Deletion of Fas protects islet beta cells from cytotoxic effects of human islet amyloid polypeptide
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- Park, Y.J., Lee, S., Kieffer, T.J. et al. Diabetologia (2012) 55: 1035. doi:10.1007/s00125-012-2451-2
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Islet amyloid, which is mainly composed of human islet amyloid polypeptide (hIAPP), is a pathological characteristic of type 2 diabetes and also forms in cultured and transplanted islets. We used islet beta cells as well as two ex vivo models of islet amyloid formation, cultured human islets and hIAPP-expressing transgenic mouse islets with or without beta cell Fas deletion, to test whether: (1) the aggregation of endogenous hIAPP induces Fas upregulation in beta cells; and (2) deletion or blocking of Fas protects beta cells from amyloid toxicity.
INS-1, mouse or human islet cells were cultured with hIAPP alone, or with amyloid inhibitor or Fas antagonist. Non-transduced islets, and human islets or hIAPP-expressing mouse islets transduced with an adenovirus that delivers a human proIAPP-specific small interfering RNA (siRNA) (Ad-ProhIAPP-siRNA) were cultured to form amyloid. Mouse islets expressing hIAPP with or without Fas were similarly cultured. Beta cell Fas upregulation, caspase-3 activation, apoptosis and function, and islet IL-1β levels were assessed.
hIAPP treatment induced Fas upregulation, caspase-3 activation and apoptosis in INS-1 and islet cells. The amyloid inhibitor or Fas antagonist reduced apoptosis in hIAPP-treated beta cells. Islet cells with Fas deletion had lower hIAPP-induced beta cell apoptosis than those expressing Fas. Ad-ProhIAPP-siRNA-mediated amyloid inhibition reduced Fas upregulation and IL-1β immunoreactivity in human and hIAPP-expressing mouse islets. Cultured hIAPP-expressing mouse islets with Fas deletion had similar amyloid levels, but lower caspase-3 activation and beta cell apoptosis, and a higher islet beta:alpha cell ratio and insulin response to glucose, compared with islets expressing Fas and hIAPP.
The aggregation of biosynthetic hIAPP produced in islets induces beta cell apoptosis, at least partially, via Fas upregulation and the Fas-mediated apoptotic pathway. Deletion of Fas protects islet beta cells from the cytotoxic effects of endogenously secreted (and exogenously applied) hIAPP.
KeywordsAmylinAmyloidBeta cell apoptosisFas receptorIslet amyloid polypeptideType 2 diabetes
An adenovirus that delivers a random non-specific small interfering RNA
An adenovirus that delivers a human proIAPP-specific small interfering RNA
Human islet amyloid polypeptide
Rat islet amyloid polypeptide
Islet amyloid formation is a pathological characteristic of the pancreas in type 2 diabetes and contributes to progressive beta cell dysfunction and death in this disease [1–3]. Islet amyloid also forms in human islets during culture  and following transplantation into mouse models of type 1 diabetes [5–7]. Importantly, widespread amyloid deposition was reported in human islets that had been transplanted into a patient with type 1 diabetes . Amyloid formation in transplanted islets is associated with beta cell dysfunction and recurrence of hyperglycaemia in animal models of type 1 diabetes [7, 9]. Thus, in addition to its role in the pathogenesis of type 2 diabetes, islet amyloid formation may also play a role in islet graft failure in patients with type 1 diabetes.
Islet amyloid is mainly formed by the aggregation of islet amyloid polypeptide (amylin) [10, 11], a 37-amino acid hormone that is co-localised and co-secreted with insulin in response to beta cell secretagogues [12, 13]. Despite considerable study during the past decade, it is still not clear why normally soluble human islet amyloid polypeptide (hIAPP) molecules form toxic aggregates in type 2 diabetes. The following have been proposed to contribute to the aggregation of hIAPP molecules in type 2 diabetes: (1) presence of an amyloidogenic sequence in the hIAPP molecule [2, 14]; (2) elevated hIAPP production and secretion from beta cells, associated with an increased demand for insulin [13, 15]; and (3) defects in trafficking and processing of proIAPP, associated with beta cell dysfunction [13, 15–17]. The rapid formation of amyloid in cultured and transplanted human islets may be related to poor clearance of released hIAPP from beta cells due to the disrupted vasculature  and/or to the impaired proIAPP processing and secretion that are associated with beta cell dysfunction induced by islet isolation, culture and transplantation [13, 19].
Aggregates of hIAPP, including small oligomeric species, are toxic to beta cells and have been shown to induce beta cell dysfunction and apoptosis in isolated human islets [4, 20]. Previous in vitro studies have suggested different mechanisms for the beta cell apoptosis mediated by hIAPP aggregates, including disruption of membrane integrity and formation of non-selective ion-channel-like structures [21–23], activation of the caspase pathways [24–27] and interaction of hIAPP fibrils with components of beta cell membranes such as heparan sulphate proteoglycans [28, 29] or touch receptors . It is, however, not clear which of these mechanism(s) contribute(s) to hIAPP-induced beta cell death in primary islets, in which the level of endogenously produced hIAPP (pmol/l) is several fold lower [1, 31] than that used in vitro (μmol/l). Recent studies suggest that endoplasmic reticulum stress [32–34], oxidative stress  and disruption in the autophagy–lysosomal pathway  due to formation of intracellular hIAPP aggregates also contribute to the cytotoxic effects of hIAPP. It therefore appears that, at least in vitro, hIAPP-induced beta cell apoptosis involves several mechanisms, some of which may share the same apoptotic signalling pathways.
Fas (CD95/APO-1) is a transmembrane receptor protein belonging to the tumour necrosis factor superfamily of receptors [37–39]. The extrinsic apoptotic pathway initiated by the interaction between Fas and Fas ligand (FasL/CD95L) has been implicated in autoimmune-mediated beta cell death in type 1 diabetes [40–42] and non-immune-mediated beta cell death in type 2 diabetes [43, 44]. Islet beta cells constitutively produce Fas ligand, but do not normally produce Fas at detectable levels [40, 43]. However, conditions associated with beta cell stress, such as exposure to elevated glucose, leptin or cytokines (e.g. IL-1β), can induce upregulation of Fas and apoptosis in isolated human islets [43–45].
In the present study, we tested whether hIAPP aggregates derived from biosynthetic hIAPP secreted from islets can induce beta cell apoptosis via upregulation of Fas. We used islets from humans and transgenic mice with beta cell-specific hIAPP expression and Fas deletion, as well as transformed and primary islet beta cells, to investigate the role of Fas in mediating islet amyloid-induced beta cell toxicity and to examine whether deletion of Fas can protect beta cells from hIAPP aggregates derived from endogenously secreted and exogenously applied hIAPP.
Synthetic hIAPP and rat islet amyloid polypeptide (rIAPP) (1-37) were from Bachem (Torrance, CA, USA) and Fas antagonist (Kp7-6) was from EMD Chemicals (Gibbstown, NJ, USA). Thioflavin S, dithizone, BSA, Congo red, HEPES buffer, 2-mercaptoethanol, sodium bicarbonate, poly-l-lysine, Triton X-100, DMSO, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), tribromoethanol and collagenase (Type XI) were obtained from Sigma-Aldrich (Oakville, ON, Canada). RPMI-1640, FBS, penicillin, streptomycin, gentamycin, cell dissociation buffer enzyme-free Hanks’-based solution, Ham’s-F10, trypsin-EDTA and glutamax were from Invitrogen Canada (Burlington, ON, Canada). CMRL culture medium was from Mediatech (Herndon, VA, USA).
Human islets isolated from deceased pancreas donors were provided by the Ike Barber Human Islet Transplant Laboratory (Vancouver, BC, Canada) in accordance with approved procedures and guidelines of the Clinical Research Ethics Board of the University of British Columbia. Hand-picked human islets (purity >90%) were cultured for 7 days at 37°C in CMRL supplemented with 11.1 mmol/l glucose, 10% (vol./vol.) FBS, 50 U/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml gentamycin. Culture procedures were in humidified 5% CO2/95% air.
Hemizygous C57BL/6 hIAPP transgenic mice with beta cell-specific hIAPP expression (hIAPP+/−) were kindly provided by S. Kahn (Department of Medicine, University of Washington, Seattle, WA, USA) and maintained by breeding with DBA/2J mice (Jackson Laboratory, Bar Harbor, ME, USA). Male hIAPP-expressing mice form islet amyloid in vivo and develop diabetes in the presence of a predisposing factor such as a high-fat diet . Mice with beta cell-specific Fas deletion (RIPcre+Fasfl/fl) were generated using the Cre/loxP recombinase system as previously described . RIPcre+Fasfl/fl mice were maintained by breeding RIPcre+Fas+/fl mice from the Ontario Cancer Institute. To generate mice with beta cell-specific hIAPP expression and Fas deletion, hIAPP+/− mice were cross-bred with RIPcre+Fasfl/fl mice to produce RIPcre+Fas+/fl/hIAPP+/− mice, which were then bred together to generate RIPcre+Fasfl/fl/hIAPP+ and RIPcre+Fas+/+/hIAPP+ mice. The first generation of these offspring was used for the studies. Islets from RIPcre+Fasfl/fl animals have normal islet mass and enhanced insulin secretion . All mice were fed mouse chow containing 9% (wt/wt) fat (Purina 5021; LabDiet, Richmond, IN, USA). Animals were cared for in accordance with the Guidelines and Principles of Laboratory Animal Care and the standard procedures established by the Canadian Council on Animal Care and the University of British Columbia’s Animal Policy and Welfare Committee.
Mouse islet isolation and culture
Islets from wild-type (hIAPP−/−) and hIAPP+/− mice (8–12 weeks old) or different genotypes of RIPcre+Fas/hIAPP mice (8–18 weeks) were isolated as previously described  with some modifications. Briefly, animals were anaesthetised with tribromoethanol (0.02 ml/g body weight, i.p.) and killed by cervical dislocation. The abdominal cavity was opened and ice-cold collagenase (Type XI; Sigma) in 2.5 ml calcium-free Hanks’ solution (final concentration 1,000 U/ml) was injected via the common bile duct. The removed pancreas was incubated with collagenase and Hanks’ solution (1,000 U/ml) in a shaker water bath (14 min, 37°C, 120 rev/min). Digestion was stopped by addition of ice-cold Hanks’ solution containing 1 mmol/l CaCl2. Digested pancreatic tissues were rinsed with the same solution, resuspended in Ham’s-F10 and filtered through a 70 μm mesh cell strainer (BD Biosciences, Oakville, ON, Canada). Hand-picked islets (purity >95%) were cultured in Ham’s-F10 supplemented with 16.7 mmol/l glucose, 0.5% (wt/vol.) BSA and antibiotics as described for human islets. For all islet studies, the culture medium was changed every 2 days.
Adenoviral transduction of human and mouse islets
As previously described , we used human islets or hIAPP-expressing mouse islets transduced overnight with an adenovirus that delivers a human proIAPP-specific small interfering RNA (Ad-ProhIAPP-siRNA) (multiplicity of infection 20), or transduced with an adenovirus that delivers a random non-specific small interfering RNA (Ad-Cont-siRNA) (multiplicity of infection 20). Adenoviral-siRNA transduced human or hIAPP-expressing mouse islets were then rinsed and cultured in CMRL (11.1 mmol/l glucose, 7 days) or Ham’s-F10 (16.7 mmol/l, 6 days), respectively.
Transformed beta cells and islet cell culture
INS-1 (832/13) cells, a transformed rat beta cell line, were a gift from C. Newgard (Duke University Medical Center, NC, USA). Cells were grown in RPMI-1640 containing 11.1 mmol/l glucose supplemented with 10% (vol./vol.) FBS, 50 U/ml penicillin, 50 μg/ml streptomycin and 50 μmol/l 2-mercaptoethanol. Human or mouse islets (~200) were dissociated as previously described  and cell viability assessed by Trypan blue. Islet cells were cultured in 8-well chamber slides (BD Biosciences) pre-coated with poly-l-lysine; the culture process was in CMRL (human) or Ham’s-F10 (mouse) containing 5.5 and 10 mmol/l glucose, respectively.
Treatment studies with hIAPP, Fas antagonist and amyloid binding dye
Lyophilised hIAPP or rIAPP was dissolved in HFIP and incubated at room temperature (1 h); aliquots were frozen (−80°C) and lyophilised. INS-1 or dispersed islet cells were treated with hIAPP or rIAPP that had been prepared freshly by dissolving lyophilised peptide in medium; they were then cultured for different times as detailed. The amyloid-binding dye Congo red or Fas antagonist Kp7-6 was added to the culture medium 30 min before addition of hIAPP at final concentrations of 25 μmol/l and 10 mmol/l, respectively.
Assessment of beta cell function in cultured islets
Islets (25 islets per mouse; duplicate, four to six mice per group) were pre-incubated (1 h) at 37°C in 300 μl KRB containing 10 mmol/l HEPES (pH 7.4), 0.25% (wt/vol.) BSA and 1.67 mmol/l glucose, followed by 1 h of incubation in 150 μl KRB containing 1.67 mmol/l glucose (basal insulin release) and by 1 h of incubation in KRB with 16.7 mmol/l glucose (stimulated insulin release). Islets were lysed in 100 μl of 1 mol/l acetic acid and 0.1% (wt/vol.) BSA lysis buffer, followed by 10 min incubation at 100°C. Incubation media and islet lysates were centrifuged and supernatant fractions frozen (−20°C) until assayed. Insulin and proinsulin levels were measured using mouse-specific insulin and proinsulin ELISA kits (ALPCO Diagnostics, Salem, NH, USA).
Immunolabelling, TUNEL and thioflavin S staining
Paraffin-embedded islet sections (5 μm) were de-waxed, rehydrated and blocked in 2% (vol./vol.) normal goat and/or donkey serum (Vector Laboratories, Burlingame, CA, USA). Fixed islet sections (following antigen retrieval with citrate buffer) or cells were incubated overnight with guinea pig anti-insulin alone (islets 1:750, cells 1:200; Dako, Carpinteria, CA, USA) or with each of rabbit anti-IL-1β (1:100; Santa Cruz, Santa Cruz, CA, USA), anti-glucagon (1:750; Dako), anti-cleaved caspase-3 (1:100; Cell Signaling, Pickering, ON, Canada) and anti-Fas (mouse islets 1:50, cells 1:100, Santa Cruz; human islets 1:100, Cell Signaling). Islet sections or cells were then incubated for 1 h with Texas red-conjugated anti-guinea pig (1:750; Jackson Laboratories, West Grove, PA, USA) and Alexa 488-conjugated anti-rabbit (1:200; Molecular Probes, Eugene, OR, USA). For triple immunostaining, islet cells were immunolabelled overnight for insulin (1:200) and Fas (1:100), followed by incubation with aminomethylcoumarin acetate-conjugated anti-guinea pig (1:200; Jackson) and Texas red-conjugated anti-rabbit (1:100; Jackson), and then with Alexa 488-conjugated cleaved caspase-3 antibody (1:10; Cell Signaling). The specificity of the Fas antibody was evaluated by immunolabelling of IL-1β- or hIAPP-treated INS-1 cells after antibody blocking using recombinant Fas (electronic supplementary material [ESM] Fig. 1). For double insulin and TUNEL or thioflavin S staining, after immunolabelling for insulin, islet sections (or cells) were incubated for 30 min at 37°C with TUNEL reaction mixture (Roche Diagnostics, Laval, QC, Canada) or for 5 min at room temperature with 0.5% (wt/vol.) thioflavin S solution.
Data are expressed as means±SEM. Statistical analyses were performed using one-way ANOVA followed by Newman–Keuls test or by Student’s t test as appropriate. A value of p < 0.05 was taken as significant. All experiments were performed in duplicate or triplicate and repeated at least three times.
Exogenously applied hIAPP induces Fas upregulation, caspase-3 activation and apoptosis in transformed INS-1 cells and in primary human and mouse islet beta cells
Preventing the interaction between hIAPP aggregates and beta cells markedly reduces beta cell apoptosis in hIAPP-treated INS-1 cells
Blocking or deletion of Fas reduces the beta cell apoptosis induced by exogenously applied hIAPP
INS-1 cells or primary islet cells were cultured with or without synthetic hIAPP in the absence or presence of a Fas antagonist (Kp7-6) for 12 h. INS-1 and primary mouse islet cells treated with hIAPP and the Fas antagonist had significantly lower beta cell apoptosis than cells treated with hIAPP in the absence of the Fas antagonist (Fig. 3b–d). Treatment with the Fas antagonist alone did not have any effect on beta cell apoptosis in non-treated cells (Fig. 3d). Furthermore, dispersed wild-type and RIPcre+Fasfl/fl mouse islet cells were treated with synthetic hIAPP. There was no significant difference in basal beta cell apoptosis between wild-type and RIPcre+Fasfl/fl mouse islet cells (Fig. 3e, f). Interestingly, hIAPP-treated RIPcre+Fasfl/fl islets had markedly lower beta cell death than hIAPP-treated wild-type islet cells, suggesting that deletion of Fas reduces beta cell death induced by the aggregation of exogenously applied hIAPP.
Inhibition of islet amyloid formation reduces IL-1β levels, Fas upregulation and beta cell apoptosis in human and hIAPP-expressing transgenic mouse islets
Beta cell-specific deletion of Fas in hIAPP-expressing transgenic mouse islets markedly reduces apoptosis induced by the aggregation of endogenously produced hIAPP during culture
Reduced amyloid-induced beta cell death in hIAPP-expressing transgenic mouse islets with Fas deletion is associated with enhanced beta cell function
Growing evidence suggests that the formation of hIAPP aggregates in pancreatic islets of patients with type 2 diabetes, cultured human islets and human islet grafts is associated with progressive beta cell dysfunction and death [1–3]. Beta cell-toxic effects of hIAPP aggregates are likely to be mediated through similar mechanisms in all conditions associated with islet amyloid formation. As yet, however, the molecular mechanisms by which the aggregation of biosynthetic hIAPP induces beta cell apoptosis are not well understood. Previous studies have shown that amyloid formation in human  and hIAPP-expressing mouse islets  increases beta cell apoptosis, and that prevention of amyloid formation enhances survival and function of human islets . We further demonstrated that prevention of caspase-3 activation protects islet beta cells from apoptosis induced by fibrillogenesis of hIAPP produced and secreted from islet beta cells .
In the present study, using two ex vivo models of islet amyloid formation, cultured human islets and hIAPP-expressing mouse islets with beta cell-specific Fas deletion, we show that Fas has a key role in the beta cell apoptosis induced by aggregation of the biosynthetic hIAPP produced by islet beta cells. We also show that prevention of hIAPP-induced Fas upregulation or deletion of Fas protects islet beta cells from the hIAPP aggregates formed during in situ islet culture. These findings suggest that aggregation of biosynthetic hIAPP induces beta cell apoptosis, at least partially, through the Fas receptor and the Fas-mediated apoptotic pathway.
Consistent with a previous in vitro study performed on beta cells , we found that micromolar concentrations of synthetic hIAPP induces Fas upregulation in INS-1 cells, as well as in human and mouse islet beta cells, in a time-dependent manner. The majority of Fas-positive islet cells were beta cells, suggesting that hIAPP-induced upregulation of Fas is beta cell-specific. Furthermore, our immunolabelling studies showed a clear co-localisation of Fas and active caspase-3 in INS-1 and dispersed islet beta cells. Finally, culture in the presence of a Fas antagonist or deletion of Fas in islet beta cells significantly reduced hIAPP-induced apoptosis. Taken together, these findings suggest that in transformed and primary islet beta cells, exposure to hIAPP aggregates derived from exogenously applied hIAPP induces upregulation of Fas and that hIAPP-induced Fas upregulation is associated with caspase-3 activation and subsequent beta cell apoptosis.
Under normal conditions, INS-1 cells did not produce Fas at levels detectable with the immunolabelling techniques used by us. Unlike INS-1 cells, however, low numbers of beta and non-beta Fas-positive islet cells were present in dispersed human and mouse islets. It is possible that cytokines or other Fas-inducing factors that are released from islet cells and other cells in the digested pancreatic tissue during islet isolation and culture contribute to the low levels of Fas detected in non-treated islet cells. Consistent with our findings, IL-1β and elevated glucose have been reported to induce Fas upregulation in islet beta cells [43, 44]. Finally, islet amyloid and elevated glucose, both of which contribute to beta cell death in type 2 diabetes, may induce Fas upregulation in an additive manner.
Treatment of INS-1 cells with the amyloid binding dye, Congo red, which prevents amyloid formation and its interaction with beta cell membranes, markedly reduced hIAPP-induced Fas upregulation and the proportion of active caspase-3 and apoptotic beta cells. These findings support the notion that interaction of extracellular hIAPP aggregates with beta cell membranes is important for Fas upregulation and subsequent beta cell apoptosis, and that inhibitors of islet amyloid may provide a way of preventing amyloid-induced Fas upregulation.
To examine whether the hIAPP aggregates formed by biosynthetic hIAPP produced and secreted from islet beta cells can induce Fas upregulation, we used human and transgenic mouse islets with beta cell hIAPP expression as two ex vivo models of islet amyloid formation [4, 16, 48]. Human and hIAPP-expressing mouse islets, but not wild-type mouse islets formed hIAPP aggregates during culture, this being associated with upregulation of Fas. Immunolabelling studies showed that Fas-positive islet areas co-localised with thioflavin S (amyloid)-positive islet areas, indicating the correlation between amyloid formation and Fas upregulation. Furthermore, the prevention of amyloid formation in human and hIAPP-expressing mouse islets by Ad-ProhIAPP-siRNA markedly reduced Fas upregulation and subsequent beta cell apoptosis.
We also generated a mouse model expressing hIAPP with beta cell-specific Fas deletion to further investigate the role of Fas in mediating the beta cell-toxic effects of endogenously produced hIAPP aggregates in islets. Islets from hIAPP-expressing mice with or without Fas deletion had comparable levels of amyloid formation, confirming that the absence of Fas as such does not have any significant effect on amyloid formation in hIAPP-expressing mouse islets. Interestingly, hIAPP-expressing mouse islets lacking Fas had significantly lower numbers of active caspase-3 and apoptotic beta cells, a higher islet insulin response to glucose and lower proinsulin release than islets expressing hIAPP and Fas, suggesting that deletion of Fas protects islet beta cells from endogenously produced hIAPP aggregates and improves their survival and function. Enhanced insulin secretion in hIAPP-expressing mouse islets with beta cell Fas deletion is likely to be due to the reduction of hIAPP-induced beta cell apoptosis mediated by Fas upregulation and to the effects of Fas deletion on beta cell secretion . Importantly, despite significant increases in the proportion of active caspase-3 and apoptotic beta cells, the number of alpha cells remained unchanged in cultured hIAPP-transgenic mouse islets expressing Fas, resulting in a lower beta:alpha cell ratio in hIAPP-expressing mouse islets with Fas expression than in those lacking Fas.
The formation of hIAPP-aggregates and upregulation of Fas in human and hIAPP-expressing mouse islets was associated with increased islet IL-1β immunoreactivity, which was reduced by adenoviral-siRNA-mediated prevention of amyloid formation, suggesting that IL-1β may contribute to hIAPP-induced Fas upregulation. The source of IL-1β detected by immunolabelling in thioflavin S (amyloid)-positive islets might be islet beta cells, non-beta cells or non-islet cells. In support of this notion, beta cells have been shown to produce IL-1β in conditions such as exposure to elevated glucose resulting in upregulation of Fas . Furthermore, two recent studies have demonstrated the release of IL-1β from non-islet cells such as macrophages activated by hIAPP aggregates [49, 50]. Taken together, these findings support a role for IL-1β in hIAPP-induced Fas upregulation, although further studies are required to identify the cell types that release IL-1β in conditions associated with islet amyloid formation.
In summary, our studies show that hIAPP-induced beta cell death is mediated, at least partially, via upregulation of Fas and activation of the Fas-mediated apoptotic pathway. We also show that deletion of Fas protects islet beta cells from the cytotoxic effects of endogenously secreted (and exogenously applied) hIAPP. Preventing the interaction between hIAPP aggregates and beta cells, or blocking Fas could constitute new ways to avoid amyloid toxicity and thereby enhance survival and function of human islets in conditions associated with islet amyloid formation, such as type 2 diabetes, islet culture, and transplantation.
Human IAPP transgenic mice for these studies were kindly provided by S. Kahn (Department of Medicine, VA Puget Sound Health Care System and University of Washington, Seattle, WA, USA). Human islets were provided by the Ike Barber Human Islet Transplant Laboratory (Vancouver, BC, Canada). We gratefully acknowledge the outstanding technical assistance of I. Barta, A. Asadi and G. Soukhatcheva to the completion of these studies.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to L. Marzban (MOP-81375) and M. Woo (MOP-201188). Y.J. Park is supported by a studentship from the CIHR Transplantation Training Program. L. Marzban is a Scholar of the Canadian Diabetes Association and T.J. Kieffer is a Michael Smith Foundation for Health Research (MSFHR) Senior Scholar. Infrastructure support was provided by grants from the Canadian Foundation for Innovation (to L. Marzban) and the MSFHR to the Centre for Human Islet Transplantation and Beta Cell Regeneration (G.L. Warnock).
All authors contributed to the conception and design, or analysis and interpretation of data, and to the drafting of the article or its critical revision for important intellectual content. All authors gave final approval of the version to be published.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.