Abstract
Type 1 diabetes mellitus is caused by T-cell-mediated autoimmune destruction of pancreatic β-cells. Systemic administration of mesenchymal stem cells (MSCs) brings about their incorporation into a variety of tissues with immunosuppressive effects, resulting in regeneration of pancreatic islets. We previously showed that human MSCs isolated from Wharton's jelly (WJ-MSCs) represent a potential cell source to treat diabetes. However, the underlying mechanisms are unclear. The purpose of this study was to discern whether undifferentiated WJ-MSCs can differentiate into pancreatic insulin-producing cells (IPCs) and modify immunological responses in nonobese diabetic (NOD) mice. Undifferentiated WJ-MSCs underwent lentiviral transduction to express green fluorescent protein (GFP) and then were injected into the retro-orbital venous sinus of NOD mice. Seven days after transplantation, fluorescent islet-like cell clusters in the pancreas were apparent. WJ-MSC-GFP-treated NOD mice had significantly lower blood glucose and higher survival rates than saline-treated mice. Systemic and local levels of autoaggressive T-cells, including T helper 1 cells and IL-17-producing T-cells, were reduced, and regulatory T-cell levels were increased. Furthermore, anti-inflammatory cytokine levels were increased, and dendritic cells were decreased. At 23 days, higher human C-peptide and serum insulin levels and improved glucose tolerance were found. Additionally, WJ-MSCs-GFP differentiated into IPCs as shown by colocalization of human C-peptide and GFP in the pancreas. Significantly more intact islets and less severe insulitis were observed. In conclusion, undifferentiated WJ-MSCs can differentiate into IPCs in vivo with immunomodulatory effects and repair the destroyed islets in NOD mice.
Keywords
Introduction
Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which CD4+ and CD8+ T-cells infiltrate the islets of Langerhans, resulting in β-cell destruction (47). Current treatments to manage the progression of T1DM are limited to the continuous replacement of insulin, which is only a palliative treatment. Therefore, the feasibility of whole pancreas or islet transplantation to treat T1DM was assessed in a number of clinical settings. However, the need for lifelong immune suppression and the limited availability of human donor organs are major drawbacks to these approaches. In addition, most reports showed that while T1DM patients achieved insulin independence during the first year after human islet transplantation, it was followed by a subsequent decline (30,36). Thus, clinical interest has recently switched toward the development of cellular therapy approaches that ideally preserve the remaining β-cells, restore the function of destroyed β-cells, and protect the replaced insulin-producing cells (IPCs) from autoimmunity.
The autoimmune diabetes-prone nonobese diabetic (NOD) mice have played an important role in the discovery of the mechanisms of T1DM. NOD mice spontaneously develop T-cell-dependent β-cell destruction that resembles human T1DM. The CD4+ T-cells in NOD mice show a T helper 1 (Th1)-cell-dominant phenotype with increased expression of interferon (IFN)-γ and decreased interleukin (IL)-4 (25). Th1 cells play a pathogenic role in the initiation of T1DM. In contrast, administration of typical T helper 2 (Th2) cytokines, such as IL-4 or IL-10, can delay the onset of autoimmune diabetes and even prohibit its progression (11,38). Th17 cells, a subset of CD4+ T-cells that secrete IL-17, have also been implicated in NOD mice as well as other autoimmune diseases, including multiple sclerosis and lupus (16). Defects in the number and function of regulatory T-cells (Tregs) may also underlie the spontaneous development of autoimmune diabetes in NOD mice, as Tregs are the primary mediators of peripheral self-tolerance, suppressing the proliferation and cytokine responses of other immune cells (41). In this study, we used NOD mice to study the basic mechanism of T1DM and to test the therapeutic effect of cell therapy.
Much research has focused on promoting the differentiation of IPCs from embryonic stem cells, perinatal stem cells [e.g., umbilical cord blood cells, placenta, and Wharton's jelly (WJ) cells], adult stem cells (e.g., bone marrow-mesenchymal stem cells and adipose tissue cells) and hepatic progenitors (20). However, current cell therapeutic approaches for T1DM have had minimal results, with only a few patients becoming insulin independent (10,32). Transplantation of mesenchymal stem cells (MSCs) to human patients with different diseases has been well tolerated with some beneficial effects (35). WJ of the human umbilical cord contains MSCs, which can differentiate into ectodermal, mesodermal, and endodermal cellular lineages (7,24). Cultures of these different cell types can be successfully expanded ex vivo and cryopreserved (1,2). WJ-MSCs are perinatal stem cells that are potential cell sources for regeneration therapy of hematopoietic reconstitution, Parkinson's disease, macular degeneration, spinal cord injuries, and diabetes mellitus (44). The main advantages of WJ-MSCs as cell sources compared to other stem cell origins include their abundant cell availability, minimal or no tissue damage to the donor, and hypoimmunologic profile (14,15). In addition, WJ-MSCs have homing capabilities, which induce them to travel to inflammatory sites and locally affect the inflammatory/immune-mediated tissue damage with subsequent support to tissue healing (8,28). Given the multipotency and immunoregulatory capacity of WJ-MSCs, they have the potential to achieve an immediate restoration of euglycemia in patients, blockade inflammatory and immune processes in the islet microenvironment, and indirectly promote the physiological replacement of β-cells from the proliferation of local precursors. Further investigation of the therapeutic mechanism in disease animal models is necessary to ensure safety and efficacy.
There are several advantages of using MSCs in T1DM patients. In vitro, WJ-MSCs can differentiate into IPCs using differentiation medium (45,51). In addition, these cells can incorporate and survive in the liver, thus contributing to glycemia normalization in NOD mice. However, the relationship of WJ-MSC homing, regeneration, and anti-inflammatory mechanisms and their putative therapeutic effect is still poorly understood. In this study, undifferentiated WJ-MSCs transduced with green fluorescent protein (GFP) were used to trace their distribution after transplantation and examine whether they can promote pancreatic IPC differentiation. The immunoprivileged and immunomodulatory characteristics of undifferentiated WJ-MSCs in NOD mice were also investigated.
Materials and Methods
Isolation and Culture of WJ-MSCs
The study protocol and procedures were approved by the appropriate Institutional Review Board approval, and all parents gave their informed consent. Fresh human umbilical cords from three male and three female babies were obtained after birth and stored in Hank's balanced salt solution (HBSS; Biological Industries, Kibbutz Beit Haemek, Israel) containing 1% antibiotic antimycotic solution (Corning Inc., Manassas, VA, USA) for 1 to 24 h before tissue processing to obtain MSCs. The isolation of MSCs was carried out as described by Wang et al. (50). Briefly, the cord was soaked in HBSS containing 10% antibiotic antimycotic solution (Corning Inc.) for 10 min and spread flat. After discarding two ends and removing blood vessels, the WJ was scraped off the inner layer and centrifuged at 250 χ g for 5 min at room temperature. The pellet was washed with serum-free Dulbecco's modified Eagle's medium (DMEM; Corning Inc.) and centrifuged at 250 χ g for 5 min twice. This pellet was resuspended in 15 ml DMEM containing 10% fetal bovine serum (FBS; Thermofisher Scientific, Swedesboro, NJ, USA) and 0.2 mg/ml collagenase (Sigma-Aldrich, St. Louis MO, USA) then incubated in a humidified atmosphere with 5% CO2 and at 37°C for 16 h, followed by the addition of 2.5% trypsin (Corning Inc.) and a further incubation for 30 min. Then cells were centrifuged at 1,000 χ g 15 min, and suspended in 10 ml HBSS (Biological Industries). The HBSS was slowly transferred to a 50-ml tube containing 10 ml Ficoll-Paque PLUS (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). After 30 min 500 χ g centrifuge, the mononuclear cells were collected in interphase and washed twice with HBSS. Finally the cells were cultured in DMEM containing 10% FBS (Thermofisher Scientific) in 5% CO2 and at 37°C. WJ-MSCs cultured in early (4-6) passages were used in the following experiments.
Transduction of WJ-MSCs with GFP
The Lentivector Packaging kit, which includes the pPackH1 Packaging Plasmid (mixture of pPACKH1-GAG, pPACKH1-REV, and pVSV-G plasmids, 0.5 μg/μl) and the transfer vector pSIF-H1-siLuc-copGFP (0.5 μg/μl) containing an enhanced GFP gene, was used to transduce GFP into WJ-MSCs (System Biosciences, Mountain View, CA, USA). Virion particles were produced in 293T cells (Invitrogen, Carlsbad, CA, USA) by transfection using the TransIT-2020 Transfection Reagent (Mirus, Madison, WI, USA). The 293T cells were seeded in 75-cm2 flasks (Corning Inc.) at an initial density of 1.3 χ 105 cells/cm2 with 10 ml of DMEM containing 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin (Corning Inc.). At 24 h posttransfection, the media was replaced with fresh DMEM with 2% FBS. The medium was changed every 24 h for 3 days. The media was removed, pooled, and filtered (pore size: 0.45 μm; Merck Millipore, Rockland, MA, USA), and centrifuged at 50,000 χ g for 90 min. The resulting pellets were resuspended in serum-free DMEM. The virus titer was determined by transducing 293T cells with the viral preparation and examining GFP expression using fluorescent-activated cell sorter (FACS; Becton Dickinson, San Jose, CA, USA) analysis. The virus titers used in later experiments were 1-2 χ 107 transducing units/ml.
Freshly isolated MSCs were first preincubated overnight in DMEM with 10% FBS at 37°C in 5% CO2 and 95% air. After virus exposure, transduced cells were cultured in serum-containing medium for 5 to 8 days, and medium was changed every 48 h. MSC transduction efficacy was assessed 5 days after transduction. Cells were rinsed with phosphate buffer solution (PBS; Gibco BRL, Grand Island, NY, USA) and incubated with 0.5% porcine trypsin (same as that mentioned earlier) and 0.2% ethyl-enediaminetetraacetic acid (Sigma-Aldrich) for 10 min to dissociate the cells. The viability of dispersed cells was evaluated by trypan blue (Sigma-Aldrich) exclusion prior to fixation in 1% paraformaldehyde (Bio Basic Inc., Markham, ON, Canada) in PBS. Cell viability was always >90%. Following centrifugation at 250 χ g for 5 min, the cell pellet was resuspended in PBS, and GFP expression was assessed in a FACSort flow cytometer (Becton Dickinson). The data obtained were further analyzed using the CellQuest (Becton Dickinson) software.
Laboratory Animals
The institutional review committee for animal experiments approved all the animal procedures. Female NOD mice 20 weeks old and weighing 24-27 g were obtained from the Laboratory Animal Center (National Defense Medical Center, Taipei, Taiwan, R.O.C.). Mice had ad libitum access to food and water and were housed on a 12-h light and 12-h dark cycle. Spontaneous diabetes started at 12 weeks of age, and the incidence of diabetes reached 80% by 20 weeks.
Transplantation and Physiological Monitoring
NOD mice were divided into two groups with 30 mice/group. After blood sugar spontaneously increased to 750-810 mg/dl, the mice were restrained, and 5 χ 105 WJ-MSCs suspended in 0.1 ml of normal saline were injected through the retro-orbital vein as previously described (39). The control, sham group, received 0.1 ml normal saline. Body weight and blood sugar were recorded twice per week before and after cell transplantation. For intraperitoneal glucose tolerance testing, NOD mice were fasted for 6 to 8 h, and 10% glucose (2 g of glucose/kg of body weight; Sigma-Aldrich) was administered intraperitoneally. Blood samples were obtained from the snipped tail and analyzed for glucose levels using the Roche ACCU-CHEK glucose meter (Roche Diagnostics, Indianapolis, IN, USA). The blood samples with glucose level above 600 mg/dl were rechecked using the Glucose Assay Kit (Abcam, Cambridge, MA, USA) following the manufacturer's instructions. After 7 or 23 days posttransplantation, animals were anesthetized and exsanguinated.
Plasma Insulin Quantification
From fasted alert animals, blood samples were collected from the tail vein. After centrifugation at 2,000 χ g, plasma human C-peptide and insulin concentrations were measured using ultrasensitive enzyme-linked immunosorbent assay (ELISA) kits (Mercodia, Uppsala, Sweden) following manufacturer's instructions.
Immunofluorescence Analysis
Mice were sacrificed at 7 or 23 days after transplantation and perfused with 4% formaldehyde (Ferak, Berlin, Germany). The pancreatic tissues were dissected and cut into 0.5- to 1.0-cm3 pieces. The samples were dehydrated and embedded in OCT (Sakura Finetek USA Inc., Torrance, CA, USA) in liquid nitrogen. Cryosections of 5 μm were washed twice with PBS, then incubated overnight at 4°C with rabbit anti-human C-peptide antibodies (1:100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-insulin antibodies (1:200; Cell Signaling Technology, Danvers, MA, USA), mouse anti-cluster of differentiation 4 (CD4) antibodies (1:500; eBioscience, San Diego, CA, USA), mouse anti-interleukin-17A (IL-17A) antibodies (1:500; BioLegend, San Diego, CA, USA), or rat anti-mouse forkhead box P3 (Foxp3) antibodies (1:100; eBioscience). After three washes in PBS, slides were incubated for 1 h at room temperature with cyanine 3-labeled goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA, USA) or fluorescein isothiocyanate (FITC)-labeled donkey anti-rat IgG (1:200; Jackson ImmunoResearch). Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; 1:5,000; Molecular Probes, Inc., Eugene, OR, USA). After the sections were mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA), microscopy was performed using a confocal microscope equipped with difference interference contrast light path (LSM 510; Zeiss, Göttingen, Germany).
Assessment of Insulitis
Pancreatic tissue obtained from the mice 23 days after transplantation was fixed in formalin (Sigma-Aldrich), embedded in paraffin, sectioned at 5 μm thickness, stained with hematoxylin and eosin (ScyTec Laboratories, Inc., West Logan, UT, USA), and examined for inflammation. The degree of insulitis in the pancreas was evaluated by scoring 15-30 islets/mouse in a blinded fashion using the following criteria: 0, normal islet; 1, peri-insulitis; 2, mononuclear cell infiltration in 25% of the islet; 3, mononuclear cell infiltration in 25-50% of the islet; 4, >50% of the islet infiltrated as previously described (48). Investigators were blind to the identity of the section. At least 200 islets from saline-treated and WJ-MSC-GFP-treated NOD mice were evaluated for the severity of insulitis.
Flow Cytometry Analysis and ELISA
Lymphocytes were harvested from the spleen or pancreatic lymph nodes (PLNs), filtered through a 100-μm strainer (BD Biosciences, Bedford, MA, USA), and treated with Tris-buffered ammonium chloride (Sigma-Aldrich) to eliminate the erythrocytes. Isolated lymphocytes were then stained with anti-mouse CD4-allophycocyanine (APC), CD8-peridinin chlorophyll protein complex, CD11c-APC, CD25-phycoerythrin (PE), and CD86-FITC antibodies (all BD Biosciences Pharmingen, San Jose, CA, USA) for 30 min at 4°C. For Foxp3 staining, surface molecules were fixed and permeabilized overnight with fixation/permeabilization working solution (eBioscience). After fixation and permeabilization, cells were stained with FITC-conjugated anti-Foxp3 (clone FJK-16S) (eBioscience). For intracellular cytokine staining, cells were stimulated for 5 h with 20 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1 μM ionomycin (Santa Cruz) in the presence of 4 μM mon-ensin (Santa Cruz). Cells were subsequently stained with anti-mouse CD4-FITC antibody (eBioscience) at 4°C for 30 min. After fixing and permeabilizing with BD Cytofix/Cytoperm kit (BD Pharmingen), cells were washed once with PBS and stained with anti-mouse IL-10-APC, anti-mouse IL-17A-APC, or anti-mouse IFNγ-PE antibodies (all from eBioscience) at 4°C for 30 min. Isotype controls (eBioscience) were used to distinguish nonspecific background staining from specific antibody staining.
Flow cytometric analysis was performed with a FACS-Calibur (BD Biosciences Pharmingen) and CellQuest software (Becton Dickinson). Data were analyzed with Summit v4.3 software (Becton Dickinson), and T-lymphocytes were gated according to their forward scatter channel, side scatter channel, and CD4 expression. Results are presented as the percentage of total CD4+ cells that are positive for each cytokine after ex vivo stimulation. To determine secreted IL-10, IL-17A, IFN-γ, TGF-β, and TNF-α levels, supernatants were harvested from splenocytes, and ELISA was carried out according to the manufacturer's instruction (eBioscience).
T-Lymphocyte Proliferation Assay
After washing, lymphocytes were resuspended at a concentration of 5 χ 106 cells/ml in RPMI-1640 medium (Gibco BRL) supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were labeled with carboxyfluorescein succinimidyl ester (10 μM; Invitrogen) for 10 min at 37°C. Cells were subsequently stimulated with plate-coated anti-CD3 (1 μg/ml) and anti-CD28 antibodies (0.5 μg/ml; BD Biosciences Pharmingen). After 5 days, the cells were incubated with anti-mouse CD4-FITC antibody (eBioscience) at 4°C for 30 min. Flow cytometric analysis was performed with a FACSCalibur (BD Biosciences Pharmingen) and CellQuest software (Becton Dickinson).
Quantitative Real-Time Polymerase Chain Reaction (PCR)
Total RNA was prepared from PLNs using TRIzol reagent (Invitrogen) in accordance with the manufacturer's protocols. Total RNA (5 μg) was reverse transcribed with oligo dT by SuperScript III Reverse Transcriptase (Invitrogen). The forward and reverse primers and annealing temperature for the PCR reaction are listed in Table 1. Real-time PCR was performed in a final volume of 20 μl containing 50 ng cDNA, Fast SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA), 3 mM MgCl2, and 0.5 μM of each primer, using Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies). To control for possible genomic DNA contamination, reactions without reverse transcription were included. Amplicons were characterized according to their size after agarose gel electrophoresis and their melting temperature determined by PCR. The threshold cycle (Ct) value was defined as the cycle number at which the fluorescence crossed a fixed threshold above baseline. For relative quantitation, fold changes were measured using the ΔΔCt method. For each sample, the Ct value of cytokine mRNA was compared with the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) endogenous control as ΔCt(ΔCt=Ctcytokine - CtGAPDH). The fold change in cytokine mRNA in experimental samples relative to the control samples was determined by 2-ΔΔCt, where ΔΔCt= ΔCtExperiment - ΔCtControl.
Primers and PCR Product Sizes
Statistical Analysis
Group comparisons were made by one-way ANOVA followed by the Dunnett's test using SPSS software, version 13.0 (Armonk, NY, USA). Survival was assessed using Kaplan-Meier survival curves. A value of p <0.05 was considered statistically significant.
Results
Transplantation of Undifferentiated Human WJ-MSCs Alleviates Hyperglycemia and Increases NOD Mouse Survival
IPCs induced from WJ-MSCs decrease blood glucose levels in diabetic rodents (45,51). To examine whether WJ-MSCs without in vitro IPC induction have a similar effect, we first transduced the cells with GFP, using a lentiviral vector, to track the transplanted cells. More than 90% of the cells expressed GFP as determined by confocal microscopy (Fig. 1A) and flow cytometry analysis (Fig. 1B).
Green fluorescent protein (GFP)-transduced Wharton's jelly mesenchymal stem cells (WJ-MSCs) decrease blood glucose level and increase survival in nonobese diabetic (NOD) mice. (A) Confocal analysis of WJ-MSCs after lentiviral-mediated transduction with GFP. GFP expression is detected in most of the GFP-transduced WJ-MSCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm. (B) Flow cytometry analysis of the GFP-transduced WJ-MSCs. Over 90% of the MSCs expressed GFP. (C) Changes in blood glucose level and Kaplan-Meier survival curves for the saline control (red), WJ-MSC (blue), WJ-MSC-GFP (green), and NOD groups (i.e., euglycemic NOD mice; black). Results are means ± SEMs. A significant decrease in blood glucose and increase in survival rate were found in both the WJ-MSC and WJ-MSC-GFP groups compared to the saline control group (*p <0.01).
After injection of 5 χ 105 WJ-MSCs (without GFP transduction), WJ-MSCs-GFP, or saline into the retro-orbital vein of NOD mice with severe hyperglycemia (750-810 mg/dl), blood glucose levels and survival were determined (Fig. 1C). WJ-MSCs and WJ-MSCs-GFP had a similar effect in reducing blood glucose levels in NOD mice. Significant decreases in blood glucose were found 3 days after transplantation compared with the saline control group (p <0.01). The decrease in blood glucose level was maintained throughout the experimental period (23 days, Fig. 1C). Increased survival rates were also found in NOD mice that received WJ-MSCs or WJ-MSCs-GFP. Most of the NOD mice that received saline died by 23 days (p <0.01).
Transplantation of WJ-MSCs Restores the Composition and Function of T-Lymphocytes and Suppresses Dendritic Cells in NOD Mice
An imbalance between Th1 and Th2 cell responses (31), increased levels of pathological dendritic cells (5,6), and reduced numbers of Treg cells (40) predispose NOD mice to developing autoimmune diabetes. To investigate whether the protective effect of WJ-MSC transplantation works through these immunomodulatory functions, we analyzed using flow cytometry the populations of lymphocytes taken from the spleen and PLNs (Fig. 2). There was a significant decrease in CD4+ T-cells in the spleen and PLNs of WJ-MSC-treated NOD mice compared with those of the control NOD mice (p <0.01) (Fig. 2A). The splenocytes harvested from WJ-MSC-treated mice displayed significantly reduced responses to plate-bound anti-CD3 stimulation of T-cell proliferation, compared with control mice (p <0.01) (Fig. 2B).
WJ-MSCs restore the systemic and local balance between autoaggressive and regulatory T-cells in NOD mice. Seven days posttransplantation, mononuclear cells were isolated from the spleen and PLNs. The lymphocyte and dendritic cell populations were examined using flow cytometry analysis for CD4 (A), CD4 after stimulation with CD3 and CD28 (B), IFN production (C), IL-10 production (D), IL-17 (E), CD4+/CD25+/FoxP3+ lymphocytes (F), CD11c+ dendritic cells (G), and CD11c+/CD86+ dendritic cells (H). Eight NOD mice were used for each WJ-MSC-treated group (black bar) and saline control group (gray bar). Data were expressed as mean ± SD. *p <0.01 compared to the saline control group. PerCP, peridinin chlorophyll protein complex; APC, allophycocyanine; CFSE, carboxyfluorescein succinimidyl ester; PE, phycoerythrin; FITC, fluorescein isothiocyanate.
Th2 cells confer protection against Th1-mediated destruction of β-cells (4). Our results revealed a shift toward the Th2 profile in WJ-MSC-treated NOD mice as evidenced by the decreased levels of IFN-γ-producing CD4+ T-cells and the increase in IL-10-producing CD4+ T-cells (p <0.01) (Fig. 2C, D).
Th17 cell population is involved in the pathogenesis of autoimmune diabetes in the NOD mice (16). In WJ-MSC-treated NOD mice, there was a significant decrease in IL-17-producing CD4+ lymphocytes in both the spleen and PLNs (p <0.01) (Fig. 2E). MSCs facilitate the generation of Tregs (12,21); therefore, we examined the effect of WJ-MSC transplantation on the Treg population and found a significant increase in the percentage of CD4+ CD25+ FoxP3+ cells in the spleen and PLN (p <0.01) (Fig. 2F).
Dendritic cells are antigen-presenting cells that play a key role in regulating the immune response. To examine the phenotype and function of dendritic cells, we assessed the level of surface expression of CD11c and the costimulatory molecule, CD86. As shown in Figure 2G, WJ-MSC-treated NOD mice had a significant decrease in CD11c+ cells in the PLN (p <0.01). Decreased CD11c+/CD86+ cells in both the spleen and PLN were also observed (p <0.01) (Fig. 2H). Taken together, these results indicate that WJ-MSC transplantation reduced systemic and pancreatic Th1 and Th17 cell levels, increased Th2 and Treg cell levels, and suppressed the activation of T-cells by decreasing the level of antigen-presenting dendritic cells.
WJ-MSCs Increase the Systemic Anti-Inflammatory Response in NOD Mice
To explore the immunomodulatory mechanism of WJ-MSCs, we examined the expression of cytokines in splenocytes by real-time PCR. In the WJ-MSC-treated NOD mice, the expression of Th1 cell cytokines, such as IFN-γ, IL-1β, TNF-α, and monocyte chemotactic protein 1 (MCP-1) were significantly downregulated (p <0.01) (Fig. 3A-D). In contrast, the expression of Th2 cell cytokines, such as IL-4 and IL-10, was significantly upregulated in the WJ-MSC-treated NOD mice (p <0.01) (Fig. 3E, F). These results are consistent with the analysis of the Th1/2 cell populations analyzed by FACS in Figure 2. Moreover, the expression of IL-17A was significantly decreased (p <0.01) (Fig. 3G), and the expression of FoxP3 (p <0.01) (Fig. 3H) was significantly increased in the MSC-treated NOD mice.
WJ-MSCs restore the pancreatic inflammatory balance in NOD mice. Seven days posttransplantation, PLN mononuclear cells were isolated, and mRNA levels of proinflammatory and anti-inflammatory markers (IFN-γ, IL-1β, TNF-α, MCP-1, IL-4, IL-10, IL-17, and FoxP3) were analyzed by real-time RT-PCR (n = 8/group). *p <0.01 compared to the saline control group.
The cytokine profile of the splenocytes was also analyzed by ELISA (Fig. 4). Splenocytes from WJ-MSC-treated NOD mice had significantly lower concentrations of Th1 cytokines, such as IFN-γ and TNF-α (p <0.01) (Fig. 4A, B) as well as the Th17 cytokine, IL-17A (p <0.01) (Fig. 4E). In contrast, the concentration of Th2 cytokines, such as IL-10 and TGF-β1 (p <0.01) (Fig. 4C, D), was much higher in WJ-MSC-treated NOD mice.
WJ-MSCs increase the pancreatic anti-inflammatory environment in NOD mice. Seven days posttransplantation, PLN mononuclear cells were isolated and cultured in DMEM with 1% FBS for 5 h. The supernatants of cultured cells were analyzed by ELISA (n = 8/group). *p <0.01 compared to the saline control group.
WJ-MSCs Differentiate Into Pancreatic IPCs and Reduce the Severity of Insulitis in NOD Mice
Human MSCs traffic to pancreatic islets once they are transplanted into diabetic mice (18). In conjunction with the blood glucose level and survival data, histological analysis of the pancreas in the WJ-MSC-GFP-treated and control NOD mice was performed. Groups of cells expressing GFP were detected in the pancreas of NOD mice 7 days after transplantation (Fig. 5A, upper panels). We estimated that about 1% of the transplanted WJ-MSC-GFP (5,120 ± 345, n=6) were found in the pancreas of NOD mice. At 23 days posttransplantation, colocalization of GFP and human C-peptide in islet-like cell clusters was found in the pancreas of NOD mice (Fig. 5A, lower panels).
WJ-MSCs differentiate into IPCs and reduce the severity of pancreatic insulitis in NOD mice. (A) Confocal analysis of the transplanted WJ-MSCs. The expression of GFP was used to track the transplanted cells. Pancreas tissues were obtained 7 days (upper panel) or 23 days (lower panel) post-cell transplantation. Clusters of cells with GFP expression were detected. In the lower panel, pancreatic tissue was double labeled with human C-peptide (red) and the nuclear stain, DAPI (blue). White arrows indicate colocalization of the C-peptide with the GFP. Scale bar: 20 μm in the upper panel and 50 μm in the lower panel. DIC, differential interference contrast microscopy. (B) The severity of insulitis was examined using hematoxylin and eosin-stained sections of pancreatic tissues from NOD mice at 23 days post-WJ-MSC transplantation. While the saline-treated control NOD mice showed severe insulitis and few intact islets, the insulitis score of the WJ-MSC-GFP group was significantly decreased (p <0.05). Scale bar: 20 μm. (C) Improvement in glucose tolerance in diabetic NOD mice transplanted with WJ-MSCs (p <0.01). At day 14 posttransplantation, mice were fasted overnight and then injected intraperitoneally with glucose (2 g/kg of body weight). Blood glucose levels were measured at the indicated time with a glucometer. Results are the mean ± SD of n=6/group.
Insulitis scoring using blinded samples showed that 50.9% of islets from WJ-MSC-GFP-treated NOD mice were free from lymphocyte infiltration with only 4.0% in saline sham controls (p <0.05). Destructive insulitis was observed in 10.4% of islets from WJ-MSC-GFP-treated NOD mice compared with 76.3% in controls (p <0.05) (Fig. 5B). Blood glucose values after intraperitoneal glucose injection showed significantly lower concentration in WJ-MSC-GFP-treated mice (p <0.01) (Fig. 5C). In contrast, blood glucose levels remained elevated in mice transplanted with saline. In addition, significant increases of both human C-peptide (52.0 ± 2.3 vs. 5.1 ± 1.5 ng/L) and mouse insulin levels (107.7 ± 12.3 vs. 30.1 ± 2.1 ng/L) in blood were found in the WJ-MSC-GFP-treated mice compared with control NOD mice (p <0.01).
Reduced Severity of Insulitis After WJ-MSC Transplantation Was Associated with an Increase in Foxp3+ Cells and a Decrease in IL-17A+ Cells
To determine the contribution of different T-cell populations in the development of insulitis, immunofluorescence staining for CD4, Foxp3, and IL-17A was carried out in combination with insulin staining and compared with control (left panels) and WJ-MSC-treated (right panels) NOD mice (Fig. 6). As shown in Figure 6A and B, insulitic lesions in the control NOD mice were composed primarily of CD4+ cells with low insulin- and Foxp3-stained cells. Examination of multiple sections of control NOD pancreatic tissue for IL-17A staining consistently showed a small number of IL-17A+ cells within the peri-islet infiltrates, regardless of the degree of insulitis (Fig. 6C). Pancreatic sections from NOD mice treated with WJ-MSCs demonstrated a reduced level of insulitis that was composed primarily of CD4+ cells and enriched with insulin- and Foxp3+ cells compared with control animals (Fig. 6A, B). In addition, few IL-17A+ cells were observed in WJ-MSC-treated animals, with only a small proportion of inflamed islets staining positive for IL-17A (Fig. 6C). Overall, these data indicate that inhibition of Th17A cells with WJ-MSC transplantation significantly reduced islet-specific inflammatory T-cell infiltration and increased the proportion of Foxp3+ cells around the islets.
WJ-MSCs reduce the degree of peri-islet T-cell infiltration, increase the levels of Foxp3+ cells, and decrease the levels of IL-17A+ cells. Pancreatic tissues were collected from NOD mice 23 days posttransplantation. Tissue sections were stained for CD4 (A), Foxp3 (B), or IL-17A (C) (each in green) in combination with insulin (red) and nuclei (DAPI in blue). WJ-MSCs reduced the frequency of both CD4+ T-cells and increased the number of Foxp3+ Tregs in the peri-islet infiltrate compared with controls. IL-17A+ staining was only visible in a small proportion of cells present within the insulitic lesion in control animals, and this frequency was further reduced after treatment with WJ-MSCs. Pancreatic tissues harvested from six to eight NOD mice per treatment group were analyzed, and representative sections from each combination of staining are shown. Scale bar: 20 μm.
Discussion
The incidence of T1DM continues to climb steadily worldwide, making it one of the most challenging health issues of the 21st century (42). Hence, a therapeutic strategy aimed to recover the lost IPCs and inhibit the autoimmune destruction of remnant and newly generated endocrine precursor cells is highly desirable (19). WJ-MSCs represent a promising source for regeneration therapy for T1DM due to their transdifferentiation potential as well as their immunomodulatory properties. We and other researchers have shown that systemic administration of WJ-MSCs results in recovery of pancreatic islets from insulitis, increased blood insulin secretion, and correction of hyperglycemia (23,45,51). Nevertheless, the relative contribution of each of these regenerative mechanisms by WJ-MSCs (i.e., transdifferentiation, immunomodulation, and proliferation/differentiation induction) underlying this therapeutic effect is poorly understood. Here we found that NOD mice had dramatic recovery from severe hyperglycemia (750-810 mg/dl) 3 days after transplantation of WJ-MSCs, which endured to the end of the study period (23 days) and was accompanied by increased survival rates. Moreover, although few intact islets and severe insulitis were found in NOD mice with blood glucose level >500 mg/dl, alleviation of hyperglycemia could also be observed as early as 3 days posttransplantation. This suggests that the transplanted cells not only restored the ability of the pancreatic microenvironment to support cell regeneration but also quickly restored the expression and secretion of paracrine cytokines that are critical for glucose homeostasis. The elevated serum human C-peptide level approximately 2 weeks after transplantation was thought to be related to the in vivo differentiation of WJ-MSCs into IPCs, as colocalization of human C-peptide and GFP was detected in the pancreas of NOD mice that received WJ-MSC-GFP transplantation. Repair of the destroyed β-cells and regeneration of the native endocrine precursor cells also likely occurred since increased mouse insulin level was detected.
In most studies using WJ-MSCs to treat T1DM, the WJ-MSCs were in vitro differentiated into IPCs and then transplanted into diabetic animals (13,45,51). There is still great variability in the differentiation protocols used in different studies, in terms of both the stimuli applied to cells and the length of the differentiation protocol (7,10). It is not well-known whether WJ-MSCs need to be differentiated in vitro prior to transplantation or whether they can be transplanted directly into the diabetic animals, allowing differentiation and engraftment to take place in vivo. We previously showed that WJ-MSCs differentiated into IPCs in the liver of NOD mice (51). In this study, we found that undifferentiated WJ-MSCs could also alleviate hyperglycemia in NOD mice and migrate to the pancreas. A similar antihyperglycemia result has been reported with bone marrow-derived MSCs to treat streptozotocin-induced diabetic mice (17). However in the earlier report, the authors were unable to detect transplanted cells that expressed insulin in the pancreas. The difference between findings may be due to the different animal models used and cell source (i.e., bone marrow vs. WJ).
Our results indicate that differentiation of IPCs from either WJ-MSCs or endogenous progenitors is important for WJ-MSC therapy for T1DM in NOD mice. We also observed glucagon expression in some of the transplanted WJ-MSCs in NOD mice (data not shown), suggesting that the transplanted WJ-MSCs in NOD mice followed the embryonic development of pancreatic endocrine cells (37).
While the regenerative potential of MSCs is a propitious feature, which has been used as a rationale to develop clinical trials, the immunomodulatory effects of MSC have recently become of interest as well. A common feature of autoimmune disease is the occurrence and persistence of autoaggressive T-cells with a concomitant breakdown in the maintenance of peripheral tolerance. In T1DM, autoaggressive T-cells promote inflammation reaction and stimulate B-cells to produce autoantibodies, which directly attack pancreatic β-cells, leading to their death (49). Both in patients and in animal models of T1DM, the levels of autoaggressive T-cells are characterized by the expression of CD4 and CD40 surface markers, which are significantly higher than those found in nondiabetic counterparts (9). Antigen-specific activation and expansion of autoaggressive T-cells in secondary lymphoid organs may be suppressed by Tregs (9). While a Treg cell functional deficiency results in T1DM, reconstitution of the Treg cell pool prevents its development (22). Therefore, the fine balance between autoaggressive and Tregs must be restored in order to successfully treat T1DM (9).
In this study, we found that the abundance of autoaggressive T-cells was reduced 7 days after transplantation; the Treg cells were increased both systemically (i.e., spleen) and locally (i.e., PLNs) in NOD mice that received WJ-MSCs. Furthermore, when we assessed the functionality of the T-lymphocytes isolated from WJ-MSC-treated diabetic mice, we observed that they were less prone to produce proinflammatory cytokines (i.e., IFN-γ, TNF-α, IL-1, and MCP-1) than the same cells isolated from saline-treated diabetic mice. WJ-MSC administration also resulted in a shift from proinflammatory to anti-inflammatory cytokine expression and secretion. Thus, our data suggest that donor WJ-MSCs inhibit the expansion of autoaggressive T-cells by inducing Tregs and modifying the cytokine profile. The former is in concordance with previous reports of other autoimmune diseases and for T1DM in other animal models (3,18,33). The latter agrees with the fact that, both in vitro and in vivo, MSCs might secrete anti-inflammatory and immunoregulatory factors that suppress Th1 activation (18,34,46). Whether the observed changes are selective for pancreatic immunogens or compromise the whole immune response is currently unknown. Nevertheless, we found no complications indicative of systemic immunosuppression (i.e., infections and tumor formation) in WJ-MSC-treated NOD mice.
The plasticity within the CD4+ Th cell population, including Th1, Th2, Th17, and Tregs, are flexible (52). Networks of cytokines are keys for determining the fates of CD4+ T-cell. IL-17 and Th17 cells participate in the development of insulitis (40), and the Th17 population is involved in the pathogenesis of autoimmune diabetes in the NOD mice (16). Moreover, both IL-17 and IFN-γ signaling may synergistically contribute to the development of diabetes in NOD mice (26). In our study, a significant decrease in the levels of IL-17-producing CD4+ lymphocytes was observed in both the spleen and PLNs. The decreased mRNA expression and secretion of IL-17A and IFN-γ was observed with WJ-MSC transplantation. Furthermore, decreased expression of IL-17A-positive cells within the peri-islet infiltrates was detected 23 days after transplantation; increased expression of TGF-β was also noted, which was thought to mediate the establishment of the Treg instead of Th17 lineage (43). Thus, our results indicate that WJ-MScs inhibit Th17 cells while promoting Tregs; therefore, they can successfully alter the course of an autoimmune response, even after overt diabetes onset. Since WJ-MSCs maintain the expression of relevant immunomodulatory molecules following in vitro differentiation (29), there are two possible roles for WJ-MSCs: one which allows the amelioration of the local inflammatory environment, and another which may directly contribute to the rescue of the diabetic phenotype by differentiating into IPCs (27).
In conclusion, undifferentiated WJ-MSCs can treat NOD mice by four possible mechanisms: 1) recovery of the expression and secretion of paracrine cytokines that are critical for glucose homeostasis; 2) immunomodulatory effects, including restoring the balance between Th1/ Th2 cells and Treg/T17 cells, as well as a reduction in antigen-presenting cells; 3) support cell regeneration and repair the destroyed islets; and 4) differentiate into pancreatic IPSs. Thus, WJ-MSC transplantation alone could represent an innovative and successful cell-based treatment regimen for T1DM.
Footnotes
Acknowledgment
This research was supported by research grants from Taipei VGH (V103D-001-1) and National Science Council (NSC-102-2314-B-010-041-MY3) to T. H. Chen; National Defense Medical Center (MAB-102-56) and Tri-Service General Hospital (TSGH-C102-124) to J. F. Shyu. We would like to thank Dr. Yann-Jang Chen for his expert technical assistance. The authors declare no conflicts of interest.