Beta Cell Count Instead of Beta Cell Mass to Assess and Localize Growth in Beta Cell Population following Pancreatic Duct Ligation in Mice

Background

Pancreatic-tail duct ligation (PDL) in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates.

Methodology

Total beta cell number and its distribution over small (<50 µm), medium, large (>100 µm) clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham.

Principal findings

PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number) which contained a higher percent proliferating beta cells (1.9% Ki67+cells) than sham tails (<0.2%); their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters.

Conclusions/significance

The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.     

Enhancing Pancreatic Beta-Cell Regeneration In Vivo with Pioglitazone and Alogliptin

Aims/Hypothesis

Pancreatic beta-cells retain limited ability to regenerate and proliferate after various physiologic triggers. Identifying therapies that are able to enhance beta-cell regeneration may therefore be useful for the treatment of both type 1 and type 2 diabetes.

Methods

In this study we investigated endogenous and transplanted beta-cell regeneration by serially quantifying changes in bioluminescence from beta-cells from transgenic mice expressing firefly luciferase under the control of the mouse insulin I promoter. We tested the ability of pioglitazone and alogliptin, two drugs developed for the treatment of type 2 diabetes, to enhance beta-cell regeneration, and also defined the effect of the immunosuppression with rapamycin and tacrolimus on transplanted islet beta mass.

Results

Pioglitazone is a stimulator of nuclear receptor peroxisome proliferator-activated receptor gamma while alogliptin is a selective dipeptidyl peptidase IV inhibitor. Pioglitazone alone, or in combination with alogliptin, enhanced endogenous beta-cell regeneration in streptozotocin-treated mice, while alogliptin alone had modest effects. In a model of syngeneic islet transplantation, immunosuppression with rapamycin and tacrolimus induced an early loss of beta-cell mass, while treatment with insulin implants to maintain normoglycemia and pioglitazone plus alogliptin was able to partially promote beta-cell mass recovery.

Conclusions/Interpretation

These data highlight the utility of bioluminescence for serially quantifying functional beta-cell mass in living mice. They also demonstrate the ability of pioglitazone, used either alone or in combination with alogliptin, to enhance regeneration of endogenous islet beta-cells as well as transplanted islets into recipients treated with rapamycin and tacrolimus.     

Impairment of rat fetal beta-cell development by maternal exposure to dexamethasone during different time-windows

Aim

Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas.

Methods

Pregnant Wistar rats received dexamethasone acetate in their drinking water (1 µg/ml) during the last week or throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal islets collected in vitro.

Results

Dexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly. Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last week, which was however the case when given throughout gestation. When given from the beginning of gestation, dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed at 15 days of fetal age.

Conclusions

GCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass.     

Epithelial-Mesenchymal Transition in Cells Expanded In Vitro from Lineage-Traced Adult Human Pancreatic Beta Cells

Background

In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.

Methodology/Principal Findings

Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.

Conclusions/Significance

These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.     

Characterisation of Age-Dependent Beta Cell Dynamics in the Male db/db Mice

Aim

To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice.

Methods

Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA_1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology.

Results

Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups.

Conclusions/Interpretation

The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.     

A Role for SPARC in the Moderation of Human Insulin Secretion

Aims/Hypothesis

We have previously shown the implication of the multifunctional protein SPARC (Secreted protein acidic and rich in cysteine)/osteonectin in insulin resistance but potential effects on beta-cell function have not been assessed. We therefore aimed to characterise the effect of SPARC on beta-cell function and features of diabetes.

Methods

We measured SPARC expression by qRT-PCR in human primary pancreatic islets, adipose tissue, liver and muscle. We then examined the relation of SPARC with glucose stimulated insulin secretion (GSIS) in primary human islets and the effect of SPARC overexpression on GSIS in beta cell lines.

Results

SPARC was expressed at measurable levels in human islets, adipose tissue, liver and skeletal muscle, and demonstrated reduced expression in primary islets from subjects with diabetes compared with controls (p< = 0.05). SPARC levels were positively correlated with GSIS in islets from control donors (p< = 0.01). Overexpression of SPARC in cultured beta-cells resulted in a 2.4-fold increase in insulin secretion in high glucose conditions (p< = 0.01).

Conclusions

Our data suggest that levels of SPARC are reduced in islets from donors with diabetes and that it has a role in insulin secretion, an effect which appears independent of SPARC’s modulation of obesity-induced insulin resistance in adipose tissue.     

Glycemic Control Promotes Pancreatic Beta-Cell Regeneration in Streptozotocin-Induced Diabetic Mice

Background

Pancreatic beta-cells proliferate following administration of the beta-cell toxin streptozotocin. Defining the conditions that promote beta-cell proliferation could benefit patients with diabetes. We have investigated the effect of insulin treatment on pancreatic beta-cell regeneration in streptozotocin-induced diabetic mice, and, in addition, report on a new approach to quantify beta-cell regeneration in vivo.

Methodology/Principal Findings

Streptozotocin-induced diabetic were treated with either syngeneic islets transplanted under the kidney capsule or subcutaneous insulin implants. After either 60 or 120 days of insulin treatment, the islet transplant or insulin implant were removed and blood glucose levels monitored for 30 days. The results showed that both islet transplants and insulin implants restored normoglycemia in the 60 and 120 day treated animals. However, only the 120-day islet and insulin implant groups maintained euglycemia (<200 mg/dl) following discontinuation of insulin treatment. The beta-cell was significantly increased in all the 120 day insulin-treated groups (insulin implant, 0.69±0.23 mg; and islet transplant, 0.91±0.23 mg) compared non-diabetic control mice (1.54±0.25 mg). We also show that we can use bioluminescent imaging to monitor beta-cell regeneration in living MIP-luc transgenic mice.

Conclusions/Significance

The results show that insulin treatment can promote beta-cell regeneration. Moreover, the extent of restoration of beta-cell function and mass depend on the length of treatment period and overall level of glycemic control with better control being associated with improved recovery. Finally, real-time bioluminescent imaging can be used to monitor beta-cell recovery in living MIP-luc transgenic mice.     

Deletion of Apoptosis Signal-Regulating Kinase 1 (ASK1) Protects Pancreatic Beta-Cells from Stress-Induced Death but Not from Glucose Homeostasis Alterations under Pro-Inflammatory Conditions

Background

Type 2 diabetes is characterized by pancreatic beta-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that apoptosis signal-regulating kinase 1 (ASK1) is involved in beta-cell death in response to different stressors. In this study, we tested whether ASK1 deficiency protects beta-cells from glucolipotoxic conditions and cytokines treatment or from glucose homeostasis alteration induced by endotoxemia.

Methodology/Principal Findings

Insulin secretion was neither affected upon shRNA-mediated downregulation of ASK1 in MIN6 cells nor in islets from ASK1-deficient mice. ASK1 silencing in MIN6 cells and deletion in islets did not prevent the deleterious effect of glucolipotoxic conditions or cytokines on insulin secretion. However, it protected MIN6 cells from death induced by ER stress or palmitate and islets from short term caspase activation in response to cytokines. Moreover, endotoxemia induced by LPS infusion increased insulin secretion during hyperglycemic clamps but the response was similar in wild-type and ASK1-deficient mice. Finally, insulin sensitivity in the presence of LPS was not affected by ASK1-deficiency.

Conclusions/Significance

Our study demonstrates that ASK1 is not involved in beta-cell function and dysfunction but controls stress-induced beta-cell death.     

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro

Background

Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.

Methodology/Principal Finding

Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.

Conclusions/Significance

These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.     

PERK Regulates the Proliferation and Development of Insulin-Secreting Beta-Cell Tumors in the Endocrine Pancreas of Mice

Background

PERK eIF2α kinase is required for the proliferation of the insulin-secreting beta- cells as well as insulin synthesis and secretion. In addition, PERK signaling has been found to be an important factor in determining growth and angiogenesis of specific types of tumors, and was attributed to PERK-dependent regulation of the hypoxic stress response. In this report we examine the role of PERK in regulating proliferation and angiogenesis of transformed beta-cells in the development of insulinomas.

Methodology

The SV40 Large T-antigen (Tag) was genetically introduced into the insulin secreting beta-cells of Perk KO mice under the control of an inducible promoter. Tumor growth and the related parameters of cell proliferation were measured. In late stage insulinomas the degree of vascularity was determined.

Principal Findings

The formation and growth of insulinomas in Perk-deficient mice was dramatically ablated with much fewer tumors, which averaged 38-fold smaller than seen in wild-type control mice. Beta-cell proliferation was ablated in Perk-deficient mice associated with reduced tumor growth. In the small number of large encapsulated insulinomas that developed in Perk-deficient mice, we found a dramatic reduction in tumor vascularity compared to similar sized insulinomas in wild-type mice. Although insulinoma growth in Perk-deficient mice was largely impaired, beta-cell mass was increased sufficiently by T-antigen induction to rescue the hypoinsulinemia and diabetes in these mice.

Conclusions

We conclude that PERK has two roles in the development of beta-cell insulinomas, first to support rapid cell proliferation during the initial transition to islet hyperplasia and later to promote angiogenesis during the progression to late-stage encapsulated tumors.     

Meta-Analysis: Prognostic Value of Survivin in Patients with Hepatocellular Carcinoma

Background

The expression of survivin is a promising prognostic indicator for some carcinomas. However, evidence for the prognostic value of survivin with respect to survival in hepatocellular carcinoma remains controversial.

Aim

To conduct a systematic review of studies evaluating survivin expression in hepatocellular carcinoma as a prognostic indicator.

Methods

The relevant literature was searched using PubMed, EMBASE, and Chinese biomedicine databases, and two meta-analyses were performed. One studied the association between survivin expression and the overall survival of patients with hepatocellular carcinoma, whereas the other studied the association between survivin expression and disease-free survival. Studies were pooled, and summary hazard ratios (HRs) were calculated. Subgroup analyses were also conducted.

Results

Fourteen eligible studies with a total of 890 patients were included in this study. Two meta-analyses were performed according to the different outcomes by which prognosis was valued. The combined HR of the overall survival studies was 2.33 (95% CI: 1.65–3.31). The combined HR of disease-free survival studies was 2.13 (95% CI: 1.65–2.75). These data appeared to be significant when stratified by detection method, the language of publication, and HR estimate. The heterogeneities were highly significant (I²>50%) when subgroup analyses of overall survival rate were conducted, whereas little heterogeneity was found when subgroup analyses of disease-free survival rate were carried out. The positive expression of survivin in the cytoplasm was significantly correlated with poor prognosis in HCC (HR>1).

Conclusions

This study showed that survivin expression was correlated with poor prognosis in patients with hepatocellular carcinoma, regardless whether they were assessed by overall survival or disease-free survival.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

Extent of beta cell destruction is important but insufficient to predict the onset of type 1 diabetes mellitus

Background

Type 1 diabetes mellitus is characterized by an inability to produce insulin endogenously. Based on a series of histopathology studies of patients with recent onset of the disease, it is commonly stated that the onset of clinical symptoms corresponds to an 80-95% reduction in beta cell mass. Motivated by the clinical importance of the degree of beta cell destruction at onset, a meta-analysis was used to determine the validity of this common wisdom.

Methods and Findings

The histopathology results identifying insulin containing islets in patients younger than 20 years of age were extracted from three different studies. The results for 105 patients were stratified by duration of diabetic symptoms and age at onset. Linear regression and a non-parametric bootstrap approach were used to determine the dependence of residual beta cell mass to age at onset. The percentage reduction in beta cell mass was highly correlated (p<0.001) with the age of onset with the greatest reduction in beta cell mass in the youngest patients. As this trend had not been previously observed, an alternative physiology-based model is proposed that captures this age-dependence.

Conclusions

The severity in beta cell reduction at onset decreased with age where, on average, a 40% reduction in beta cell mass was sufficient to precipitate clinical symptoms at 20 years of age. The observed trend was consistent with a physiology-based model where the threshold for onset is based upon a dynamic balance between insulin-production capacity, which is proportional to beta cell mass, and insulin demand, which is proportional to body weight.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1

Aim

We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (βTSC2^−/−) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells.

Methods

Isolated islets from βTSC2^−/− mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes.

Results

Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of βTSC2^−/− mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of βTSC2^−/− mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, βTSC2^−/− mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1.

Conclusion

Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.     

Co-Graft of Allogeneic Immune Regulatory Neural Stem Cells (NPC) and Pancreatic Islets Mediates Tolerance,while Inducing NPC-Derived Tumors in Mice

Background

Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model.

Methodology/Principal Finding

Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP⁺ NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2Rα chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4⁺CD25⁺FoxP3⁺ T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion.

Conclusion

These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment.