups, after MSC
and MSC-CM injections (Fig.7d–f). MSC-IP, CM-IP and
MSC-IV groups induced CD4+ CD25+ FOXP3+ cells in
both CD4+ and splenocyts.
Detection oftheGFP+ MSC cells inthepancreas
andmesenteric lymph nodes
To investigate the invivo fate of AD-MSCs after trans-
plantation, the GFP+ MSCs were traced in the pancreas
and mesenteric lymph nodes using anti-GFP antibody. The
GFP+ MSCs could be found in the mesenteric lymph nodes
of those animals received the MSCs intraperitoneally (MSC-
IP). No GFP+ MSCs were found in the mesenteric lymph
nodes following IV injection of MSCs (MSC-IV). Similarly,
a few GFP+ MSCs were observed in the pancreas sections
of both MSC-IP and MSC-IV groups (Fig.8).
Splenocyte cytokine measurement
The concentration of inflammatory and anti-inflammatory
cytokines in supernatant of splenocytes after MSC or CM
administration was measured at 72h following incubation
with PHA (nonspecific stimulator) and pancreatic lysates
(specific stimulator).
Higher levels of TGFβ and IL-10 were observed in CM-IP
groups that stimulated with lysates as compared with T1D
groups (P < 0.05). Moreover, the TGFβ and IL-10 concen-
tration increased remarkably in CM-IP and MSC-IP groups
that stimulated with PHA. After stimulation with PHA, the
IL-4 production was significantly (P < 0.05) higher in the
MSC-IP group than in the T1D group, but no significant
changes were observed between the test and control groups
that stimulated with lysates. The secretion of IL-17 was sig-
nificantly reduced in MSC-IP, CM-IP and MSC-IV groups
after stimulation with PHA, but there was no significant dif-
ference between the test and T1D groups that incubated with
lysates (Fig.9).
Serum cytokine measurement
Blood samples were collected from MSC or CM-treated
mice for the measurement of serum IL-4, IL-10, IL-17,
IFNγ and TGFβ cytokines. The IL-17 serum concentration
was significantly decreased in MSC-IP, CM-IP and MSC-IV
groups (P < 0.05, Fig.10). The IL-4, IL-10, IFNγ and TGFβ
serum levels were not statistically different between the test
and T1D groups. Moreover, the IFNγ/IL-4 cytokine secre-
tion ratio decreased significantly (P < 0.05) in the CM-IP
and MSC-IP groups (Fig.10).
Fig. 3 Clinical improvement after injection of the MSC and MSC-
CM in diabetic mice. a Blood glucose measurement of test and con-
trol groups at 1-week intervals after MSC and MSC-CM injection. b
Blood glucose measurement in test and control groups at 0, 15, 30, 90
and 120min after peritoneal glucose injection. c Body weight in test
and control groups after initial administration of MSC and MSC-CM.
Level of significance was considered < 0.05(*P < 0.05). Quantitative
data are shown as mean ± SD for seven animals per experimental
group (N = 7)

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Fig. 4 Hematoxylin–eosin-stained pancreas sections. Pancreas sec-
tions of normal (control-IP and control-IV) and treated groups
(MSCs-IP, MSCs-IV, CM-IP and CM-IV) 6 weeks after MSCs and
MSC-CM infusion. Islets structures indicated by circles. Magnifica-
tion, ×40 and ×100 (N = 7)

Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
1 3
Fig. 5 Insulin immunohistochemical staining of pancreatic islets.
a Normal, control and treated groups (MSCs-IP, MSCs-IV, CM-IP
and CM-IV) 6 weeks after MSCs and MSC-conditioned medium
(MSC-CM) therapies. Magnification × 40 and × 100. b Number of
islets after MSC and MSC-CM therapies after hematoxylin–eosin
staining. c Number of insulin-producing pancreatic islets per section
of normal, control and test groups after anti-insulin staining. Level
of significance was considered < 0.05(*P < 0.05). Data are shown as
mean ± SD for seven animals per experimental group (N = 7)

S.M.Hashemi et al.
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Discussion
Stem cell therapy is a promising tool for the treatment of
autoimmune diseases. In the present study, we aimed to
investigate different aspects of cell therapy for T1D, the
route of administration and immunosuppressive effects
of AD-MSC in comparison with AD-MSC-conditioned
medium. Our results demonstrated that both MSC and
MSC-CM therapies improved the clinical symptoms of
diabetes in a mouse model. AD-MSC and CM therapy led
to a significant decrease in the blood glucose levels, recon-
struction of pancreas islets and weight gain. Previous stud-
ies also indicated that MSC and MSC-CM had significant
effects on clinical features of type 1 and type 2 diabetes
(Jiang 2011; Lee etal. 2006). In our study, infusion of
MSC and MSC-CM significantly (P < 0.05) ameliorated
hyperglycemia at 6weeks after injection. Due to the anti-
inflammatory and immunomodulatory capacity of MSCs
(Chhabra and Brayman 2013), one of the major effects
of MSCs transplantation was regeneration of β pancre-
atic structures and increasing number of pancreatic islets
(Lee etal. 2006). In agreement with previous studies, our
results showed that MSCs-treated mice (MSC-IP, MSC-IV
groups) had an increase in the number of islets. There was
also a significant increase in number of insulin-positive
islets in CM-IP group. These results indicated that AD-
MSC and AD-MSC-CM have cytoprotective properties.
The regeneration capacity of MSCs may relate to their
anti-inflammatory properties (Mackenzie and Flake 2001).
MSCs produce anti-inflammatory cytokines and trophic
factors that modulate the inflammatory environment and
protect damaged tissues from inflammatory responses.
Our result suggests a direct correlation between the islet
reconstruction and hyperglycemia reduction in T1D
animals.
To allow invivo tracking of MSCs, we used GFP+ mice.
Using IHC, we found a small number of GFP+ MSCs in
mesenteric lymph node and pancreas tissue in MSC-IP and
MSC-IV groups.
Numerous studies indicated that MSCs have immu-
nomodulatory effects (Soleymaninejadian etal. 2012;
Caplan and Sorrell 2015; Hossein-khannazer etal. 2019b).
The exact mechanisms by which the MSCs inhibit immune
system responses are still unknown. It is well established
that MSCs are able to suppress T cell proliferation (Svo-
bodova etal. 2012; Djouad etal. 2007; Sioud etal. 2011).
Our result showed the reduced proliferation of splenocytes
after administration of AD-MSCs, when splenocytes were
stimulated using antigen and mitogen. Moreover, the inhibi-
tory effect of AD-MSCs on splenocytes was significantly
(P < 0.05) higher than AD-MSCs CM. We also compared the
immunosuppressive properties of MSCs when injected IPor
IV. Results indicated that IP-injected MSCs had a greater
impact on splenocyte suppression than IV-injected MSCs.
It has been shown that Th1 and Th17 populations
increase in T1D and T reg cells decrease due to the
chronic inflammation conditions (Alnek etal. 2015).
Other studies suggested MSCs potential to induce T regs
cells (Wang etal. 2009; Kavanagh and Mahon 2011).
T reg population has a great impression on maintain-
ing the pancreas structure. T reg also plays an important
role in MSCs immunosuppressive effects (Augello etal.
2007; Du etal. 2011; Yaochite etal. 2015). In the pre-
sent study, we examined CD4+ CD25+ FOXP3+ T cells
in the C57BL/6 mice after MSC and CM injections using
flow cytometry analysis. We found that the percentage
of CD4+ CD25+ FOXP3+ splenic T reg cells increased
significantly in the MSC and MSC-CM-treated groups as
compared with control groups. These data are in line with
previous reports showing the expansion of Treg cells in
T1D after MSC administration (Madec etal. 2009; Bassi
etal. 2012). Several studies revealed that the suppres-
siveeffectsofMSCs are mediated by cell–cell contact
and soluble mediators such as anti-inflammatory cytokines
(Chen etal. 2015; Shim etal. 2016; Ciccocioppo etal.
2015; Kyurkchiev 2014). Anti-inflammatory cytokines
inhibit Th1 and Th17 activation (Boumaza etal. 2009;
Tse etal. 2003). In the present study, we assessed the AD-
MSCs ability in modulating the splenocytes cytokine pro-
duction. Our results showed the AD-MSCs able to alter
the secretion of cytokine by splenocytes that stimulated
with antigen or mitogen. AD-MSCs showed increase in
the levels of anti-inflammatory cytokines, IL-10, IL-4 and
TGFβ. In agreement with previous reports, higher levels
of anti-inflammatory cytokine were detected in MSC-IP
groups in comparison with MSC-IV groups (Zhang etal.
Fig. 6 In vitro splenocytes proliferation assay 6weeks after MSCs
and MSC-CM infusion. Splenocytes proliferation measured 48 h
after PHA (nonspecific stimulator) or pancreatic lysates (specific
antigen stimulator) stimulation in test and control groups. Level of
significance was considered < 0.05(*P < 0.05). Data are shown as
mean ± SD for seven animals per experimental group (N = 7)

Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
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Fig. 7 Detection of spleen T CD4+ CD25+ FOXP3+ after MSC
and MSC-CM therapies in STZ-diabetic mice. a, b Representa-
tive dot plots showing CD4+ CD25+ FOXP3+ splenic T cell subset
after MSC or CM injection. Data are gated on total splenic T cells.
c Showing percentage of CD4+ CD25+ FOXP3+ splenic T cell sub-
set within gated total splenic lymphocytes after MSC or MSC-CM
therapies. Detection of T CD4+ CD25+ FOXP3+ after MSC and
MSC-CM infusion in diabetic mice. d, e Representative dot plots
showing CD4+ CD25+ FOXP3+ T cell subset in diabetic mice after
MSC or CM injection. Data are gated on CD4+ T cells. f Percentage
of CD4+ CD25+ FOXP3+ T cell subset within gated total CD4+ lym-
phocytes in diabetic mice after MSC or MSC-conditioned medium
(MSC-CM) therapies. Data are shown as mean ± SD for seven ani-
mals per experimental group (N = 7)

S.M.Hashemi et al.
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Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
1 3
2013; Tanaka etal. 2008). Moreover, the higher levels of
IL-10, IL-4 and TGFβ were observed in CM-IP groups.
A previous study by Zheng etal. (2008) showed that AD-
MSCs administration decreased the amount of inflamma-
tory cytokines, IL-17 and IFN-γ, in Rheumatoid arthritis
(RA) animal model. In this study, we demonstrated that
the levels of IFN-γ and IL-17 were significantly reduced
in the MSC-IP, MSC-IV and CM-IP-treated groups. We
also analyzed serum cytokine levels after AD-MSC injec-
tion and found that the level of IL-17 was decreased in
the treated groups in comparison with control groups.
But, no significant change was observed in the levels of
anti-inflammatory cytokines in the blood serum. The T1D
is a local autoimmune disorder and various factors influ-
ence the levels of cytokines levels in the peripheral blood,
and this may explain why circulating cytokines did not
change after MSC therapy. The impacts of MSC treat-
ment on Th1/Th2 cytokine pattern were also assessed and
showed the cytokines shift toward TH2 profile with ele-
vated levels of IL-4 and decreased levels of IFNγ. Moreo-
ver, the IFNγ/IL-4 cytokine secretion ratio was decreased
in the MSC-treated animals. Increased TH2 cytokine pro-
duction in MSC-treated animals had protective effect on
T1D. Altogether, current data suggest that MSC and MSC-
CM treatment could modulate the imbalance between Th1,
Th2, Th17 and T reg cells and recover the inflammatory
status.
Additionally, current results showed that the AD-MSCs
CM had clinical effect by reducing the hyperglycemia and
increasing the number of insulin-producing pancreas islets
Fig. 8 Immunostaining of mesenteric lymph node and pancreas sec-
tions using anti-GFP antibody. a Anti-GFP antibody in mesenteric
lymph node in MSC-IP and control groups. b Anti-GFP antibody
in pancreas sections in MSC-IP and MSC-IV and control groups.
Arrows indicate GFP+ MSC
◂
Fig. 9 Cytokine production by splenocytes after MSC and CM therapies. TGFβ, IL-10, IL-4 and IL-17 production by splenocyets that incubated
72h with PHA or pancreatic lysates. Data represent the mean of three independent experiments ± SD. *P < 0.05 considered significant

S.M.Hashemi et al.
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Investigating theroute ofadministration andefficacy ofadipose tissue‑derived mesenchymal…
1 3
as well as suppressing the immune cell responses through
Treg cell induction, production of anti-inflammatory
cytokines and inhibition of IL-17 production. Since MSC-
CM includes several soluble mediators and exosomes,
previous studies proved the immunomodulatory effects of
MSC-CM in autoimmune diseases (Yousefi etal. 2016). The
current study further demonstrated that AD-MSC-CM has
protective effects on T1D. The immunosuppressive effect
of AD-MSC-conditioned medium was determined in mouse
model of acute colitis. AD-MSC-CM increased the IL-10
and TGF-b levels and decreased IL-17 cytokine productions
(Pouya etal. 2018). The impacts of AD-MSC and AD-MSC-
CM in improving the inflammation response were also dem-
onstrated in chronic colitis animal model. It is demonstrated
that both AD-MSC and AD-MSC-CM therapies increased
the anti-inflammatory cytokines and percentage of Treg cells
besides decreases the inflammatory cytokines (Heidari etal.
2018). In terms of clinical applications, MSC-CM possesses
several advantages such as easy production and transport in
comparison with stem cells application. Moreover, the host
immune system does not reject the CM.
Another critical aspect of stem cell therapy that analyzed
in this study was the route of stem cell and CM administra-
tion. In the previous study, we demonstrated that IP admin-
istration was more effective in maintaining the Tregs cells
and increasing the anti-inflammatory cytokines as compared
with I.V. route in experimental autoimmune encephalomy-
elitis (EAE) (Yousefi etal. 2013). The current study proved
that the IP injection of MSC and MSC-CM had beneficial
effects on pancreas regeneration, induction of Treg cells and
anti-inflammatory cytokines in a diabetic mouse model. In
comparison with IV injection, IP administration of MSCs
was more effective in increasing the pancreas islets, reduc-
tion of splenocyte proliferation and induction of regulatory
T cells. Similar result was reported by Andrea Augello etal.
(2007) arthritis animal model. Another study showed that
IP transplantation of amniotic fluid stem cells was safer and
resulted in higher migration and homing of transplanted
cells to the tissues (Pierro etal. 2009). One explanation
for these findings is that systemic transplanted AD-MSCs
trapped in the lungs and could not access to other organs
whereas peritoneally infused MSCs took up with lymphat-
ics and migrated through liver, lungs and kidney (Wagner
etal. 2013).
In conclusion, current data demonstrated that both intra-
peritoneal and intravenous injections had beneficial effects
on T1D animal model. However, intraperitoneal adminis-
tration of AD-MSC and AD-MSC-CM was more effective
in induction of immunosuppressive effects than systemic
administration.
Acknowledgements This work was supported by research grants from
Tarbiat Modarres University.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All applicable national and institutional guidelines
for the care and use of animals were followed.
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