Human C-peptide Research Articles

Enhancing human islet xenotransplant survival and function in diabetic immunocompetent mice through LRH-1/NR5A2 pharmacological activation.

The intricate etiology of type 1 diabetes mellitus (T1D), characterized by harmful interactions between the immune system and insulin-producing beta cells, has hindered the development of effective therapies including human islet transplantation, which requires strong immunosuppressants that impair beta cell survival and function. As such alternative immunomodulating therapies are required for successful transplantation. The discovery that pharmacological activation of the nuclear receptor LRH-1/NR5A2 can reverse hyperglycemia in mouse models of T1D by altering, and not suppressing the autoimmune attack, prompted us to investigate whether LRH-1/NR5A2 activation could improve human islet function/survival after xenotransplantation in immunocompetent mice. Human islets were transplanted under the kidney capsule of streptozotocin (STZ)-induced diabetic mice, and treatment with BL001 (LRH-1/NR5A2 agonist) or vehicle was administered one week post-transplant. Our study, encompassing 3 independent experiments with 3 different islet donors, revealed that mice treated for 8 weeks with BL001 exhibited lower blood glucose levels correlating with improved mouse survival rates as compared to vehicle-treated controls. Human C-peptide was detectable in BL001-treated mice at both 4 and 8 weeks indicating functional islet beta cells. Accordingly, in mice treated with BL001 for 8 weeks, the beta cell mass was preserved, while a significant decrease in alpha cells was observed compared to mice treated with BL001 for only 4 weeks. In contrast, vehicle-treated mice exhibited a reduction in insulin-expressing cells at 8 weeks compared to those at 4 weeks. These results suggest that BL001 significantly enhances the survival, engraftment, and functionality of human islets in a STZ-induced diabetic mouse model.

Optimizing expression, purification, structural and functional assessments of a novel dimeric incretin (GLP-1cpGLP-1)

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces postprandial glycemic excursions by enhancing insulin secretion. In this study, a new dimeric GLP-1 analogue (GLP-1cpGLP-1) was designed by inserting human insulin C-peptide (CP) in the middle of a dimer of [Gly8] GLP-1 (7–36). Then, the dimeric incretin (GLP-1cpGLP-1) was ligated to human αB-crystallin (αB-Cry) to create a hybrid protein, abbreviated as αB-GLP-1cpGLP-1. The constructed gene was well expressed in the bacterial host system. After specific chemical release from the hybrid protein, the dimeric incretin was purified by size exclusion chromatography (SEC). Finally, the RP-HPLC analysis indicated a purity of >99 % for the dimeric incretin. The secondary structure assessments by various spectroscopic methods, and in silico analysis suggested that the dimeric incretin has α-helical rich structure. The dynamic light scattering (DLS) analysis indicates that our dimeric incretin forms large oligomeric structures. This incretin analogue significantly reduced blood glucose levels in both healthy and diabetic mice while effectively triggering insulin release. The size exclusion HPLC also indicates the interaction of the new incretin analogue with human serum albumin, the main carrier protein in the bloodstream. Consistent with the results obtained from the biological activity assessments, this significant interaction indicates its potential as a viable therapeutic agent with a long-lasting effect. The results of our research represent a significant breakthrough in the successful design of an active incretin dimer capable of effectively controlling blood sugar levels and inducing insulin secretion in the realm of diabetes treatment.

855-P: Evolution of KT-A112, an AAV-Mediated Gene Therapy for the Treatment of Type 1 Diabetes

Type 1 Diabetes (T1D) is a chronic, life-threatening autoimmune disorder characterized by profound insulin deficiency that results from progressive destruction of insulin-producing pancreatic β-cells. For over a century, exogenous insulin delivery was the only option to help T1D patients manage persistent hyperglycemia and reduce the risk of severe microvascular and macrovascular complications. Recent exploratory treatments use complex cell-based therapies requiring long-term immunosuppression. This abstract describes a unique AAV-mediated gene therapy approach to drive skeletal muscle expression of human glucokinase (GcK) and human insulin (INS). One-time IM administration to the quadriceps, gastrocnemius and tibialis anterior muscles of STZ-induced diabetic mice resulted in dose-related increases of pharmacologically active INS at fasting levels (~1 ng/mL) and human C-peptide that decreased fed and fasted blood glucose, lowered HbA1c by >5% at the highest dose, and reduced AUCGlu upon oral glucose challenge. Elevated triglycerides seen in control STZ-treated animals were normalized. No hypoglycemia or other adverse effects were observed through 8 weeks. No immunosuppression was required. Next steps in evaluation of KT-A112 involve a rolling dose-escalation study in STZ-treated NHPs. Disclosure C.Smith: Consultant; Horizon Therapeutics plc, Employee; Kriya Therapeutics. F.Bosch: None. M.Rhodes: None. W.W.Harrington: Consultant; Kriya Therapeutics. M.Stone: None. U.Karadge: None. J.Liu: None. W.J.Quinn: Employee; Kriya Therapeutics. A.Bassiri: None. J.Mallory: Employee; Kriya Therapeutics. V.Jimenez: None.

VPIF-1 is an insulin-like antiferroptotic viral peptide

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.

Light-stimulated insulin secretion from pancreatic islet-like organoids derived from human pluripotent stem cells

Optogenetic techniques permit non-invasive, spatiotemporal, and reversible modulation of cellular activities. Here, we report a novel optogenetic regulatory system for insulin secretion in human pluripotent stem cell (hPSC)-derived pancreatic islet-like organoids using monSTIM1 (monster-opto-Stromal interaction molecule 1), an ultra-light-sensitive OptoSTIM1 variant. The monSTIM1 transgene was incorporated at the AAVS1 locus in human embryonic stem cells (hESCs) by CRISPR-Cas9-mediated genome editing. Not only were we able to elicit light-induced intracellular Ca2+ concentration ([Ca2+]i) transients from the resulting homozygous monSTIM1+/+-hESCs, but we also successfully differentiated them into pancreatic islet-like organoids (PIOs). Upon light stimulation, the β-cells in these monSTIM1+/+-PIOs displayed reversible and reproducible [Ca2+]i transient dynamics. Furthermore, in response to photoexcitation, they secreted human insulin. Light-responsive insulin secretion was similarly observed in monSTIM1+/+-PIOs produced from neonatal diabetes (ND) patient-derived induced pluripotent stem cells (iPSCs). Under LED illumination, monSTIM1+/+-PIO-transplanted diabetic mice produced human c-peptide. Collectively, we developed a cellular model for the optogenetic control of insulin secretion using hPSCs, with the potential to be applied to the amelioration of hyperglycemic disorders.

Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes

BackgroundHyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin.MethodsInsulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney.ResultsWe found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage.ConclusionsOverall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

The Impact of Different Implantation Sites and Sex on the Differentiation of Human Pancreatic Endoderm Cells Into Insulin-Secreting Cells InVivo.

Few studies have directly compared the differentiation of human embryonic stem cell-derived progenitors in different implantation sites in male and female recipients. We investigated whether the site of implantation and/or the sex of the recipient influenced the differentiation of pancreatic progenitors invivo in mice. Mice implanted with cells in macroencapsulation devices contained fewer off-target structures and developed stimulated insulin release faster than other implant sites, while females implanted with cells under the kidney capsule developed stimulated insulin release before males. Macroencapsulation devices reduced the formation of off-target cells from human embryonic stem cell-derived progenitors, a useful characteristic for clinical applications.

Therapeutic effect of ultra-long-lasting human C-peptide delivery against hyperglycemia-induced neovascularization in diabetic retinopathy.

Rationale: Neovascularization is a hallmark of the late stages of diabetic retinopathy (DR) leading to blindness. The current anti-DR drugs have clinical disadvantages including short circulation half-lives and the need for frequent intraocular administration. New therapies with long-lasting drug release and minimal side effects are therefore needed. We explored a novel function and mechanism of a proinsulin C-peptide molecule with ultra-long-lasting delivery characteristics for the prevention of retinal neovascularization in proliferative diabetic retinopathy (PDR). Methods: We developed a strategy for ultra-long intraocular delivery of human C-peptide using an intravitreal depot of K9-C-peptide, a human C-peptide conjugated to a thermosensitive biopolymer, and investigated its inhibitory effect on hyperglycemia-induced retinal neovascularization using human retinal endothelial cells (HRECs) and PDR mice. Results: In HRECs, high glucose conditions induced oxidative stress and microvascular permeability, and K9-C-peptide suppressed those effects similarly to unconjugated human C-peptide. A single intravitreal injection of K9-C-peptide in mice resulted in the slow release of human C-peptide that maintained physiological levels of C-peptide in the intraocular space for at least 56 days without inducing retinal cytotoxicity. In PDR mice, intraocular K9-C-peptide attenuated diabetic retinal neovascularization by normalizing hyperglycemia-induced oxidative stress, vascular leakage, and inflammation and restoring blood-retinal barrier function and the balance between pro- and anti-angiogenic factors. Conclusions: K9-C-peptide provides ultra-long-lasting intraocular delivery of human C-peptide as an anti-angiogenic agent to attenuate retinal neovascularization in PDR.

Glutamine deamidation does not increase the immunogenicity of C-peptide in people with type 1 diabetes

Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease in which the insulin-producing beta cells are destroyed. While it is clear that full-length C-peptide, derived from proinsulin, is a major antigen in human T1D it is not clear how and why C-peptide becomes a target of the autoimmune CD4+ T-cell responses in T1D. Neoepitopes formed by the conversion of glutamine (Q) residues to glutamic acid (E) by deamidation are central to the immune pathogenesis of coeliac disease and have been implicated in autoimmune responses in T1D. Here, we asked if the immunogenicity of full-length C-peptide, which comprises four glutamine residues, was enhanced by deamidation, which we mimicked by substituting glutamic acid for glutamine residue. First, we used a panel of 18 well characterized CD4+ T-cell lines specific for epitopes derived from human C-peptide. In all cases, when the substitution fell within the cognate epitope the response was diminished, or in a few cases unchanged. In contrast, when the substitution fell outside the epitope recognized by the TCR responses were unchanged or slightly augmented. Second, we compared CD4+ T-cell proliferation responses, against deamidated and unmodified C-peptide, in the peripheral blood of people with or without T1D using the CFSE-based proliferation assay. While, as reported previously, responses were detected to unmodified C-peptide, no deamidated C-peptide was consistently more stimulatory than native C-peptide. Overall responses were weaker to deamidated C-peptide compared to unmodified C-peptide. Hence, we conclude that deamidated C-peptide does not play a role in beta-cell autoimmunity in people with T1D.

Human C-peptide is a ligand of the elastin-receptor-complex and therewith central to human vascular remodelling and disease in metabolic syndrome

This hypothesis ties together various causal risk factors from our “Western-lifestyle” (elevated blood-glucose, smoking, processed red-meat-products, obesity, and inflammation) in a unifying perspective on the cause of human vascular disease in metabolic syndrome.When, in response to enhanced blood-glucose levels, insulin is excreted, it is always excreted together with C-peptide. I identified an elastin-receptor-complex (ERC) binding amino-acid motif xGxxPG in C-peptide and pose that C-peptide’s earlier unknown receptor is this ERC. This receptor is involved in vascular remodelling, commonly activated by vascular-lesion-derived elastin-peptides with said motif xGxxPG. Systemic elastin-peptides may arise from smoking or from consuming elastin-rich processed red-meat-products, therewith interfering with vascular remodelling. Both excess elastin-peptides as well as excess C-peptide associate with core-manifestations of metabolic syndrome: insulin resistance, angiogenesis, and vascular remodelling. C-peptide fragments as well as elastin peptide-fragments with motif xGxxPG are angiogenic and blocked by antagonists of ERC. Many other bioactive peptides with an xGxxPG motif exist, a third example discussed here is galectin-3, elevated in obesity and inflammation, and again associated with insulin resistance, angiogenesis, and vascular remodelling in metabolic syndrome.On the one side of this hypothesis, a lack of ERC-mediated vascular remodelling through a lack of C-peptide is central to microvascular disease in T1D. On the other side, excess ERC-activity through excess activation (by systemic C-peptide, elastin-peptide, galectin-3, or others), is centrally involved in metabolic syndrome and T2D, inducing insulin resistance with excess and maladaptive macrovascular remodelling. Systemic C-peptide is increased in a “Western-diet” lifestyle, systemic elastin peptides are increased after smoking and possibly after elastin-consumption, and systemic galectin-3 increases in chronic inflammation and obesity. As all these risk-factors associate with life-style mediated metabolic syndrome, this hypothesis poses one up-stream cause: excess activation of ERC stands central in causing metabolic syndrome.

Immunological parameters in hyperglycemic COVID19 patients of wasit-Iraq

Background: A number of studies have also reported that preexisting diabetes as well as newly diagnosed diabetes with a first glucose measurement on hospital admission are both associated with an increased risk of all-cause mortality. This study aimed to investigate the immunological characteristics of patients with severe COVID-19 with diabetes mellitus. Methods: This study was carried out in a period between November 2021 and March 2022 in Iraq. A total of samples were subjected to laboratory examinations including Human Glutamic acid decarboxylase (GAD) ELISA Kit, Tumor necrosis factor α using specific kit ELISA Kit, Human Islet cell antibody (ICA)ELISA Kit and Human C-Peptide ELISA Kit using specific ELISA Kit. Results: Regarding mean difference of variables among diabetic and non-diabetic COVID-19 patients all the investigated parameters has been showed significant differences between diabetic and non-diabetic patients with a P-value less than 0.001 except for CRP, correlation between age and COVID-19 patients immunological parameters showed that there was no significant correlation between age and the studied variables, mean differences of COVID-19 patients between males and females did not revealed a significant differences,,,,

78-OR: LRH-1/NR5A2 Agonism Triggers an Anti-Inflammatory Phenotypic Switch of Immune Cells from Patients with Type 1 Diabetes and Improves Islet Engraftment/Function

Administration of the LRH-1 agonist BL0refrains hyperglycemia in 3 mouse models of type 1 diabetes (T1DM) . This antidiabetes action correlated with beta cell mass preservation and the presence of anti, rather than pro, inflammatory immune cells, indicating tolerization. Herein, we sought to translate these data to human immune cells of T1DM patients to demonstrate BL0benefits to re-educate the immune system and to assess its therapeutic value on human islet engraftment/function. Blood mononuclear cells purified from blood samples (healthy and T1DM) were derived in culture into 1) pro-inflammatory macrophages (MDM1) , 2) mature dendritic cells (mDCs) , 3) T-cells and 4) B-cells. Cells were then treated with BL0 and processed for 1) cell surface marker profiling, 2) cytokine secretome profiling, 3) T-cell proliferation and 4) RNAseq. In T1DM samples, BL0reduced the expression of pro-inflammatory markers on MDM1 (CD80) and mDCs (CD54 and CXCR4) and the secretion of the pro-inflammatory cytokines, CXCL6 and CCL24 (MDM1) as well as IL7 (mDCs) . Moreover, in these samples, BL0also increased Tregs, but not B-cells. T-cell proliferation was blunted by BL001-treated mDCs and Tregs. RNAseq analysis of BL001-treated MDM1 revealed increased expression of genes encoding components of complex I, III, IV, and V of the respiratory chain consistent with a transition from a glycolytic driven MDM1 to an oxidative phosphorylation driven MDM2 anti-inflammatory phenotype. BL0administration to STZ-treated mice transplanted with human islet improved survival correlating with reduced hyperglycemia, increased circulating human C-peptide, preserved beta cell mass, and reduced CD4+ T-cell infiltration. Our results establish that BL0can induce a pro-to-anti-inflammatory phenotypic switch to human immune cells, and improve human islet engraftment/function, supporting the therapeutic benefits of BL0in human. Disclosure N. Cobo-Vuilleumier: None. S. Rodriguez-Fernandez: Employee; Ahead Therapeutics SL. P.I. Lorenzo: None. A. Dorronsoro: None. C.C. Lachaud: None. L. Almenara-Fuentes: Employee; Ahead Therapeutics SL. M. Ramos-Rodríguez: None. I. Merida: None. M. Aguilar-Diosdado: None. L. Piemonti: None. L. Pasquali: None. F. Martin: None. I. Garcia-Sanchez: None. M. Martinez-Brocca: None. M. Vives-Pi: Research Support; Ahead Therapeutics S.L. B.R. Gauthier: None. Funding JDRF (2-SRA-2019-837-S-B) Consejeria de Salud y Familia, junta de Andalucia (PI-0001-2020)

Enhanced Differentiation Capacity and Transplantation Efficacy of Insulin-Producing Cell Clusters from Human iPSCs Using Permeable Nanofibrous Microwell-Arrayed Membrane for Diabetes Treatment.

Although pancreatic islet transplantation is a potentially curative treatment for insulin-dependent diabetes, a shortage of donor sources, low differentiation capacity, and transplantation efficacy are major hurdles to overcome before becoming a standard therapy. Stem cell-derived insulin-producing cells (IPCs) are a potential approach to overcoming these limitations. To improve the differentiation capacity of the IPCs, cell cluster formation is crucial to mimic the 3D structure of the islet. This study developed a biodegradable polycaprolactone (PCL) electrospun nanofibrous (NF) microwell-arrayed membrane permeable to soluble factors. Based on the numerical analysis and experimental diffusion test, the NF microwell could provide sufficient nutrients, unlike an impermeable PDMS (polydimethylsiloxane) microwell. The IPC clusters in the NF microwells showed higher gene expression of insulin and PDX1 and insulin secretion than the PDMS microwells. The IPC clusters in the NF microwell-arrayed membrane could be directly transplanted. Transplanted IPC clusters in the microwells survived well and expressed PDX1 and insulin. Additionally, human c-peptide was identified in the blood plasma at two months after transplantation of the membranes. The NF microwell-arrayed membrane can be a new platform promoting IPC differentiation capacity and realizing an in situ transplantation technique for diabetic patients.

Inhibition of SGLT2 Preserves Function and Promotes Proliferation of Human Islets Cells In Vivo in Diabetic Mice.

Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor used for the treatment of diabetes. This study examines the effects of dapagliflozin on human islets, focusing on alpha and beta cell composition in relation to function in vivo, following treatment of xeno-transplanted diabetic mice. Mouse beta cells were ablated by alloxan, and dapagliflozin was provided in the drinking water while controls received tap water. Body weight, food and water intake, plasma glucose, and human C-peptide levels were monitored, and intravenous arginine/glucose tolerance tests (IVarg GTT) were performed to evaluate islet function. The grafted human islets were isolated at termination and stained for insulin, glucagon, Ki67, caspase 3, and PDX-1 immunoreactivity in dual and triple combinations. In addition, human islets were treated in vitro with dapagliflozin at different glucose concentrations, followed by insulin and glucagon secretion measurements. SGLT2 inhibition increased the animal survival rate and reduced plasma glucose, accompanied by sustained human C-peptide levels and improved islet response to glucose/arginine. SGLT2 inhibition increased both alpha and beta cell proliferation (Ki67+glucagon+ and Ki67+insulin+) while apoptosis was reduced (caspase3+glucagon+ and caspase3+insulin+). Alpha cells were fewer following inhibition of SGLT2 with increased glucagon/PDX-1 double-positive cells, a marker of alpha to beta cell transdifferentiation. In vitro treatment of human islets with dapagliflozin had no apparent impact on islet function. In summary, SGLT2 inhibition supported human islet function in vivo in the hyperglycemic milieu and potentially promoted alpha to beta cell transdifferentiation, most likely through an indirect mechanism.

402.1: Towards a GMP-Compliant Protocol for the Differentiation of Human Pluripotent Stem Cells to Beta-like Cells for the Treatment of Type 1 Diabetes

Background: β-cell replacement therapy by allogeneic pancreas or islet transplantation is a promising approach for patients with type 1 diabetes. However, there is a scarcity of organ donors. The generation of insulin-producing pancreatic β-cells from human pluripotent stem cells (hPSC) in vitro would provide an unlimited cell source for drug discovery and cell replacement therapy for diabetes. Here we aim to generate β-cells under GMP (Good Manufacturing Practice)-compliant conditions that can be used for clinical application. Methods: We applied a modified seven-stage (30-day) differentiation protocol to generate hPSC-derived insulin-producing β-like cells in a 3D microwell culture system. Next, in order to scale up the production of hPSC-derived β-cells, we adapted the protocol to spinner flask culture. Differentiation was assessed by qPCR, immunocytochemistry and flowcytometry for stage-specific marker expression. β-cell function was evaluated in vitro by GSIS (Glucose-Stimulated Insulin Secretion), and in vivo by intraperitoneal glucose tolerance tests after transplantation of stage-7 cell clusters in immunodeficient mice and human C-peptide secretion was determined by ELISA. Results: In the microwell set-up, cells acquired a definitive endoderm phenotype at stage 1 (day 3), characterized by expression of SOX17 (87.2% ± 4.6; n=5), FOXA2 (82.8% ± 4.6; n=3), co-expression of c-KIT and CXCR4 (93.3% ± 5.6; n=4), and silencing of pluripotency marker OCT4 (2.8% ± 6; n=5). At the end of stage 4 (day 12), pancreatic progenitor cells were identified by the co-expression of PDX1/NKX6.1 (46.7% ± 3.5; n=3). Finally, at the end of stage 7 (day 30), C-peptide-positive β-like cells (49.5% ± 10.5; n=3) and glucagon-positive α-like cells (18.4%± 6.1; n=3) were present. The stimulation index upon exposure to glucose was 4.4 ± 3.1(n=5). Following transplantation of day-30 clusters into mice, stimulated human C-peptide levels reached 46 pmol/L (n=21), 70.5 pmol/L (n=19) and 327.4 pmol/L (n=11) at day 14, 28 and 60 respectively, indicating further maturation of the cells in vivo after transplantation. Next, we scaled up the cell culture in disposable spinner flasks, and obtained comparable differentiation efficiency. Conclusion: We can generate functional hPSCs-derived β-like cells in GMP-compliant conditions in vitro. We will apply these protocols on a clinical-grade PSC line that is currently in expansion (banking) in the GMP facility of our institute. hPSCs-derived β-like cells are a promising future alternative to donor islets for the treatment of type 1 diabetes.

307.2: Bioprinted Immune-protective Islet-containing Tissues Successfully Regulate Blood Glucose in Rodent Models of Type 1 Diabetes

Introduction: Type 1 diabetes (T1D) is a disease characterized by elevated blood glucose due to insufficient insulin release from pancreatic β-cells. Transplantation of cadaveric islets demonstrates that cell therapy can fully reverse hyperglycemia. However, limited cell supply, immune rejection of implanted allogeneic cells, and cell survival represent major challenges. Cell encapsulation has great potential to overcome these challenges by blocking immune cell access to the grafts while allowing nutrient exchange and secreted products from the implanted cells to be delivered to the body. In this study, we use a unique microfluidic bioprinting technology to precisely control the placement of cells and biomaterials within 3D tissues with micro-architectures optimized for cellular fitness and immune protection. Methods: Living tissues consisting of fibres with a cell-containing core and immune-protective alginate-based shell were generated using Aspect Biosystems’ RX1 bioprinter technology. Core-shell fibres with reaggregated primary human pancreatic islets or embryonic stem cell-derived β-cells were tested in vitro using viability and functional (glucose-stimulated insulin secretion, GSIS) assays. Bioprinted tissues were then implanted into the IP space or omentum of streptozotocin (STZ)-induced diabetic mice and rats, respectively. Glucose homeostasis, body weight, and human C-peptide secretion were monitored for up to 3 months following implantion. Retrieved grafts were fixed and analyzed by histology (H&E, Masson’s trichrome stain) and immunohistochemistry (α-SMA, CD45) to quantify fibrotic encapsulation and immune cell infiltration. Results: Bioprinted tissues supported viability and dynamic insulin secretion of cells in vitro up to 28 days. When transplanted into immunodeficient and immunocompetent diabetic rodents, bioprinted tissues containing reaggregated human islets successfully regulated blood glucose for up to 3 months (Figure 1), although normoglycemia was only sustained in a subset of immunocompetent animals and associated with variable fibrosis. Post-retrieval viability stain, assessment of GSIS, and histology revealed high viability and functionality of implanted cells, and the absence of leukocyte infiltration through the shell. Discussion: This is the first study showing a fully 3D bioprinted tissue composed of a core/shell fibre can successfully deliver a therapeutic dose of xenogeneic cells into a diabetic animal (Figure 2). The unique features of the microfluidic technology were leveraged to bioprint an implantable and retrievable tissue patch that merges the benefits of a single fibre (cell fitness, access to nutrients, immune-protection, perm-selectivity) with those provided by a 3D structure (retrievability, ease of implant, structural integrity). The promising results obtained in rodent studies warrant further investigation to minimize fibrosis and explore the scaling-up of bioprinted tissues in large animal models of T1D.NRC-IRAP. NSERC. Stem Cell Network. Genome BC.

The potential for isotope dilution-LC-MS/MS to improve laboratory measurement of C-peptide: Reasons and critical determinants

Human C-peptide is secreted in equimolar amounts with insulin by pancreatic beta-cells. Measurement of C-peptide plays an important role in the diagnosis and treatment of diabetes where it is used to evaluate the function of islet cells. However, C-peptide measurement results across different laboratories vary considerably and there is an urgent need to improve comparability between laboratories. As it is sensitive and specific, isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) has made a major contribution and will continue to play a significant role in the standardization of C-peptide measurement. Here, we reviewed the application of ID-LC-MS/MS in C-peptide measurement by discussing the biochemical properties of C-peptide, common sample preparation procedures, and the sensitivity problems often encountered with ID-LC-MS/MS C-peptide measurement. Collectively, these factors are crucial for the development of ID-LC-MS/MS methods for C-peptide measurement. We also discussed the advantages, disadvantages, and progress of implementing ID-LC-MS/MS as a routine measurement tool for C-peptide in clinical laboratories. Finally, we summarized the existing reference system and the status of C-peptide measurement in clinical laboratories to convey the necessity of improving the comparability of C-peptide measurement in clinical laboratories using ID-LC-MS/MS.

Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS.

The increasing importance to determine bioactive peptide hormones such as insulin, its synthetic analogs, and C-peptide in urine samples represents an analytical challenge. The physiological concentrations of insulin in urine are commonly found at sub-ng/mL levels and thus represent a complex analytical task. C-peptide concentrations, on the other hand, tend to be in the moderate ng/mL range and are hence much easier to determine. Insulin and C-peptide are important in the diagnostics and management of metabolic disorders such as diabetes mellitus and are also particularly relevant target analytes in professional sports and forensics. All insulins are classified on the World Anti-Doping Agency’s (WADA) list of prohibited substances and methods in sports with a minimum required performance level (MRPL) of 50 pg/mL. Until now, methods combining immunoextraction and subsequent mass spectrometric detection have mostly been used for this purpose. With the method developed here, sample preparation has been simplified considerably and does not require an antibody-based sample purification. This was achieved by a sophisticated mixed-mode solid-phase extraction and subsequent separation with liquid chromatography coupled to high-resolution mass spectrometry. Included target insulins were human, lispro, glulisine, aspart, glargine metabolite, degludec, and additionally, human C-peptide. The method was validated for the synthetic insulin analogs considering WADA requirements including specificity, limit of detection (10–25 pg/mL), limit of identification, recovery (25–100%), robustness, carry over (<2%), and matrix effects. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely, [[2H10] LeuB6, B11, B15, B17]-insulin and [[13C6] Leu26, 30] C-peptide. Finally, the method was applied to samples from patients with diabetes mellitus treated with synthetic insulins.

Selection for CD26- and CD49A+ Cells From Pluripotent Stem Cells-Derived Islet-Like Clusters Improves Therapeutic Activity in Diabetic Mice.

BackgroundCell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the β-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells.MethodsFunctional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice.ResultsCD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A+ cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26+ cells followed by enrichment for CD49A+ cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26-/CD49A+ enriched ILC exhibited improved function over non-sorted ILC or CD49A+ cells in diabetic mice and maintain prolonged blood C-peptide levels.ConclusionsRefining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

Development of radioimmunoassay for estimation of C-peptide in human serum

C-Peptide radioimmunoassay (RIA) is an invaluable aid in diagnosis of diabetes, insulinoma and hypoglycaemia. An RIA for C-peptide was developed by using a polyclonal antibody for human C-peptide raised in rabbits and validated using human serum samples. Tyrosyated C-peptide was radioiodinated to a specific activity of around 283 µCi/µg by conventional chloramine-T method. The detection limit of the assay (90% binding) was 0.43 ng/mL and the ED50 value was 8.5 ng/mL. The assay with the standard range of 0.3–18 ng/mL and low non-specific binding provide superior assay features. Separation of the bound from free complex was carried out using 22% polyethylene glycol. Intra-assay and inter-assay variations were found to be < 11.5% along with analytical recovery of 90–104%. No cross reactivity was observed with proinsulin, glucagon, somatostatin, insulin and pancreatic polypeptide.The developed assay can be used for the routine analysis of clinical samples.