DSpace at VNU: Improving the efficacy of type 1 diabetes therapy by transplantation of immunoisolated insulin-producing...
Trang 1R E S E A R C H A R T I C L E
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
differenti-ated 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
moni-tored 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
algi-nate 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
Keywords Mesenchymal stem cells Insulin-producing cells Encapsulation Allograft Xenograft Diabetes Diabetic mouse model Umbilical cord blood 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 pro-vide 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 per-formed 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 immunosup-pressive 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 unex-pected 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 physi-cally protects the grafts against the host’s immune cells
P K Ngoc P V Phuc (&) T H Nhung
D T Thuy 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: pvphuc@hcmuns.edu.vn
DOI 10.1007/s13577-011-0018-z
Trang 2while 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
encap-sulated islets of Langerhans cells have been shown to
restore normal blood glucose levels in animals in which
diabetes was induced by autoimmune diseases or
chemi-cal 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
differen-tiated from MSCs and tested their efficacy in type 1
dia-betes To evaluate the capabilities of the encapsulated IPCs,
we conducted both allogeneic and xenogeneic
transplanta-tion The former was conducted using IPCs derived from
mouse bone marrow MSCs and the latter using cells
pro-duced by mesenchymal tissue from human umbilical cord
blood
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
mononu-clear cells (MNCs), each unit of blood was diluted to 1:1
with phosphate-buffered saline (PBS) and loaded onto
Fi-coll–Hypaque solution (1.077 g/ml, Sigma-Aldrich, St
Louis, MO, USA) After density gradient centrifugation at
8009g for 16 min at room temperature, MNCs were har-vested 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 Sigma-Aldrich) 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–ethy-lenediaminetetraacetate (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/strep-tomycin Bone marrow cells were collected by flushing the femurs and tibias with DMEM/F12 plus 10% FBS (Sigma-Aldrich) The bone marrow cell suspension was plated on a T-25 flask at a density of 4 9 106cells/cm2 The culture media were replaced 2 days later to remove nonadherent cells The cells were maintained for 3–4 weeks and sub-cultured following harvest with 0.25% trypsin–EDTA to yield mouse (m)MSCs
Characterization of MSCs Adipogenic differentiation assays were performed as pre-viously described [23] Briefly, MSCs were incubated in medium supplemented with 10-8M dexamethasone and
10-4M 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 CD13, CD14; CD34, CD45, CD44, anti-CD90, 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
Trang 380–90% 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
(Sigma-Aldrich), 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
sup-plemented 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%
paraformal-dehyde, 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
pri-mary 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
(Sigma-Aldrich) 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
mechan-ical strength, the capsules were incubated in 30 ml of
20 mM barium chloride (BaCl2) for 2 min The
differen-tiated IPCs derived from mouse and human MSCs were
labeled as mIPCs and hIPCs, respectively
Measurement of insulin secretion in vitro Insulin secretion was measured in vitro by radioimmuno-assay (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 mag-nesium 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 superna-tant was collected into new 1.5-ml tubes for insulin mea-surement 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 transplanta-tion The mice were considered to be diabetic if two con-secutive blood glucose readings were [250 mg/dl Mice were anesthetized with ketamine (50 mg/kg) and 200 ll PBS containing 200–300 capsules, or 105IPCs 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 col-lected 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
Trang 4using Student’s two-sample t test or analysis of variance
(ANOVA), as appropriate Values of P \ 0.05 were
con-sidered statistically significant
Results
hMSCs and mMSCs expressed MSC markers
and successfully differentiated into adipocytes
Although there were some slight differences in the
mor-phology 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,
characteriza-tion for specific markers by flow cytometry revealed
sim-ilar 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)
Differentiation of MSCs into IPCs and capsulation
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
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 superna-tant 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
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)
Trang 5more pronounced in mice that received encapsulated
mI-PCs (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
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 Fig 2 Cell-surface markers expressed on human (hMSC) and mouse (mMSC) mesenchymal stem cells
Trang 6377.8 ± 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 unen-capsulated IPCs was unable to reduced blood glucose levels, whereas mice given unencapsulated hIPCs experi-enced greater increases in glucose levels compared with untreated diabetic mice In contrast, transplantation of encapsulated IPCs delivered positive effects on glucose
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
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)
insulin-producing 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)
Trang 7control 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
rela-tively 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
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 encapsu-lated the cells with a biocompatible membrane composed of alginate To determine the efficacy of this technique, we compared transplantation of encapsulated or nonencapsu-lated 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 hematopoi-etic 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 three-step protocol, as previously described [23] The induced cells exhibited a change in morphology and aggregated in islet-like clusters As reported elsewhere [13,23], we confirmed the differentiation of MSCs into IPCs by immunocyto-chemistry After staining, we observed that the induced cells
Fig 5 Changes in blood
glucose levels of mice treated
with unencapsulated and
encapsulated human- (hIPCs) or
mouse-derived (mIPCs)
insulin-producing 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-saline-treated diabetic mice (negative
control)
Trang 8expressed C-peptide, confirming that the MSCs were
dif-ferentiated 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
meaing insulin secretion from the IPC capsules to the
sur-rounding 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]
demon-strated that liver cells encapsulated in alginate exerted
nor-mal 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
lev-els, 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.,
mI-PCs) yielded greater treatment efficacy compared with
transplantation of unencapsulated IPCs Indeed, the body
weight of mice given encapsulated mIPCs increased
stea-dily and was similar to that of control/nondiabetic mice,
whereas no weight gain was noted in mice given
unen-capsulated 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 improve-ments 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 allo-geneic 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, encapsu-lated 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 short-term immunosuppression to avoid rejection This approach
Fig 6 Immune responses in
mice treated with
unencapsulated and
encapsulated human- (hIPCs) or
mouse-derived (mIPCs)
insulin-producing 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-saline-treated diabetic mice (negative
control)
Trang 9was 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
im-munoisolated 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
dif-ferentiated into IPCs and encapsulated in alginate
Trans-plantation 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
enor-mous 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
dia-betes 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
sup-plying umbilical cord blood samples to perform this research.
Conflict of interest None.
References
1 Alejandro R, Feldman EC, Bloom AD, Kenyon NS Effects of
cyclosporin on insulin and C-peptide secretion in healthy beagles.
Diabetes 1989;6:698–703.
2 Bani-Sacchi T, Bani D, Filipponi F, Michel A, Houssin D Immunocytochemical and ultrastructural changes of islet cells in rats treated long-term with cyclosporine at immunotherapeutic doses Transplantation 1990;5:982–7.
3 Chandra VGS, Phadnis S, Nair PD, Bhonde RR Generation of pancreatic hormone-expressing islet-like cell aggregates from murine adipose tissue-derived stem cells Stem Cells 2009;8: 1941–53.
4 David B, Dufresne M, Nagel MD, Legallais C In vitro assess-ment of encapsulated C3A hepatocytes functions in a fluidized bed bioreactor Biotechnol Prog 2004;4:1204–12.
5 De Vos P, De Haan BJ, Wolters GH, Strubbe JH, Van Schilfg-aarde R Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets Diabetologia 1997;3:262–70.
6 Dean SK, Scott H, Keogh GW, Roberts S, Tuch BE Effect of immunosuppressive doses of cyclosporine on pancreatic beta cell function in pigs Am J Vet Res 2002;11:1501–6.
7 Dong QY, Chen L, Gao GQ, Wang L, Song J, Chen B, Xu YX, Sun L Allogeneic diabetic mesenchymal stem cells transplanta-tion in streptozotocin-induced diabetic rat Clin Invest Med 2008;6:E328–37.
8 Dufrane D, Steenberghe M, Goebbels RM, Saliez A, Guiot Y, Gianello P The influence of implantation site on the biocom-patibility and survival of alginate encapsulated pig islets in rats Biomaterials 2006;17:3201–8.
9 Duvivier-Kali VF, Omer A, Parent RJ, O’Neil JJ, Weir GC Complete protection of islets against allorejection and autoim-munity by a simple barium–alginate membrane Diabetes 2001;8:1698–705.
10 Fan MY, Lum ZP, Fu XW, Levesque L, Tai IT, Sun AM Reversal of diabetes in BB rats by transplantation of encapsulated pancreatic islets Diabetes 1990;4:519–22.
11 Figliuzzi M, Plati T, Cornolti R, Adobati F, Fagiani A, Rossi
L, Remuzzi G, Remuzzi A Biocompatibility and function of microencapsulated pancreatic islets Acta Biomater 2006;2: 221–7.
12 Gabr MM, Sobh MM, Zakaria MM, Refaie AF, Ghoneim MA Transplantation of insulin-producing clusters derived from adult bone marrow stem cells to treat diabetes in rats Exp Clin Transplant 2008;3:236–43.
13 Gao F, Wu DQ, Hu YH, Jin GX, Li GD, Sun TW, Li FJ In vitro cultivation of islet-like cell clusters from human umbilical cord blood-derived mesenchymal stem cells Transl Res 2008;6:293–302.
14 Gillison SL, Bartlett ST, Curry DL Inhibition by cyclosporine of insulin secretion–a beta cell-specific alteration of islet tissue function Transplantation 1991;5:890–5.
15 Hahn HJ, Dunger A, Laube F, Besch W, Radloff E, Kauert C, Kotzke G Reversibility of the acute toxic effect of cyclosporin A
on pancreatic B cells of Wistar rats Diabetologia 1986;8: 489–94.
16 Kadam S, Muthyala S, Nair P, Bhonde R Human placenta-derived mesenchymal stem cells and islet-like cell clusters gen-erated from these cells as a novel source for stem cell therapy in diabetes Rev Diabet Stud 2010;2:168–82.
17 Kim SC, Han DJ, Lee JY Adipose tissue derived stem cells for regeneration and differentiation into insulin-producing cells Curr Stem Cell Res Ther 2010;2:190–4.
18 Koblas T, Zacharovova´ K, Berkova´ Z, Leontovic I, Dovolilova´ E, Za´mecnı´k L, Saudek F In vivo differentiation of human umbil-ical cord blood-derived cells into insulin-producing beta cells Folia Biol (Praha) 2009;6:224–32.
19 Li M, Abraham NG, Vanella L, Zhang Y, Inaba M, Hosaka N, Hoshino S, Shi M, Ambrosini YM, Gershwin ME, Ikehara S Successful modulation of type 2 diabetes in db/db mice with
Trang 10intra-bone marrow–bone marrow transplantation plus concurrent
thymic transplantation J Autoimmun 2010;4:414–23.
20 Neshati Z, Matin MM, Bahrami AR, Moghimi A Differentiation
of mesenchymal stem cells to insulin-producing cells and their
impact on type 1 diabetic rats J Physiol Biochem 2010;2:181–7.
21 Omer A, Keegan M, Czismadia E, De Vos P, Van Rooijen N,
Bonner-Weir S, Weir GC Macrophage depletion improves
sur-vival of porcine neonatal pancreatic cell clusters contained in
alginate macrocapsules transplanted into rats
Xenotransplanta-tion 2003;3:240–51.
22 Parekh VS, Joglekar MV, Hardikar AA Differentiation of human
umbilical cord blood-derived mononuclear cells to endocrine
pancreatic lineage Differentiation 2009;4:232–40.
23 Phuc PV, Nhung TH, Loan DT, Chung DC, Ngoc PK
Differ-entiating of banked human umbilical cord blood-derived
mes-enchymal stem cells into insulin-secreting cells In Vitro Cell Dev
Biol Anim 2011;1:54–63.
24 Shao S, Gao Y, Xie B, Xie F, Lim SK, Li G Correction of
hyperglycemia in Type 1 diabetic models by transplantation of
encapsulated insulin-producing cells derived from mouse embryo
progenitor J Endocrinol 2011 (epub ahead of print).
25 Soon-Shiong P, Feldman E, Nelson R, Heintz R, Merideth N,
Sandford P, Zheng T, Komtebedde J Long-term reversal of
diabetes in the large animal model by encapsulated islet
trans-plantation Transplant Proc 1992;6:2946–7.
26 Soon-Shiong P, Feldman E, Nelson R, Heintz R, Yao Q, Yao Z,
Zheng T, Merideth N, Skjak-Braek G, Espevik T Long-term
reversal of diabetes by the injection of immunoprotected islets.
Proc Natl Acad Sci USA 1993;12:5843–7.
27 Soon-Shiong P, Feldman E, Nelson R, Komtebedde J, Smidsrod
O, Skjak-Braek G, Espevik T, Heintz R, Lee M Successful
reversal of spontaneous diabetes in dogs by intraperitoneal
mi-croencapsulated islets Transplantation 1992;5:769–74.
28 Sun Y, Ma X, Zhou D, Vacek I, Sun AM Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immu-nosuppression J Clin Invest 1996;6:1417–22.
29 van Schilfgaarde R, van der Burg MP, van Suylichem PT, Fro¨lich
M, Gooszen HG, Moolenaar AJ Interference by cyclosporine with the endocrine function of the canine pancreas Transplan-tation 1987;1:13–6.
30 Wang HS, Shyu JF, Shen WS, Hsu HC, Chi TC, Chen CP, Huang
SW, Shyr YM, Tang KT, Chen TH Transplantation of insulin producing cells derived from umbilical cord stromal mesenchy-mal stem cells to treat NOD mice Cell Transplant 2010 (epub ahead of print).
31 Wu LF, Wang NN, Liu YS, Wei X Differentiation of Wharton’s jelly primitive stromal cells into insulin-producing cells in com-parison with bone marrow mesenchymal stem cells Tissue Eng Part A 2009;10:2865–73.
32 Xie QP, Huang H, Xu B, Dong X, Gao SL, Zhang B, Wu YL Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro Differentiation 2009;5:483–91.
33 Zhang Y, Ren Z, Zou C, Wang S, Luo B, Li F, Liu S, Zhang YA Insulin-producing cells from human pancreatic islet-derived progenitor cells following transplantation in mice Cell Biol Int.
2010 (Epub ahead of print).
34 Zhang Y, Shen W, Hua J, Lei A, Lv C, Wang H, Yang C, Gao Z, Dou Z Pancreatic islet-like clusters from bone marrow mesen-chymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes Rejuvenation Res 2010;6: 695–706.