Determination of insulin secretion from stem cell-derived islet organoids with liquid chromatography-tandem mass spectrometry

Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100–200 µm), which can be grown from the patient́s own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: ≤0.2 pg/µL, relative error: ≤±10%, relative standard deviation: <10%), and was well suited for measuring 20 µL amounts of Krebs buffer containing insulin secreted from islet organoids.publishedVersio

Tissue Engineering Strategies for Improving Beta Cell Transplantation Outcome

Abstract Purpose of Review Beta cell replacement therapy as a form of islet transplantation is a promising alternative therapy with the possibility to make selected patients with type 1 diabetes (T1D) insulin independent. However, this technique faces challenges such as extensive activation of the host immune system post-transplantation, lifelong need for immunosuppression, and the scarcity of islet donor pancreas. Advancement in tissue engineering strategies can improve these challenges and allow for a more widespread application of this therapy. This review will discuss the recent development and clinical translation of tissue engineering strategies in beta cell replacement therapy. Recent Findings Tissue engineering offers innovative solutions for producing unlimited glucose responsive cells and fabrication of appropriate devices/scaffolds for transplantation applications. Generation of pancreatic organoids with supporting cells in biocompatible biomaterials is a powerful technique to improve the function of insulin-producing cell clusters. Fabrication of physical barriers such as encapsulation strategies can protect the cells from the host immune system and allow for graft retrieval, although this strategy still faces major challenges to fully restore physiological glucose regulation. Summary The three main components of tissue engineering strategies including the generation of stem cell-derived insulin-producing cells and organoids and the possibilities for therapeutic delivery of cell-seeded devices to extra-hepatic sites need to come together in order to provide safe and functional insulin-producing devices for clinical beta cell replacement therapy

Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis

One of the key limitations to successful human islet transplantation is loss of islets due to stress responses pre- and post-transplantation. Nutrient deprivation and ER stress have been identified as important mechanisms leading to apoptosis. Glial Cell-line Derived Neurotrophic Factor (GDNF) has recently been found to promote islet survival after isolation. However, whether GDNF could rescue human islets from nutrient deprivation and ER stress-mediated apoptosis is unknown. Herein, by mimicking those conditions in vitro, we have shown that GDNF significantly improved glucose stimulated insulin secretion, reduced apoptosis and proinsulin: insulin ratio in nutrient deprived human islets. Furthermore, GDNF alleviated thapsigargin-induced ER stress evidenced by reduced expressions of IRE1 alpha and BiP and consequently apoptosis. Importantly, this was associated with an increase in phosphorylation of PI3K/AKT and GSK3B signaling pathway. Transplantation of ER stressed human islets pre- treated with GDNF under kidney capsule of diabetic mice resulted in reduced expressions of IRE1 alpha and BiP in human islet grafts with improved grafts function shown by higher levels of human C-peptide post-transplantation. We suggest that GDNF has protective and anti-apoptotic effects on nutrient deprived and ER stress activated human islets and could play a significant role in rescuing human islets from stress responses

The effects of exendin-4 treatment on graft failure: An animal study using a novel revascularized minimal human islet transplant model

Islet transplantation has become a viable clinical treatment, but is still compromised by long-term graft failure. Exendin-4, a glucagon-like peptide 1 receptor agonist, has in clinical studies been shown to improve insulin secretion in islet transplanted patients. However, little is known about the effect of exendin-4 on other metabolic parameters. We therefore aimed to determine what influence exendin-4 would have on revascularized minimal human islet grafts in a state of graft failure in terms of glucose metabolism, body weight, lipid levels and graft survival. Introducing the bilateral, subcapsular islet transplantation model, we first transplanted diabetic mice with a murine graft under the left kidney capsule sufficient to restore normoglycemia. After a convalescent period, we performed a second transplantation under the right kidney capsule with a minimal human islet graft and allowed for a second recovery. We then performed a left-sided nephrectomy, and immediately started treatment with exendin-4 with a low (20μg/kg/day) or high (200μg/kg/day) dose, or saline subcutaneously twice daily for 15 days. Blood was sampled, blood glucose and body weight monitored. The transplanted human islet grafts were collected at study end point and analyzed. We found that exendin-4 exerts its effect on failing human islet grafts in a bell-shaped dose-response curve. Both doses of exendin-4 equally and significantly reduced blood glucose. Glucagon-like peptide 1 (GLP-1), C-peptide and pro-insulin were conversely increased. In the course of the treatment, body weight and cholesterol levels were not affected. However, immunohistochemistry revealed an increase in beta cell nuclei count and reduced TUNEL staining only in the group treated with a low dose of exendin-4 compared to the high dose and control. Collectively, these results suggest that exendin-4 has a potential rescue effect on failing, revascularized human islets in terms of lowering blood glucose, maintaining beta cell numbers, and improving metabolic parameters during hyperglycemic stress

Pancreas-on-a-Chip Technology for Transplantation Applications

Abstract Purpose of Review Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. Recent Findings PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Summary Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions

Encapsulation boosts islet-cell signature in differentiating human induced pluripotent stem cells via integrin signalling

Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process

In vivo hyperglycemia exposure elicits distinct period-dependent effects on human pancreatic progenitor differentiation, conveyed by oxidative stress

Aim: The loss of insulin‐secreting β‐cells, ultimately characterizing most diabetes forms, demands the development of cell replacement therapies. The common endpoint for all ex vivo strategies is transplantation into diabetic patients. However, the effects of hyperglycaemia environment on the transplanted cells were not yet properly assessed. Thus, the main goal of this study was to characterize global effect of brief and prolonged in vivo hyperglycaemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors. Methods: To rigorously study the effect of hyperglycaemia, in vitro differentiated human‐induced pluripotent stem cells (hiPSC)‐derived pancreatic progenitors were xenotransplanted in normoglycaemic and diabetic NSG rat insulin promoter (RIP)‐diphtheria toxin receptor (DTR) mice. The transplants were retrieved after 1‐week or 1‐month exposure to overt hyperglycaemia and analysed by large‐scale microscopy or global proteomics. For this study we pioneer the use of the NSG RIP‐DTR system in the transplantation of hiPSC, making use of its highly reproducible specific and absolute β‐cell ablation property in the absence of inflammation or other organ toxicity. Results: Here we show for the first time that besides the presence of an induced oxidative stress signature, the cell fate and proteome landscape response to hyperglycaemia was different, involving largely different mechanisms, according to the period spent in the hyperglycaemic environment. Surprisingly, brief hyperglycaemia exposure increased the bihormonal cell number by impeding the activity of specific islet lineage determinants. Moreover, it activated antioxidant and inflammation protection mechanisms signatures in the transplanted cells. In contrast, the prolonged exposure was characterized by decreased numbers of hormone + cells, low/absent detoxification signature, augmented production of oxygen reactive species and increased apoptosis. Conclusion: Hyperglycaemia exposure induced distinct, period‐dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival

Chronically elevated exogenous glucose elicits antipodal effects on the proteome signature of differentiating human iPSC-derived pancreatic progenitors

The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature

Encapsulation boosts islet-cell signature in differentiating human induced pluripotent stem cells via integrin signalling

Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process

In vivo hyperglycemia exposure elicits distinct period-dependent effects on human pancreatic progenitor differentiation, conveyed by oxidative stress

Aim The loss of insulin‐secreting β‐cells, ultimately characterizing most diabetes forms, demands the development of cell replacement therapies. The common endpoint for all ex vivo strategies is transplantation into diabetic patients. However, the effects of hyperglycaemia environment on the transplanted cells were not yet properly assessed. Thus, the main goal of this study was to characterize global effect of brief and prolonged in vivo hyperglycaemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors. Methods To rigorously study the effect of hyperglycaemia, in vitro differentiated human‐induced pluripotent stem cells (hiPSC)‐derived pancreatic progenitors were xenotransplanted in normoglycaemic and diabetic NSG rat insulin promoter (RIP)‐diphtheria toxin receptor (DTR) mice. The transplants were retrieved after 1‐week or 1‐month exposure to overt hyperglycaemia and analysed by large‐scale microscopy or global proteomics. For this study we pioneer the use of the NSG RIP‐DTR system in the transplantation of hiPSC, making use of its highly reproducible specific and absolute β‐cell ablation property in the absence of inflammation or other organ toxicity. Results Here we show for the first time that besides the presence of an induced oxidative stress signature, the cell fate and proteome landscape response to hyperglycaemia was different, involving largely different mechanisms, according to the period spent in the hyperglycaemic environment. Surprisingly, brief hyperglycaemia exposure increased the bihormonal cell number by impeding the activity of specific islet lineage determinants. Moreover, it activated antioxidant and inflammation protection mechanisms signatures in the transplanted cells. In contrast, the prolonged exposure was characterized by decreased numbers of hormone + cells, low/absent detoxification signature, augmented production of oxygen reactive species and increased apoptosis. Conclusion Hyperglycaemia exposure induced distinct, period‐dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival