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

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

Human Cell (2011) 24:86–95 DOI 10.1007/s13577-011-0018-z RESEARCH ARTICLE Improving the efficacy of type 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 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 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: pvphuc@hcmuns.edu.vn 123 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 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 and 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 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 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 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 87 8009g for 16 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 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 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 106 cells/cm2 The culture media were replaced 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 days After 30 days, the cultures were fixed in 3% formaldehyde in PBS for 10 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 were allowed to reach 123 88 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 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 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 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 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 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 The differentiated IPCs derived from mouse and human MSCs were labeled as mIPCs and hIPCs, respectively 123 P K Ngoc et al 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 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 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 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) 89 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 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 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 to 29.46 ± 0.17 g at day 30—whereas weight gain was Fig 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 123 90 P K Ngoc et al Fig 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 123 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 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 to Improving the efficacy of type diabetes therapy Fig 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 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 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 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 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 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 123 92 P K Ngoc et al Fig 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 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 123 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 diabetes therapy 93 Fig 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 (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 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 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 123 94 P K Ngoc et al 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 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 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 diabetes We believe that the approach described here is not only suitable for treating type 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 References Alejandro R, Feldman EC, Bloom AD, Kenyon NS Effects of cyclosporin on insulin and C-peptide secretion in healthy beagles Diabetes 1989;6:698–703 123 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 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 David B, Dufresne M, Nagel MD, Legallais C In vitro assessment of encapsulated C3A hepatocytes functions in a fluidized bed bioreactor Biotechnol Prog 2004;4:1204–12 De Vos P, De Haan BJ, Wolters GH, Strubbe JH, Van Schilfgaarde R Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets Diabetologia 1997;3:262–70 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 Dong QY, Chen L, Gao GQ, Wang L, Song J, Chen B, Xu YX, Sun L Allogeneic diabetic mesenchymal stem cells transplantation in streptozotocin-induced diabetic rat Clin Invest Med 2008;6:E328–37 Dufrane D, Steenberghe M, Goebbels RM, Saliez A, Guiot Y, Gianello P The influence of implantation site on the biocompatibility and survival of alginate encapsulated pig islets in rats Biomaterials 2006;17:3201–8 Duvivier-Kali VF, Omer A, Parent RJ, O’Neil JJ, Weir GC Complete protection of islets against allorejection and autoimmunity 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 placentaderived mesenchymal stem cells and islet-like cell clusters generated 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 umbilical 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 diabetes in db/db mice with Improving the efficacy of type diabetes therapy 20 21 22 23 24 25 26 27 intra-bone marrow–bone marrow transplantation plus concurrent thymic transplantation J Autoimmun 2010;4:414–23 Neshati Z, Matin MM, Bahrami AR, Moghimi A Differentiation of mesenchymal stem cells to insulin-producing cells and their impact on type diabetic rats J Physiol Biochem 2010;2:181–7 Omer A, Keegan M, Czismadia E, De Vos P, Van Rooijen N, Bonner-Weir S, Weir GC Macrophage depletion improves survival of porcine neonatal pancreatic cell clusters contained in alginate macrocapsules transplanted into rats Xenotransplantation 2003;3:240–51 Parekh VS, Joglekar MV, Hardikar AA Differentiation of human umbilical cord blood-derived mononuclear cells to endocrine pancreatic lineage Differentiation 2009;4:232–40 Phuc PV, Nhung TH, Loan DT, Chung DC, Ngoc PK Differentiating of banked human umbilical cord blood-derived mesenchymal stem cells into insulin-secreting cells In Vitro Cell Dev Biol Anim 2011;1:54–63 Shao S, Gao Y, Xie B, Xie F, Lim SK, Li G Correction of hyperglycemia in Type diabetic models by transplantation of encapsulated insulin-producing cells derived from mouse embryo progenitor J Endocrinol 2011 (epub ahead of print) 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 transplantation Transplant Proc 1992;6:2946–7 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 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 microencapsulated islets Transplantation 1992;5:769–74 95 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 immunosuppression J Clin Invest 1996;6:1417–22 29 van Schilfgaarde R, van der Burg MP, van Suylichem PT, Froălich M, Gooszen HG, Moolenaar AJ Interference by cyclosporine with the endocrine function of the canine pancreas Transplantation 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 mesenchymal 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 comparison 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 mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes Rejuvenation Res 2010;6: 695–706 123 ... the other groups In the negative control diabetic group, the blood glucose level increased from 318 ± 25.43 mg/dl on day to Improving the efficacy of type diabetes therapy Fig Encapsulation of. .. modulation of type diabetes in db/db mice with Improving the efficacy of type diabetes therapy 20 21 22 23 24 25 26 27 intra-bone marrow–bone marrow transplantation plus concurrent thymic transplantation. .. autoimmunity by a simple barium–alginate membrane Diabetes 20 01; 8 :16 98–705 10 Fan MY, Lum ZP, Fu XW, Levesque L, Tai IT, Sun AM Reversal of diabetes in BB rats by transplantation of encapsulated pancreatic