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

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

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

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

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

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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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

91

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,

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

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

93

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

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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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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