DSpace at VNU: Improving the efficacy of type 1 diabetes therapy by transplantation of immunoisolated insulin-producing cells

Human Cell (2011) 24:86–95
DOI 10.1007/s13577-011-0018-z

RESEARCH ARTICLE

Improving the efficacy of type 1 diabetes therapy
by transplantation of immunoisolated insulin-producing cells
Phan Kim Ngoc • Pham Van Phuc •
Truong Hai Nhung • Duong Thanh Thuy
Nguyen Thi Minh Nguyet

•

Received: 14 February 2011 / Accepted: 19 April 2011 / Published online: 13 May 2011
Ó Japan Human Cell Society and Springer 2011

Abstract Type 1 diabetes occurs when pancreatic islet
b-cells are damaged and are thus unable to secrete insulin.
Pancreas- or islet-grafting therapy offers highly efficient
treatment but is limited by inadequate donor islets or
pancreases for transplantation. Stem-cell therapy holds
tremendous potential and promises to enhance treatment
efficiency by overcoming the limitations of traditional
therapies. In this study, we evaluated the efficiency of
preclinical diabetic treatment. Diabetes was induced in
mice by injections of streptozotocin. Mesenchymal stem
cells (MSCs) were derived from mouse bone marrow or
human umbilical cord blood and subsequently differentiated into insulin-producing cells. These insulin-producing
cells were encapsulated in an alginate membrane to form
capsules. Finally, these capsules were grafted into diabetic
mice by intraperitoneal injection. Treatment efficiency was

evaluated by monitoring body weight and blood glucose
levels. Immune reactions after transplantation were monitored by counting total white blood cells. Allografting or
xenografting of encapsulated insulin-producing cells
(IPCs) reduced blood glucose levels and increased body
weight following transplantation. Encapsulation with alginate conferred immune isolation and prevented graft
rejection. These results provide further evidence supporting
the use of allogeneic or xenogeneic MSCs obtained from
bone marrow or umbilical cord blood for treating type 1
diabetes.

P. K. Ngoc Á P. V. Phuc (&) Á T. H. Nhung Á
D. T. Thuy Á N. T. M. Nguyet
Laboratory of Stem Cell Research and Application,
University of Science, Vietnam National University,
227 Nguyen Van Cu, District 5, Ho Chi Minh, Vietnam
e-mail:

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Keywords Mesenchymal stem cells Á Insulin-producing
cells Á Encapsulation Á Allograft Á Xenograft Á Diabetes Á
Diabetic mouse model Á Umbilical cord blood Á Bone
marrow

Introduction
Transplantation of insulin-producing cells (IPCs) offers a
potential cell replacement therapy for patients with type 1
diabetes. However, because of the inadequate number of
cells obtained from donors, sources of stem cells to provide IPCs have drawn much attention from many research
groups. Various studies proved that IPCs could be derived

from mesenchymal stem cells (MSCs) from bone marrow,
umbilical cord, fresh or frozen umbilical cord blood, and
fat tissue. Moreover, numerous studies have been performed to test the efficacy of these cell types, as well as
IPCs, in type 1 and 2 diabetes in preclinical and clinical
settings [3, 7, 12, 16–20, 22–24, 30–34]. However, the
efficacy of these approaches has remained limited because
they typically necessitate administration of immunosuppressive agents to prevent rejection of transplanted cells.
The use immunosuppressive drugs can lead to deleterious
side effects, such as increased susceptibility to infection,
liver and kidney damage, and increased risk of cancer.
In addition, immunosuppressive drugs may have unexpected effects on transplanted tissues, as some reports
have shown that cyclosporine A (CsA) can inhibit insulin
secretion from pancreatic cells [1, 2, 6, 14, 15, 29].
Immunoisolation is a promising technique to protect
implanted tissues from rejection. One of the most common
immunoisolation techniques is to encapsulate cells in a
semipermeable membrane, such as alginate, which physically protects the grafts against the host’s immune cells


Improving the efficacy of type 1 diabetes therapy

while allowing nutrients and metabolic products to diffuse
into or out of the capsule. To achieve this, the cells are
encapsulated within a hydrogel or alginate membrane using
gravity, electrostatic forces, or coaxial airflow to form the
capsule.
Allogeneic and xenogeneic transplantation of encapsulated islets of Langerhans cells have been shown to
restore normal blood glucose levels in animals in which
diabetes was induced by autoimmune diseases or chemical injury—mice [8, 10, 21], dog [25–27], and nonhuman
primates [28]—without relying on immunosuppressive

agents. In most of these studies, the transplantations were
performed by intraperitoneal injection of islets. Recently,
however, Dufrane et al. [8] reported the generation of
encapsulated porcine islets in a Ca–alginate material and
implanted these capsules under the kidney capsule of
nondiabetic Cynomolgus maccacus. In that study, the
implanted porcine islets survived for up to 6 months after
implantation without immunosuppression, even in animals
administered with porcine immunoglobulin G (IgG).
Moreover, C-peptide was detected in 71% of the animals.
After 135 and 180 days, the explanted capsules still
synthesized insulin and responded to glucose stimulation
[8].
In this study, we encapsulated IPCs that were differentiated from MSCs and tested their efficacy in type 1 diabetes. To evaluate the capabilities of the encapsulated IPCs,
we conducted both allogeneic and xenogeneic transplantation. The former was conducted using IPCs derived from
mouse bone marrow MSCs and the latter using cells produced by mesenchymal tissue from human umbilical cord
blood.

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8009g for 16 min at room temperature, MNCs were harvested from the interphase, washed twice with PBS, and
resuspended in Iscove’s modified Dulbecco’s medium
(IMDM). Next, the cell suspension was transferred to a
T-25 culture flask (Nunc, Roskilde, Denmark) containing
3 ml of IMDM, 20% FBS, 10 ng/ml fibroblast growth
factor, 20 ng/ml epidermal growth factor (EGF), and 1%
antibiotic/antimycotic solution (all purchased from SigmaAldrich). The cultures were then maintained at 37°C in a
humidified atmosphere containing 5% carbon dioxide
(CO2), and the medium was changed 2 days later. When
the fibroblast-like cells at the base of the flask reached high

confluence, they were harvested with 0.25% trypsin–ethylenediaminetetraacetate (EDTA) (Sigma-Aldrich) and
subcultured at a 1:3 dilution as passage one to yield human
(h)MSCs.
Isolation of MSCs from mouse bone marrow
To obtain bone marrow, 6- to 8-week-old mice were
euthanized by cervical dislocation. The hind limbs were
dissected and stored on ice in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 19 penicillin/streptomycin. Bone marrow cells were collected by flushing the
femurs and tibias with DMEM/F12 plus 10% FBS (SigmaAldrich). The bone marrow cell suspension was plated on a
T-25 flask at a density of 4 9 106 cells/cm2. The culture
media were replaced 2 days later to remove nonadherent
cells. The cells were maintained for 3–4 weeks and subcultured following harvest with 0.25% trypsin–EDTA to
yield mouse (m)MSCs.
Characterization of MSCs

Materials and methods
Isolation of MSCs from human umbilical cord blood
MSCs were isolated as previously described [23]. Briefly,
human umbilical cord blood was obtained from the
umbilical cord vein of mothers attending Hung Vuong
Hospital (Ho Chi Minh City, Vietnam) with informed
consent from the mother. All donors must have signed an
agreement with our laboratory prior to donation. All blood
sample procedures and manipulations were approved by
our Institutional Ethical Committee (Laboratory of Stem
cell Research and Application, University of Science,
VNU-HCM, VN) and the Hospital Ethical Committee
(Hung Vuong Hospital, HCM, VN). To isolate mononuclear cells (MNCs), each unit of blood was diluted to 1:1
with phosphate-buffered saline (PBS) and loaded onto Ficoll–Hypaque solution (1.077 g/ml, Sigma-Aldrich, St
Louis, MO, USA). After density gradient centrifugation at

Adipogenic differentiation assays were performed as previously described [23]. Briefly, MSCs were incubated in
medium supplemented with 10-8 M dexamethasone and
10-4 M L-ascorbic acid 2-phosphate (Sigma-Aldrich) with
changes in media every 5 days. After 30 days, the cultures
were fixed in 3% formaldehyde in PBS for 10 min and
stained with Oil Red O. The phenotype of MSCs was
analyzed by flow cytometry using a FACSCalibur flow
cytometer (BD Biosciences, NJ, USA). The following
monoclonal antibodies (mAbs) (BD Biosciences, NJ, USA)
were used: fluorescein isothiocyanate (FITC)-labelled antiCD13, anti-CD14; anti-CD34, anti-CD45, anti-CD44, antiCD90, anti-c-kit, and anti-CD73. Isotype controls were
used in all cases.
Differentiation of hMSCs and mMSCs into IPCs
To differentiate cells into a pancreatic endocrine lineage,
the expanded MSCs from passage 5 were allowed to reach

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80–90% confluence and induced to differentiate into IPCs
by an enhanced three-step protocol [13, 23]. Briefly, in step
1, the cell monolayer was treated for 24 h with high-glucose
DMEM (H-DMEM, 25 mmol/L glucose) supplemented
with 10% FBS and 10-6 mol/L retinoic acid (SigmaAldrich), followed by H-DMEM containing 10% FBS alone
for a further 2 days. In step 2, the medium was replaced
with low-glucose DMEM (L-DMEM, 1,000 mg glucose/L)
supplemented with 10% FBS, 10 mmol/L nicotinamide
(Sigma-Aldrich), and 20 ng/ml EGF for 6 days. In step 3, to

mature the IPCs, cells were cultured with L-DMEM supplemented with 10% FBS and 10 nmol/L exendin-4
(Sigma-Aldrich) for 6 days.
Characterization of differentiated IPCs
Cellular differentiation was monitored by observing the
3D formation of islet-like cell clusters, the expression of
insulin detected by immunocytochemistry. As a control
group, cells were cultured in L-DMEM containing only
10% FBS. Immunocytochemistry was also performed.
Briefly, the induced cells were fixed in 4% paraformaldehyde, washed three times with PBS, permeabilized with
PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and
blocked with 10% normal serum for 40 min at room
temperature. The cells were then incubated with the primary antibody (mouse anti-human C-peptide antibody)
followed by FITC-conjugated goat anti-mouse IgG. In all
immunocytochemistry assays, negative staining controls
were established by omitting the primary antibody.
Nuclei were detected using Hoechst 33342 (SigmaAldrich) staining. Images were captured using a Carl
Zeiss Cell Observer microscope with a monochromatic
cool-charged coupled camera (Carl Zeiss AG, Jena,
Germany).
Encapsulation of IPCs
Sodium alginate was dissolved in sterile water at 2.2% w/v,
followed by the addition of sterile 0.9% sodium chloride
(NaCl) (0.2 ml per 1.8 ml alginate solution). The solution
was mixed and centrifuged at 1,000 rpm for 5 min. The
IPCs were washed twice with 0.9% saline and pelleted by
centrifugation. The alginate was mixed evenly with the
cells at a volume of 800 ll alginate per 100 ll of cell
suspension. This mixture was then loaded into a 1-ml
syringe connected to a 32.5-gauge needle. The capsules
were formed by pushing the syringe. To provide mechanical strength, the capsules were incubated in 30 ml of

20 mM barium chloride (BaCl2) for 2 min. The differentiated IPCs derived from mouse and human MSCs were
labeled as mIPCs and hIPCs, respectively.

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Measurement of insulin secretion in vitro
Insulin secretion was measured in vitro by radioimmunoassay (RIA) after static stimulating. Briefly, 30 capsules or
IPCs (mIPCs or hIPCs) were picked and transferred into
1.5-ml tubes. The capsules or IPCs were left to settle for a
few minutes and the supernatant was then discarded.
Samples were incubated with 250 ll of the stimulation
buffers [oxygenated Krebs–Ringer bicarbonate buffer:
137 mM NaCl, 20 mM potassium chloride (KCl), 1.2 mM
potassium di-hydrogen phosphate(KH2PO4),1.2 mM magnesium sulfate water (MgSO4-7H2O), 2.5 mM calcium
chloride (CaCl2-2H2O), 25 mM sodium bicarbonate
(NaHCO3), 0.25% bovine serum albumin (BSA)] for 1 h at
37°C and 5% CO2, with each sample prepared in triplicate.
After lightly mixing the samples a few times, the supernatant was collected into new 1.5-ml tubes for insulin measurement using RIA. IPCs were used as control samples.
Transplantation of encapsulated IPCs
Male Swiss mice were obtained from the Pasteur Institute
(Ho Chi Minh City, Vietnam). All procedures were
approved by the Animal Care and Ethics Committee of our
university and laboratory. Diabetes was induced in these
mice by intraperitoneal injection of 50 mg/kg streptozotocin
(Sigma-Aldrich) once daily for 5 days before transplantation. The mice were considered to be diabetic if two consecutive blood glucose readings were [250 mg/dl. Mice
were anesthetized with ketamine (50 mg/kg) and 200 ll
PBS containing 200–300 capsules, or 105 IPCs were injected
directly into the portal vein using a 14-gauge catheter. A

negative control, diabetic group received PBS alone.
Evaluation of immune responses, body weight,
and blood glucose and insulin levels
To monitor immune responses, peripheral blood was collected on days 7, 15, and 30, suspended in PBS, and
counted using a Nucleocounter (Chemomotec, Denmark).
Briefly, blood samples were lysed with lysis solution to
permeabilize the cell membrane and then neutralized using
neutralization solution. The samples were then loaded onto
a cassette, stained with propidium iodide, and counted.
Blood glucose was evaluated by measuring glucose levels
in tail-vein blood using an Accu-ChekÒ glucose monitor
(Hoffmann-La Roche Inc). Body weight was measured
every 2–3 days.
Statistical analysis
All data are presented as means ± standard error (SE).
Comparisons between the two groups were performed


Improving the efficacy of type 1 diabetes therapy

using Student’s two-sample t test or analysis of variance
(ANOVA), as appropriate. Values of P \ 0.05 were considered statistically significant.

Results
hMSCs and mMSCs expressed MSC markers
and successfully differentiated into adipocytes
Although there were some slight differences in the morphology of MSCs obtained from the umbilical cord blood
and bone marrow—the hMSCs tended to be larger than the
mMSCs (Fig. 1a, d)—the fibroblast-like shape was still
recognizable in both cell lines. Furthermore, characterization for specific markers by flow cytometry revealed similar profiles of both cell lines. Both lines were positive for

CD13, CD44, CD90, and CD166 but negative for CD14,
CD34, CD45, and HLA-DR (Fig. 2), whereas mMSCs
showed higher expression of CD90 and CD44. Overall,
90.23 ± 1.25%, 85 ± 1.95%, 92 ± 2.15% and 50 ± 3.29%
(n = 3) of mMSCs and 72 ± 1.34%, 75 ± 2.18%, 83 ±
2.52%, and 73 ± 4.32% of hMSCs were positive for
CD13, CD44, CD90, and CD166, respectively (Fig. 2). All
MSCs from both sources could be induced into adipocytes
(Fig. 1b, c, e, f).

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and formed islet-like clusters by days 7–9 (Fig. 3a, b, d, e).
Immunocytochemistry analysis confirmed that the cells
expressed insulin protein (Fig. 3c, f). During capsulation,
we measured the size of 100 capsules per preparation, and
the mean capsule size was 325 ± 30.5 lm (n = 5).
Although detectable insulin was measured in the supernatant after encapsulation, its secretion was significantly
reduced compared with that of controls after stimulation
(3.2 ± 1.5 vs. 21.3 ± 9.1 lU/h; P = 0.05).
Effects of IPC transplantation on the body weight
of diabetic mice

Following exposure to the differentiation media, hMSCs
and mMSCs differentiated IPCs using the same culture
conditions. Both cell types started to aggregate by day 5

As shown in Fig. 4, the body weight of the control group
increased gradually over the 30-day study period, from
33.2 ± 1.03 to 43.7 ± 0.97 g, whereas that in the negative

control/diabetic group that received PBS decreased from
25.16 ± 1.00 to 15.66 ± 0.64 g. Furthermore, only two (of
five) mice in the negative control group survived to day 30.
Significant differences in body weight were observed
among the other experimental groups. Noticeably, the body
weight of mice given unencapsulated hIPCs showed the
lowest treatment efficacy, with a slight decrease in body
weight from 20.88 ± 0.68 g on day 1 to 20.28 ± 1.63 g on
day 30. Similarly, the body weight of mice treated with
unencapsulated mIPCs decreased from 25.14 ± 1.00 to
24.62 ± 0.96 g. Despite the absence of weight gain, four
(of five) mice in both groups survived until day 30, which
was higher than that in the negative control group. The
body weight of mice increased significantly over mice that
received encapsulated hIPCs—from 26.82 ± 0.68 g at day
1 to 29.46 ± 0.17 g at day 30—whereas weight gain was

Fig. 1 Isolation and differentiation of mesenchymal stem cells
(MSCs) isolated from human umbilical cord blood (a, hMSCs) and
mouse bone marrow (d, mMSCs) were capable of differentiation into

adipocytes (b, c for hMSCs and e, f for mMSCs). The differentiated
MSCs stored triglyceride in the cytoplasm (b, e) and the lipid
vacuoles turned red following Oil Red O staining (c, f)

Differentiation of MSCs into IPCs and capsulation

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P. K. Ngoc et al.

Fig. 2 Cell-surface markers expressed on human (hMSC) and mouse (mMSC) mesenchymal stem cells

more pronounced in mice that received encapsulated mIPCs (from 23.34 ± 0.88 to 34.16 ± 0.65 g), corresponding
to a mean weight gain of 10.82 g over 30 days. This
increase in weight was comparable with that observed in
the control group, in which mean weight gain was 10.5 g.
Accordingly, these changes in body weight confirmed the
beneficial effects of IPC transplantation, particularly
encapsulated IPCs, on the status of diabetic mice, and also
confirmed that encapsulated mIPCs had the greatest effects
on body weight.

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Effects of IPC transplantation on blood glucoses levels
in diabetic mice
As would be expected, the blood glucose level of the
control (nondiabetic) mice was broadly stable during the
study, being 98 ± 9.20 mg/dl on day 1 and 105.8 ±
9.26 mg/dl on day 30. On the other hand, marked changes
in blood glucose levels were noted in the other groups.
In the negative control diabetic group, the blood glucose
level increased from 318 ± 25.43 mg/dl on day 1 to


Improving the efficacy of type 1 diabetes therapy

Fig. 3 Encapsulation of insulin-producing cells (IPCs). Human (a–
c) and mouse (d–f) mesenchymal stem cells were differentiated into
IPCs, which resulted in marked changes in shape (a, d: before

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differentiation; b, e: after differentiation). The resulting IPCs were
stained with C-peptide antibody (c, f), confirming insulin production

Fig. 4 Changes in body weight
of mice treated with
unencapsulated and
encapsulated human- (hIPCs) or
mouse-derived (mIPCs) insulinproducing cells. Diabetic mice
were injected with
unencapsulated or encapsulated
IPCs derived from human or
mouse mesenchymal stem cells.
Control nondiabetic mice (no
transplantation of IPCs). PBS
PBS-treated diabetic mice
(negative control)

377.8 ± 21.96 mg/dl on day 30. Interestingly, the greatest
increase in blood glucose was observed in mice treated
with unencapsulated hIPCs, increasing from 281.8 ±
21.19 mg/dl on day 1 to 464.8 ± 21.03 mg/dl on day 30.
An increase, although with a slightly smaller increment,
was also noted in mice treated with unencapsulated mIPCs,

with blood glucose strongly increasing from 217.4 ±
14.63 mg/dl on day 1 to 408.8 ± 18.20 mg/dl on day 30.
In contrast, the blood glucose level in the encapsulated
hIPC group tended to slightly increase over time
(263.3 ± 17.64 mg/dl on day 1 to 299.2 ± 32 mg/dl on

day 30), whereas a more pronounced decrease was noted in
the encapsulated mIPC group (from 277.4 ± 15.11 mg/dl
on day 1 to 144.8 ± 6.57 mg/dl on day 30) (Fig. 5). These
results mean that transplantation of IPCs derived from
different sources may have different effects on glucose
levels in diabetic mice. Of note, transplantation of unencapsulated IPCs was unable to reduced blood glucose
levels, whereas mice given unencapsulated hIPCs experienced greater increases in glucose levels compared with
untreated diabetic mice. In contrast, transplantation of
encapsulated IPCs delivered positive effects on glucose

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Fig. 5 Changes in blood
glucose levels of mice treated
with unencapsulated and
encapsulated human- (hIPCs) or
mouse-derived (mIPCs) insulinproducing cells. Diabetic mice
were injected with
unencapsulated or encapsulated

IPCs derived from human or
mouse mesenchymal stem cells.
Control nondiabetic mice (no
transplantation of IPCs). PBS
phosphate-buffered-salinetreated diabetic mice (negative
control)

control in diabetic mice, with stabilization of blood glucose
levels in the hIPC group and a marked decrease in the
mIPC group.
Immune responses in mice treated with IPCs
The immune response showed differences between the
individual groups. As shown in Fig. 6, the white blood cell
count in untreated control and in PBS-treated diabetic mice
showed a small but nonsignificant change over time. In the
PBS-treated diabetic mice, the white blood cell count had
decreased slightly on day 15 but returned to the baseline
level on day 30. In mice given unencapsulated IPCs, the
white blood cell count increased over time in the hIPC
group by day 15, indicating marked immune activity at this
time, whereas a further increase was noted by day 30.
Increases in white blood cell counts between days 1 and 15
were similar in both groups of mice given encapsulated
IPCs, although there was a gradual reduction in the hIPC
group versus a slight increase in the mIPC group between
days 15 and 30. Among the four groups of mice given
IPCs, those given the encapsulated mIPC exhibited the
lowest immune response, with a moderate but statistically
insignificant increase in white blood cell count compared
with the PBS-treated diabetic group. This indicates relatively little antigen presentation following implantation of

encapsulated IPCs, particularly mIPCs, by preventing the
cells’ surface antigens from being detected by the host.

Discussion
MSCs are an important source of stem cells, with enormous
potential for use in regenerative medicine. MSCs have long
been considered for treating several diseases, including
diabetes, by implanting MSCs and IPCs derived from MSCs.

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A major hurdle, however, when using these cells, is tissue
rejection by the host. Although immunosuppressant drugs
can be used, they are associated with potentially serious side
effects, such as infection, cancer, and kidney and liver
damage. Therefore, to overcome these issues, we encapsulated the cells with a biocompatible membrane composed of
alginate. To determine the efficacy of this technique, we
compared transplantation of encapsulated or nonencapsulated IPCs derived from allogeneic or xenogeneic sources
into diabetic mice. To date, several studies have evaluated
allogeneic and xenogeneic transplantation of encapsulated
islets to improve grafting efficiency animals with diabetes
induced by autoimmune disease or chemical induction,
including in mice [9, 10, 21], dogs [25–27] and monkeys [28]
in the absence of immunosuppression.
Based on these earlier studies, we proposed a novel
encapsulation technique to protect from rejection the
implanted IPCs derived from mMSCs (for allografting) or
hMSCs (for xenografting). Cells harvested from mouse
bone marrow or human umbilical cord blood expressed
typical characteristics of MSCs. Their shape was similar to

that of fibroblasts, and they were positive for CD13, CD44,
CD90, and CD166 and negative for hematopoietic markers
such as CD14 (a monocyte marker), CD34 (a hematopoietic stem cell marker), CD45 (a white blood cell marker),
and HLA-DR (a leucocyte marker). The differentiation
potency of these MSCs was also confirmed by in vitro
adipogenesis following culture in an inducing medium.
These results indicate that we successfully isolated MSCs
from mouse bone marrow and human umbilical cord blood.
Next, we differentiated the MSCs into IPCs using a threestep protocol, as previously described [23]. The induced cells
exhibited a change in morphology and aggregated in isletlike clusters. As reported elsewhere [13, 23], we confirmed
the differentiation of MSCs into IPCs by immunocytochemistry. After staining, we observed that the induced cells


Improving the efficacy of type 1 diabetes therapy

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Fig. 6 Immune responses in
mice treated with
unencapsulated and
encapsulated human- (hIPCs) or
mouse-derived (mIPCs) insulinproducing cells. Diabetic mice
were injected with
unencapsulated or encapsulated
IPCs derived from human or
mouse mesenchymal stem cells.
The white blood cell count was
determined on day 7 (blue), day
15 (red), and day 30 (green).
Control nondiabetic mice (no

transplantation of IPCs). PBS
phosphate-buffered-salinetreated diabetic mice (negative
control)

expressed C-peptide, confirming that the MSCs were differentiated into IPCs and were capable of producing insulin.
The resulting cells were then encapsulated in the alginate
solution. In this step, we encapsulate the IPCs in an alginate
membrane to achieve a size suitable for transplantation. The
in vitro efficacy of encapsulation was evaluated by measuring insulin secretion from the IPC capsules to the surrounding environment. Following stimulation with KCl for
1 h, we measured the concentration of insulin in the medium
by RIA. Insulin could go out the membrane. Using the RIA
method, we detected the presence of insulin in inducible
medium supernatant. Of course, the insulin quantity was
lower compared with controls. The results were consistent
with those reported elsewhere, as David et al. [4] demonstrated that liver cells encapsulated in alginate exerted normal metabolic activity.
To demonstrate that transplantation of encapsulated
cells will help avoid immune rejection, we next compared
the efficacy of allogeneic and xenogeneic IPCs with or
without encapsulation on body weight, blood glucose levels, and white blood cell count of diabetic mice without
immunosuppression. The mice that received encapsulated
IPC showed significant differences in these parameters
compared with mice that received unencapsulated IPCs.
Allogeneic transplantation of encapsulated IPCs (i.e., mIPCs) yielded greater treatment efficacy compared with
transplantation of unencapsulated IPCs. Indeed, the body
weight of mice given encapsulated mIPCs increased steadily and was similar to that of control/nondiabetic mice,
whereas no weight gain was noted in mice given unencapsulated IPCs. Meanwhile, the blood glucose level of
mice given an allogeneic transplantation of encapsulated

IPCs decreased after day 6, reaching a level similar to that
in nondiabetic mice on day 30. In contrast, no improvements in blood glucose levels were noted in the two groups

of mice given unencapsulated IPCs. We explain these
findings in terms of the host’s immune responses, as allogeneic transplantation of encapsulated IPCs ameliorated
the effects of rejection compared with unencapsulated
cells. Thus, allogeneic transplantation of encapsulated IPCs
derived from mMSCs helped protect the grafts from
rejection and enhanced treatment efficiency in diabetic
mice. These results are consistent with a study reported by
De Vos [5], who allografted encapsulated islets in diabetic
mice and achieved normal blood glucose levels 5 days
after transplantation.
With xenografting, as with allografting, the effects of
encapsulation of IPCs were also evident on body weight and
blood glucose levels. Indeed, compared with unencapsulated
hIPCs, the implantation of encapsulated hIPCs enabled
weight gain, stabilized blood glucose levels, and reduced
rejection via the immune response. Accordingly, the mice
given encapsulated hIPCs showed a remarkable recovery,
although the magnitude of these effects was less than those
achieved with encapsulated mIPCs. Nevertheless, encapsulated IPCs derived from xenogeneic and allogeneic sources
had beneficial effects on the diabetic step, indicating that
encapsulation plays a critical role in reducing immune
rejection and thus improving treatment efficiency.
Because implantation of encapsulated IPCs derived
from a xenogeneic source did not completely overcome
rejection, if xenotransplantation is necessary, it may be
prudent to use encapsulation in combination with shortterm immunosuppression to avoid rejection. This approach

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was investigated by Figliuzzi et al. [11], who showed that
xenotransplantation of encapsulated islets in combination
with short-term immunosuppression prolong the life of the
implanted islets.
Taken together, the results of our study indicate that
MSCs can be induced to differentiate into IPCs, thus
offering an important source of cells for treating diabetes.
Allogeneic or xenogeneic transplantation of induced IPCs
confers higher treatment efficacy when the cells are immunoisolated by encapsulation in an alginate membrane.
Our findings shed light on the potential use of stem cells,
particularly MSCs, for treating diabetes. Because the use of
autografts faces many technical problems, particularly the
limited availability of stem cells, immunoisolating the cells
by encapsulation before transplantation may offer a better
choice to treat diabetes and other diseases using stem cells.
Moreover, these findings open a new direction to treat
diabetes using stem cells preserved in a stem cell bank or
blood bank for patients themselves or for their relatives.

Conclusions
In conclusion, immunoisolated MSCs can be used with
high efficacy to treat type 1 diabetes. MSCs can be differentiated into IPCs and encapsulated in alginate. Transplantation of the encapsulated IPCs obtained from
allogeneic or xenogeneic sources had greater efficacy than
unencapsulated cells for treating type 1 diabetes in a mouse
model. Encapsulation of cells in an alginate membrane
reduced IPC rejection by the host’s immune response. The

treated mice achieved normal blood glucose levels and
gained weight by 30 days after transplantation of the
encapsulated IPCs. These results demonstrate the enormous potential of using cells induced from stem cells to
treat type 1 diabetes. We believe that the approach
described here is not only suitable for treating type 1 diabetes but also other diseases in which differentiated stem
cells can be used.
Acknowledgments This work was funded by grants from Vietnam
National University, Ho Chi Minh City (VNU-HCM), Laboratory of
Stem Cell Research and Application, University of Science (SCL),
GeneWorld Ltd company. We thank Hung Vuong Hospital for supplying umbilical cord blood samples to perform this research.
Conflict of interest

None.

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