Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.

Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Function and composition of pancreatic islet cell implants in omentum of type 1 diabetes patients.

Intraportal (IP) islet cell transplants can restore metabolic control in type 1 diabetes patients, but limitations raise the need for establishing a functional beta cell mass (FBM) in a confined extrahepatic site. This study reports on function and composition of omental (OM) implants after placement of islet cell grafts with similar beta cell mass as in our IP-protocol (2-5.106 beta cells/kg body weight) on a scaffold. Four of seven C-peptide-negative recipients achieved low beta cell function (hyperglycemic clamp [HGC] 2-8 percent of controls) until laparoscopy, 2-6 months later, for OM-biopsy and concomitant IP-transplant with similar beta cell dose. This IP-transplant increased HGC-values to 15-40 percent. OM-biopsies reflected the composition of initial grafts, exhibiting varying proportions of endocrine-cell-enriched clusters with more beta than alpha cells and leucocyte pole, non-endocrine cytokeratin-positive clusters surrounded by leucocytes, and scaffold remnants with foreign body reaction. OM-implants on a polyglactin-thrombin-fibrinogen-scaffold presented larger endocrine clusters with infiltrating endothelial cells and corresponded to the higher HGC-values. No activation of cellular immunity to GAD/IA2 was measured post-OM-transplant. Establishment of a metabolically adequate FBM in omentum may require a higher beta cell number in grafts but also elimination of their immunogenic non-endocrine components as well as local conditioning that favors endocrine cell engraftment and function.

Formation of amyloid in encapsulated human pancreatic and human stem cell-generated beta cell implants.

Detection of amyloid in intraportal islet implants of type 1 diabetes patients has been proposed as cause in their functional decline. The present study uses cultured adult human islets devoid of amyloid to examine conditions of its formation. After intraportal injection in patients, amyloid deposits <15 µm diameter were identified in 5%-12% of beta cell containing aggregates, 3-76 months posttransplant. Such deposits also formed in glucose-controlling islet implants in the kidney of diabetic mice but not in failing implants. Alginate-encapsulated islets formed amyloid during culture when functional, and in all intraperitoneal implants that corrected diabetes in mice, exhibiting larger sizes than in functioning nonencapsulated implants. After intraperitoneal injection in a patient, retrieved single capsules presented amyloid near living beta cells, whereas no amyloid occurred in clustered capsules with dead cells. Amyloid was also demonstrated in functional human stem cell-generated beta cell implants in subcutaneous devices of mice. Deposits up to 35 µm diameter were localized in beta cell-enriched regions and related to an elevated IAPP over insulin ratio in the newly generated beta cells. Amyloid in device-encapsulated human stem cell-generated beta cell implants marks the formation of a functional beta cell mass but also an imbalance between its activated state and its microenvironment.

Immunogenicity of human embryonic stem cell-derived beta cells.

AIMS/HYPOTHESIS: To overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells. METHODS: We comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice. RESULTS: We found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells. CONCLUSIONS/INTERPRETATION: hESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.

A combination of cytokines EGF and CNTF protects the functional beta cell mass in mice with short-term hyperglycaemia.

AIMS/HYPOTHESIS: When the beta cell mass or function declines beyond a critical point, hyperglycaemia arises. Little is known about the potential pathways involved in beta cell rescue. As two cytokines, epidermal growth factor (EGF) and ciliary neurotrophic factor (CNTF), restored a functional beta cell mass in mice with long-term hyperglycaemia by reprogramming acinar cells that transiently expressed neurogenin 3 (NGN3), the current study assesses the effect of these cytokines on the functional beta cell mass after an acute chemical toxic insult. METHODS: Glycaemia and insulin levels, pro-endocrine gene expression and beta cell origin, as well as the role of signal transducer and activator of transcription 3 (STAT3) signalling, were assessed in EGF+CNTF-treated mice following acute hyperglycaemia. RESULTS: The mice were hyperglycaemic 1 day following i.v. injection of the beta cell toxin alloxan, when the two cytokines were applied. One week later, 68.6 ± 4.6% of the mice had responded to the cytokine treatment and increased their insulin(+) cell number to 30% that of normoglycaemic control mice, resulting in restoration of euglycaemia. Although insulin(-) NGN3(+) cells appeared following acute EGF+CNTF treatment, genetic lineage tracing showed that the majority of the insulin(+) cells originated from pre-existing beta cells. Beta cell rescue by EGF+CNTF depends on glycaemia rather than on STAT3-induced NGN3 expression in acinar cells. CONCLUSIONS/INTERPRETATION: In adult mice, EGF+CNTF allows the rescue of beta cells in distress when treatment is given shortly after the diabetogenic insult. The rescued beta cells restore a functional beta cell mass able to control normal blood glucose levels. These findings may provide new insights into compensatory pathways activated early after beta cell loss.

Direct effect of glucocorticoids on glucose-activated adult rat ß-cells increases their cell number and their functional mass for transplantation.

Compounds that increase ß-cell number can serve as ß-cell replacement therapies in diabetes. In vitro studies have identified several agents that can activate DNA synthesis in primary ß-cells but only in small percentages of cells and without demonstration of increases in cell number. We used whole well multiparameter imaging to first screen a library of 1,280 compounds for their ability to recruit adult rat ß-cells into DNA synthesis and then assessed influences of stimulatory agents on the number of living cells. The four compounds with highest ß-cell recruitment were glucocorticoid (GC) receptor ligands. The GC effect occurred in glucose-activated ß-cells and was associated with increased glucose utilization and oxidation. Hydrocortisone and methylprednisolone almost doubled the number of ß-cells in 2 wk. The expanded cell population provided an increased functional ß-cell mass for transplantation in diabetic animals. These effects are age dependent; they did not occur in neonatal rat ß-cells, where GC exposure suppressed basal replication and was cytotoxic. We concluded that GCs can induce the replication of adult rat ß-cells through a direct action, with intercellular differences in responsiveness that have been related to differences in glucose activation and in age. These influences can explain variability in GC-induced activation of DNA synthesis in rat and human ß-cells. Our study also demonstrated that ß-cells can be expanded in vitro to increase the size of metabolically adequate grafts.

Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

ß-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in ß-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20-30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and ß-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived ß-cells at PT weeks 20-30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation.

Comparison of Omentum and Subcutis as Implant Sites for Device-Encapsulated Human iPSC-Derived Pancreatic Endoderm in Nude Rats.

Subcutaneous implants of device-encapsulated stem cell-derived pancreatic endoderm (PE) can establish a functional beta cell mass (FBM) with metabolic control in immune-compromised mice. In a study with human-induced pluripotent stem cell-PE, this outcome was favored by a preformed pouch which allowed lesion-free insertion of devices in a pre-vascularized site. This was not reproduced in nude rats, known to exhibit a higher innate reactivity than mice and therefore relevant as preclinical model: a dense fibrotic capsule formed around subcutis (SC) implants with virtually no FBM formation. Placement in omentum (OM) of nude rats provided a less fibrous, better vascularized environment than SC. It resulted in less donor cell loss (56% recovery at post-transplant-PT week 3 versus 16% in SC) allowing FBM-formation. At PT week 30, 6/13 OM-recipients exhibited glucose-induced plasma hu-C-peptide to 0.1-0.4 ng/ml, versus 0/8 in SC-recipients. These levels are more than 10-fold lower than in a state of metabolic control. This shortcoming is not caused by inadequate glucose responsiveness of the beta cells but by their insufficient number. The size of the formed beta cell mass (0.4 ± 0.2 µl) was lower than that reported in mice receiving the same cell product subcutaneously; the difference is attributed to a lower expansion of pancreatic progenitor cells and to their lower degree of differentiation to beta cells. This study in the nude rat model demonstrates that OM provides a better environment for formation of beta cells in device-encapsulated PE-implants than SC. It also identified targets for increasing their dose-efficacy.

Insulitis and lymphoid structures in the islets of Langerhans of a 66-year-old patient with long-standing type 1 diabetes.

Insulitis is a characteristic inflammatory lesion consisting of immune cell infiltrates around and within the pancreatic islets of patients with recent-onset type 1 diabetes (T1D). The infiltration is typically mild, both in terms of the number of infiltrating cells and the number of islets affected. Here, we present an unusual histopathological case study of a 66-year-old female patient with long-standing T1D, insulitis, and islet-associated lymphoid tissue. Most islets in the head of the pancreas of this patient were insulin-deficient, whereas the islets in the tail appeared normal. Insulitis was present in 0.84% of the insulin-containing islets and three islets had large lymphocytic infiltrates resembling tertiary lymphoid structures (TLS). Of note, this is the first description of potential TLS in the endocrine pancreas of a patient with T1D. Their association with a marked residual beta cell mass is of interest and may hint at new insights into disease progression and regulation of autoimmunity.

The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts.

Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.

Insulitis in the pancreas of non-diabetic organ donors under age 25 years with multiple circulating autoantibodies against islet cell antigens.

Autoantibodies against islet cell antigens are routinely used to identify subjects at increased risk of symptomatic type 1 diabetes, but their relation to the intra-islet pathogenetic process that leads to positivity for these markers is poorly understood. We screened 556 non-diabetic organ donors (3 months to 24 years) for five different autoantibodies and found positivity in 27 subjects, 25 single- and two double autoantibody-positive donors. Histopathological screening of pancreatic tissue samples showed lesion characteristic for recent-onset type 1 diabetes in the two organ donors with a high-risk profile, due to their positivity for multiple autoantibodies and HLA-inferred risk. Inflammatory infiltrates (insulitis) were found in a small fraction of islets (<5%) and consisted predominantly of CD3+CD8+ T-cells. Islets with insulitis were found in close proximity to islets devoid of insulin-positivity; such pseudo-atrophic islets were present in multiple small foci scattered throughout the pancreatic tissue or were found to be distributed with a lobular pattern. Relative beta cell area in both single and multiple autoantibody-positive donors was comparable to that in autoantibody-negative controls. In conclusion, in organ donors under age 25 years, insulitis and pseudo-atrophic islets were restricted to multiple autoantibody-positive individuals allegedly at high risk of developing symptomatic type 1 diabetes, in line with reports in older age groups. These observations may give further insight into the early pathogenetic events that may culminate in clinically overt disease.

Evidence of Tissue Repair in Human Donor Pancreas After Prolonged Duration of Stay in Intensive Care.

M2 macrophages play an important role in tissue repair and regeneration. They have also been found to modulate ß-cell replication in mouse models of pancreatic injury and disease. We previously reported that ß-cell replication is strongly increased in a subgroup of human organ donors characterized by prolonged duration of stay in an intensive care unit (ICU) and increased number of leukocytes in the pancreatic tissue. In the present study we investigated the relationship between duration of stay in the ICU, M2 macrophages, vascularization, and pancreatic cell replication. Pancreatic organs from 50 donors without diabetes with different durations of stay in the ICU were analyzed by immunostaining and digital image analysis. The number of CD68+CD206+ M2 macrophages increased three- to sixfold from ≥6 days' duration of stay in the ICU onwards. This was accompanied by a threefold increased vascular density and a four- to ninefold increase in pancreatic cells positive for the replication marker Ki67. A strong correlation was observed between the number of M2 macrophages and ß-cell replication. These results show that a prolonged duration of stay in the ICU is associated with an increased M2 macrophage number, increased vascular density, and an overall increase in replication of all pancreatic cell types. Our data show evidence of marked levels of tissue repair in the human donor pancreas.

Retraction Note: Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.

This article has been retracted; see accompanying Retraction Note, which can be accessed via a link at the top of the paper.

Glucocorticoids and checkpoint tyrosine kinase inhibitors stimulate rat pancreatic beta cell proliferation differentially.

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids.

Cell Mass Increase Associated with Formation of Glucose-Controlling ß-Cell Mass in Device-Encapsulated Implants of hiPS-Derived Pancreatic Endoderm.

Device-encapsulated human stem cell-derived pancreatic endoderm (PE) can generate functional ß-cell implants in the subcutis of mice, which has led to the start of clinical studies in type 1 diabetes. Assessment of the formed functional ß-cell mass (FBM) and its correlation with in vivo metabolic markers can guide clinical translation. We recently reported ex vivo characteristics of device-encapsulated human embryonic stem cell-derived (hES)-PE implants in mice that had established a metabolically adequate FBM during 50-week follow-up. Cell suspensions from retrieved implants indicated a correlation with the number of formed ß cells and their maturation to a functional state comparable to human pancreatic ß cells. Variability in metabolic outcome was attributed to differences in number of PE-generated ß cells. This variability hinders studies on processes involved in FBM-formation. This study reports modifications that reduce variability. It is undertaken with device-encapsulated human induced pluripotent stem cell-derived-PE subcutaneously implanted in mice. Cell mass of each cell type was determined on intact tissue inside the device to obtain more precise data than following isolation and dispersion. Implants in a preformed pouch generated a glucose-controlling ß-cell mass within 20 weeks in over 60% of recipients versus less than 20% in the absence of a pouch, whether the same or threefold higher cell dose had been inserted. In situ analysis of implants indicated a role for pancreatic progenitor cell expansion and endocrine differentiation in achieving the size of ß- and α-cell mass that correlated with in vivo markers of metabolic control. Stem Cells Translational Medicine 2019;8:1296&1305.

Stromal-targeting radioimmunotherapy mitigates the progression of therapy-resistant tumors.

Radioimmunotherapy (RIT) aims to deliver a high radiation dose to cancer cells, while minimizing the exposure of normal cells. Typically, monoclonal antibodies are used to target the radionuclides to cancer cell surface antigens. However, antibodies face limitations due to their poor tumor penetration and suboptimal pharmacokinetics, while the expression of their target on the cancer cell surface may be gradually lost. In addition, most antigens are expressed in a limited number of tumor types. To circumvent these problems, we developed a Nanobody (Nb)-based RIT against a prominent stromal cell (stromal-targeting radioimmunotherapy or STRIT) present in nearly all tumors, the tumor-associated macrophage (TAM). Macrophage Mannose Receptor (MMR) functions as a stable molecular target on TAM residing in hypoxic areas, further allowing the delivery of a high radiation dose to the more radioresistant hypoxic tumor regions. Since MMR expression is not restricted to TAM, we first optimized a strategy to block extra-tumoral MMR to prevent therapy-induced toxicity. A 100-fold molar excess of unlabeled bivalent Nb largely blocks extra-tumoral binding of 177Lu-labeled anti-MMR Nb and prevents toxicity, while still allowing the intra-tumoral binding of the monovalent Nb. Interestingly, three doses of 177Lu-labeled anti-MMR Nb resulted in a significantly retarded tumor growth, thereby outcompeting the effects of anti-PD1, anti-VEGFR2, doxorubicin and paclitaxel in the TS/A mammary carcinoma model. Together, these data propose anti-MMR STRIT as a valid new approach for cancer treatment.

Combined Analysis of GAD65, miR-375, and Unmethylated Insulin DNA Following Islet Transplantation in Patients With T1D.

Aim: Several biomarkers have been proposed to detect pancreatic ß cell destruction in vivo but so far have not been compared for sensitivity and significance. Methods: We used islet transplantation as a model to compare plasma concentrations of miR-375, 65-kDa subunit of glutamate decarboxylase (GAD65), and unmethylated insulin DNA, measured at subpicomolar sensitivity, and study their discharge kinetics, power for outcome prediction, and detection of graft loss during follow-up. Results: At 60 minutes after transplantation, GAD65 and miR-375 consistently showed near-equimolar and correlated increases proportional to the number of implanted ß cells. GAD65 and miR-375 showed comparable power to predict poor graft outcome at 2 months, with areas under the curve of 0.833 and 0.771, respectively (P = 0.53). Using receiver operating characteristic analysis, we defined likelihood ratios (LRs) for rationally selected result intervals. In GADA-negative recipients (n = 28), GAD65 <4.5 pmol/L (LR = 0.15) and >12.2 pmol/L (LR = ∞) predicted good and poor outcomes, respectively. miR-375 could be used in all recipients irrespective of GAD65 autoantibody status (n = 46), with levels <1.4 pmol/L (LR = 0.14) or >7.6 pmol/L (LR = 9.53) as dual thresholds. The posttransplant surge of unmethylated insulin DNA was inconsistent and unrelated to outcome. Combined measurement of these three biomarkers was also tested as liquid biopsy for ß cell death during 2-month follow-up; incidental surges of GAD65, miR-375, and (un)methylated insulin DNA, alone or combined, were confidently detected but could not be related to outcome. Conclusions: GAD65 and miR-375 performed equally well in quantifying early graft destruction and predicting graft outcome, outperforming unmethylated insulin DNA.

CD99 as surface anchor for human islet endocrine cell purification.

Rat and human beta cell proteomes were quantified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), searching for cell surface markers. In human beta cells, CD99 (cluster of differentiation 99) was ranked among the plasma membrane proteins that combine a high molar abundance with a relative degree of selectivity for the endocrine cells of the islets of Langerhans. Therefore, the applicability of CD99 as anchor for islet endocrine cell purification was investigated. The CD99 gene and protein expression were studied using microarray, LC-MS/MS, western blotting, flow cytometry and immunofluorescence, and a protocol was developed for magnetic bead-mediated beta cell enrichment from human pancreas digests using available anti-CD99 antibodies. In human, but not in rat, CD99 protein and mRNA were abundantly expressed by islet endocrine cells but undetectable in exocrine pancreas. The extracellular CD99 epitopes appeared to be trypsin-resistant, enabling the binding of anti-CD99 antibodies to an insulin+/TSQ+ cell subset and efficient coupling of magnetic beads for positive selection of CD99+ cells. A MACS-CD99 purification of human pancreas fractions with low endocrine purity consistently yielded a fourfold enrichment of insulin+/TSQ+ cells and formation of viable and functional endocrine aggregates after 24 h of culture. It is concluded that CD99 is a human beta cell surface marker that, by virtue of its high molar abundance and resistance to tryptic digestion, can be used as anchor for upscalable magnetic bead-mediated islet endocrine cell purification. Copyright © 2016 John Wiley & Sons, Ltd.

An analytical comparison of three immunoassay platforms for subpicomolar detection of protein biomarker GAD65.

A disproportional increase of circulating GAD65 within hours from an intraportal islet allotransplantation has been validated as biomarker of beta cell loss and poor functional outcome. More sensitive assays are, however, needed to allow detection of episodes of subtle beta cell loss during late-stage graft rejection or in the peri-onset period of type 1 diabetes. We applied the same sandwich monoclonal antibody couple reactive towards the C- and N-terminus of GAD65 on three advanced immunoassay platforms-the Cytometric Bead Array (CBA, Becton, Dickinson and Company), ElectroChemiLuminescence ImmunoAssay (ECLIA, Meso Scale Discovery) and digital ELISA technology (Single Molecule Array-SIMOA, Quanterix. We then compared analytical performance (linearity, imprecision, limit of detection and functional sensitivity), correlation of results, and practicality. All evaluated techniques showed linearity up to at least 500 ng/dL (76.9 pmol/L). SIMOA achieved the lowest imprecision. The 3 platforms correlate well with each other and could all detect subpicomolar concentrations of GAD65 in plasma, but only SIMOA and CBA could quantify down to that range. SIMOA can achieve the highest sample throughput. The three methods tested allow sensitive detection of GAD65, but SIMOA appears best suited for automated quantification of subpicomolar concentrations.

Functional Beta Cell Mass from Device-Encapsulated hESC-Derived Pancreatic Endoderm Achieving Metabolic Control.

Human stem cells represent a potential source for implants that replace the depleted functional beta cell mass (FBM) in diabetes patients. Human embryonic stem cell-derived pancreatic endoderm (hES-PE) can generate implants with glucose-responsive beta cells capable of reducing hyperglycemia in mice. This study with device-encapsulated hES-PE (4 × 106 cells/mouse) determines the biologic characteristics at which implants establish metabolic control during a 50-week follow-up. A metabolically adequate FBM was achieved by (1) formation of a sufficient beta cell number (>0.3 × 106/mouse) at >50% endocrine purity and (2) their maturation to a functional state comparable with human pancreatic beta cells, as judged by their secretory responses during perifusion, their content in typical secretory vesicles, and their nuclear NKX6.1-PDX1-MAFA co-expression. Assessment of FBM in implants and its correlation with in vivo metabolic markers will guide clinical translation of stem cell-derived grafts in diabetes.