Source Of Islets Research Articles

Enhancing differentiation and functionality of insulin-producing cells derived from iPSCs using esterified collagen hydrogel for cell therapy in diabetes mellitus

BackgroundIslet transplantation is a recommended treatment for type 1 diabetes but is limited by donor organ shortage. This study introduces an innovative approach for improving the differentiation and functionality of insulin-producing cells (IPCs) from iPSCs using 3D spheroid formation and hydrogel matrix as an alternative pancreatic islet source. The extracellular matrix (ECM) is crucial for pancreatic islet functionality, but finding the ideal matrix for β-cell differentiation has been challenging. We aimed to advance IPC differentiation and maturation through an esterified collagen hydrogel, comparing its effectiveness with conventional basement membrane extract (BME) hydrogels.MethodsiPSCs were differentiated into IPCs using a small molecule-based sequential protocol, followed by spheroid formation in concave microwells. Rheological analysis, scanning electron microscopy, and proteomic profiling were used to characterize the chemical and physical properties of each matrix. IPCs, both in single-cell form and as spheroids, were embedded in either ionized collagen or BME hydrogels, which was followed by assessments of morphological changes, pancreatic islet-related gene expression, insulin secretion, and pathway activation using comprehensive analytical techniques.ResultsEsterified collagen hydrogels markedly improved the structural integrity, insulin expression, and cell-cell interactions in IPC spheroids, forming densely packed insulin-expressing clusters, in contrast to the dispersed cells observed in BME cultures. Collagen hydrogel significantly enhanced the mRNA expression of crucial endocrine markers and maturation factors, with IPC spheroids showing accelerated differentiation from day 5, suggesting a faster differentiation compared to single cells in hydrogel encapsulation. Insulin secretion in response to glucose in collagen environments, with a GSIS index of 2.46 ± 0.05, exceeded those in 2D and BME, demonstrating superior pancreatic islet functionality. Pathway analysis highlighted enhanced insulin secretion capabilities, evidenced by the upregulation of genes like Secretogranin III and Chromogranin A in collagen cultures. In vivo transplantation results showed that collagen hydrogel enhanced cluster integrity, tissue integration, and insulin secretion compared to non-embedded IPCs and BME groups.ConclusionEsterified collagen hydrogels demonstrated superior efficacy over 2D and BME in promoting IPC differentiation and maturation, possibly through upregulation of the expression of key secretion pathway genes. Our findings suggest that using collagen hydrogels presents a promising approach to enhance insulin secretion efficiency in differentiating pancreatic β-cells, advancing cell therapy in diabetes cell therapy.

Suspension culture improves iPSC expansion and pluripotency phenotype

BackgroundInduced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol. MethodsSendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs.ResultsExpansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D (p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67+ expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4+Nanog+Sox2+) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67+ expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype.ConclusionThis study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation.

Heterogenous expression of endocrine and progenitor cells within the neonatal porcine pancreatic lobes-Implications for neonatal porcine islet xenotransplantation.

Neonatal porcine islets (NPIs) are a source of islets for xenotransplantation. In the pig, the pancreatic lobes remain separate, thus, when optimizing NPI isolation, the pancreatic lobes included in the pancreatic digest should be specified. These lobes are the duodenal (DL), splenic (SL) and connecting (CL) lobe that correspond to the head, body-tail, and uncinate process of the human pancreas. In this study we are the first to evaluate all three neonatal porcine pancreatic lobes and NPIs isolated from these lobes. We report, a significant difference in endocrine and progenitor cell composition between lobes, and observed pancreatic duct glands (PDG) within the mesenchyme surrounding exocrine ducts in the DL and CL. Following in vitro differentiation, NPIs isolated from each lobe differed significantly in the percent increase of endocrine cells and final cell composition. Compared to other recipients, diabetic immunodeficient mice transplanted with NPIs isolated from the SL demonstrated euglycemic control as early as 4 weeks (p < 0.05) and achieved normoglycemia by 6 weeks post-transplant (p < 0.01). For the first time we report significant differences between the neonatal porcine pancreatic lobes and demonstrate that NPIs from these lobes differ in xenograft function.

The Outcomes and Quality of Pancreatic Islet Cells Isolated from Surgical Specimens for Research on Diabetes Mellitus.

Isolating a large quantity of high-quality human islets is a prerequisite for diabetes research. Human islets are typically isolated from the pancreases of brain-dead donors, making research difficult due to low availability. Pancreas tissue discarded after surgical resection may be a good alternative source of islet cells. To test this hypothesis, we isolated islets from discarded surgical specimens and evaluated the islet yield and quality as well as islet cell preparations. Eighty-two segmental pancreases were processed using the Ricordi automated method, and islet yield and quality were investigated. The mean age of patients was 54.6, and the cohort included 32 diabetes patients. After purification, partially resected pancreases yielded an average of 59,593 ± 56,651 islet equivalents (IEQs) and 2546 IEQ/g of digested pancreas, with 71.5 ± 21% purity. Multivariate analysis revealed that diabetes (p = 0.0046) and the lobe used (p = 0.0156) significantly altered islet yield. Islets transplanted into diabetic mice displayed good viability and in vitro glucose responses, DNA/RNA quality, mitochondrial function, and glucose control, even though these results were dependent on islet quality. Isolated cells also maintained high viability and function even after cryopreservation. Our findings indicate that pancreatic tissue discarded after surgery can be a valuable source of islets for diabetes research.

255-LB: Defining Functional Phenotype in Human Islets across Endocrine Cell Composition, Donor Age, Sex, and Ancestry

The Integrated Islet Distribution Program (IIDP) is currently the largest source of human islets for research in the U.S. The IIDP Human Islet Phenotyping Program (HIPP) provides standardized islet assessment and makes these data available to researchers. Since 2016, HIPP has phenotyped 337 islet samples from nondiabetic organ donors (40%F / 60%M, 45±12 years, BMI 29.0±5.4; 52% European, 26% Latino, 8% African, 3% Asian, and 11% other ancestry) . To define the islet structure-function relationships, we integrated physiological profiling of hormone secretion with islet cell composition measured by immunohistochemistry­. We found that % β cells varied significantly (range 34 - 92%; CV 21%) and had a strong negative correlation with % α (range 3 - 59%; CV 33%; r -0.95; p&amp;lt;1.78E-163) and % δ cells (range 1 - 19%; CV 44%; r -0.32, p=2.69E-09) . This variation did not impact basal insulin secretion at 5.6 mM glucose, but insulin secretory responses stimulated by high 16.7 mM glucose, cAMP-evoked potentiation, and KCl-mediated depolarization, were all negatively correlated with % α and δ cells. By contrast, glucagon response to basal 5.6 mM glucose had the strongest negative correlation with % β cells (r -0.31, p=4.25E-09) , but not % δ cells (r 0.03, p=0.63) . Furthermore, the β cell composition did not correlate with age or BMI, but was higher in males vs. females (59.1±12.2% vs. 55.7±11.0%, p=0.0107) and in islets from donors of Asian ancestry (71.2±14.7%, p&amp;lt;0.01) . Finally, we noted that insulin response to KCl-mediated depolarization was significantly lower in islets from donors of African vs. European or Asian ancestry (8.3±4.0 vs. 16.5±8.8 and 13.0±7.0ng/IEQ; p&amp;lt;0.01) . Thus, these data highlight the importance of endocrine cell composition in islet hormone secretion possibly influencing β-α cell paracrine interactions and setpoint of glucagon secretion in response to low glucose. In addition, they suggest that β cell composition and insulin secretion are influenced by sex and ancestry. Disclosure M. Brissova: None. G. Poffenberger: None. D. C. Saunders: None. A. C. Powers: None. J. C. Niland: None. C. Evans-molina: Advisory Panel; Avotres Inc., DiogenX, Isla Technologies, MaiCell Therapeutics, Provention Bio, Inc., Other Relationship; Astellas Pharma Inc., Dompé, Lilly, Research Support; BMS, Nimbus Therapeutics. H. H. Durai: None. S. Mei: None. A. Coldren: None. C. Davis: None. C. Reihsmann: None. A. L. Hopkirk: None. D. Gibson: None. R. Aramandla: None.

Current Topics of Relevance to the Xenotransplantation of Free Pig Islets.

Pig islet xenotransplantation is a potential treatment for patients with type 1 diabetes. Current efforts are focused on identifying the optimal pig islet source and overcoming the immunological barrier. The optimal age of the pig donors remains controversial since both adult and neonatal pig islets have advantages. Isolation of adult islets using GMP grade collagenase has significantly improved the quantity and quality of adult islets, but neonatal islets can be isolated at a much lower cost. Certain culture media and coculture with mesenchymal stromal cells facilitate neonatal islet maturation and function. Genetic modification in pigs affords a promising strategy to prevent rejection. Deletion of expression of the three known carbohydrate xenoantigens (Gal, Neu5Gc, Sda) will certainly be beneficial in pig organ transplantation in humans, but this is not yet proven in islet transplantation, though the challenge of the ‘4th xenoantigen’ may prove problematic in nonhuman primate models. Blockade of the CD40/CD154 costimulation pathway leads to long-term islet graft survival (of up to 965 days). Anti-CD40mAbs have already been applied in phase II clinical trials of islet allotransplantation. Fc region-modified anti-CD154mAbs successfully prevent the thrombotic complications reported previously. In this review, we discuss (I) the optimal age of the islet-source pig, (ii) progress in genetic modification of pigs, (iii) the immunosuppressive regimen for pig islet xenotransplantation, and (iv) the reduction in the instant blood-mediated inflammatory reaction.

Adult Pig Islet Isolation.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic β cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.

Angiopoietins stimulate\xa0pancreatic islet development from stem cells

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and β cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs.

Human postnatal Mesenchymal Stem Cell Derived Islets as a Model for Diabetes Research.

The scarcity of human cadaver islets for transplantation in patients with Diabetes Mellitus (DM) has necessitated the search for alternative islet sources. With advancing islet biology research, Islet-Like Clusters (ILCs) derived from stem cells have demonstrated potential for treating DM and in novel drug discovery programs for drug and cytotoxicity testing. In vitro differentiation of ILCs from stem cells also provides an opportunity to mimic the in vivo islet developmental pathways. In vitro derived ILCs are often considered immature as they do not respond to glucose challenges efficiently. However, the in vitro and in vivo performance of ILCs can be improved by pharmacological preconditioning. In this review, we discuss how ILCs generated from human postnatal tissues can be utilized as an in-vitro model to study cytotoxicity, drug screening and enhancement of transplantation efficacy. The use of human cadaver islets is not permitted for research purposes in India. Under these restrictions, the application of ILCs in drug screening and their role in complementing, reducing, and replacing animal testing will evolve as a reliable platform for in vitro screening as well as for stem cell-based treatment in DM.

Improvement of the therapeutic capacity of insulin-producing cells trans-differentiated from human liver cells using engineered cell sheet

BackgroundAlthough pancreatic islet transplantation therapy is ideal for diabetes patients, several hurdles have prevented it from becoming a standard treatment, including donor shortage and low engraftment efficacy. In this study, we prepared insulin-producing cells trans-differentiated from adult human liver cells as a new islet source. Also, cell sheet formation could improve differentiation efficiency and graft survival.MethodsLiver cells were expanded in vitro and trans-differentiated to IPCs using adenovirus vectors carrying human genes for PDX1, NEUROD1, and MAFA. IPCs were seeded on temperature-responsive culture dishes to form cell sheets. Differentiation efficiency was confirmed by ß cell-specific gene expression, insulin production, and immunohistochemistry. IPC suspension was injected by portal vein (PV), and IPC sheet was transplanted on the liver surface of the diabetic nude mouse. The therapeutic effect of IPC sheet was evaluated by comparing blood glucose control, weight gain, histological evaluation, and hepatotoxicity with IPC injection group. Also, cell biodistribution was assessed by in vivo/ex vivo fluorescence image tagging.ResultsInsulin gene expression and protein production were significantly increased on IPC sheets compared with those in IPCs cultured on conventional culture dishes. Transplanted IPC sheets displayed significantly higher engraftment efficiency and fewer transplanted cells in other organs than injected IPCs, and also lower liver toxicity, improved blood glucose levels, and weight gain. Immunohistochemical analyses of liver tissue revealed positive staining for PDX1 and insulin at 1, 2, and 4 weeks after IPC transplantation.ConclusionsIn conclusion, cell sheet formation enhanced the differentiation function and maturation of IPCs in vitro. Additionally, parameters for clinical application such as distribution, therapeutic efficacy, and toxicity were favorable. The cell sheet technique may be used with IPCs derived from various cell sources in clinical applications.

Pancreatic Islet Composition Affects Hormone Secretion in Isolated Alpha and Beta cells

Background/Objective: &#x0D; The Integrated Islet Distribution Program (IIDP) distributes islets from five isolation Centers and serves as the main source of human islets for research in the U.S. In 2016, the IIDP initiated the Human Islet Phenotyping Program (HIPP), which provides standardized post-shipment assessment of islet hormone secretion and endocrine cell composition for each IIDP-supported islet isolation. To date, islets from 276 non-diabetic donors have been analyzed. We hypothesized that analysis of this unique resource will provide novel insights into how demographic and clinical features impact islet health. &#x0D; &#x0D; Methods: &#x0D; Relationships between insulin and glucagon secretion assessed by perifusion and islet composition (% of β- and α-cells) were analyzed using SAS Version 9.4. For each analysis, the isolation center was used as a covariate. &#x0D; &#x0D; Results: &#x0D; Of the 276 donors, 60% were male; 59% of donors were Caucasian, 28% were Hispanic, 9% were African-American; 4% were Asian; and 0.36% were American Indian. The % of β-cells was moderately correlated with insulin responses to 16.7 mM glucose (r=0.2785; p&lt;0.0001) and 20 mM KCL (r=0.3109, p&lt;0.0001). Similarly, the α-cell% was moderately correlated with total glucagon content (r=0.3362, p&lt;0.0001) and glucagon responses to 1.7 mM glucose + 1mM epinephrine (r=0.2015, p=0.0001). The % of β -cells was negatively correlated with glucagon total content (r=-.243,p=.0001), while the α-cell% was negatively correlated with insulin stimulation index to KCL (r=-0.2573, p&lt;0.0001). Notably, Asian donors exhibited a significantly higher β-cell% compared to other groups (p&lt;0.05). Consistent with this, glucose-stimulated insulin secretion was higher in Asian donors compared to responses observed in islets from African American donors (p&lt;0.01). &#x0D; &#x0D; Conclusion: &#x0D; These data indicate that islet cell composition influences insulin and glucagon secretory responses and suggests that race may impact islet composition and hormone secretion. Continued analysis of the HIPP dataset may aid in our understanding of risk factors for the development islet dysfunction in diabetes.

Spheroid Fabrication Using Concave Microwells Enhances the Differentiation Efficacy and Function of Insulin-Producing Cells via Cytoskeletal Changes

Pancreatic islet transplantation is the fundamental treatment for insulin-dependent diabetes; however, donor shortage is a major hurdle in its use as a standard treatment. Accordingly, differentiated insulin-producing cells (DIPCs) are being developed as a new islet source. Differentiation efficiency could be enhanced if the spheroid structure of the natural islets could be recapitulated. Here, we fabricated DIPC spheroids using concave microwells, which enabled large-scale production of spheroids of the desired size. We prepared DIPCs from human liver cells by trans-differentiation using transcription factor gene transduction. Islet-related gene expression and insulin secretion levels were higher in spheroids compared to those in single-cell DIPCs, whereas actin–myosin interactions significantly decreased. We verified actin–myosin-dependent insulin expression in single-cell DIPCs by using actin–myosin interaction inhibitors. Upon transplanting cells into the kidney capsule of diabetic mouse, blood glucose levels decreased to 200 mg/dL in spheroid-transplanted mice but not in single cell-transplanted mice. Spheroid-transplanted mice showed high engraftment efficiency in in vivo fluorescence imaging. These results demonstrated that spheroids fabricated using concave microwells enhanced the engraftment and functions of DIPCs via actin–myosin-mediated cytoskeletal changes. Our strategy potentially extends the clinical application of DIPCs for improved differentiation, glycemic control, and transplantation efficiency of islets.

Current Assessment of Clinical Pancreatic Islet Allotransplantation

Clinical islet allotransplantation represents a minimally invasive, efficacious alternative to pancreas transplantation for restoring glycemic control and insulin independence in select patients with type 1 diabetes that is complicated by intractable impaired hypoglycemia awareness and/or severe hypoglycemic events refractory to stabilization by other means. Over the last decade, islet transplantation outcomes have steadily improved in part due to refinements in the selection of optimal donors, islet isolation techniques, safer engraftment methods, and effective immunomodulatory and anti-inflammatory therapies. Insulin independence rates at five years post-transplantation at select centers have reached parity with pancreas alone transplantation, and marked progress has been achieved in islet transplantation outcomes using single-donor pancreas. However, widespread application of the procedure is still hindered due to a limited supply of donor pancreases, inadequate engraftment, and the harmful side effects of chronic immunosuppression. Strategies to address some of these challenges involve the use of alternative sources of beta cells or islets, extrahepatic sites of implantation, encapsulation of islets and novel therapies to induce tolerance. While several countries have now transitioned islet transplantation from experimental status to a funded clinical cure for patients with brittle type 1 diabetes that cannot be stabilized by more conventional means, in the US it still awaits regulatory approval and a financial mechanism for sustainable reimbursement. This review details the history and the current status of clinical islet allotransplantation while summarizing improvements that have been made in techniques involving isolation, purification, culture and assessment of human islets as well as the islet transplantation process itself. Furthermore, it discusses the limitations encountered that prevent its widespread application, strategies that address those limitations, and last but not least, clinical trials being conducted that will help position islet transplantation as a mainstay treatment for the cure of type 1 diabetes.

Transplant strategies for type 1 diabetes: whole pancreas, islet and porcine beta cell therapies.

Whole-organ pancreas and islet transplantations are performed in a highly selected group of patients with diabetes mellitus, primarily those with type 1 diabetes mellitus, complicated by recurrent severe hypoglycaemia or renal failure requiring kidney transplantation. Clinical accessibility to pancreases or islets, and patient characteristics and therapeutic goals, may dictate choice of procedure. Pancreas transplantation is most often performed simultaneous with a kidney transplant, but patients with particularly labile type 1 diabetes may be considered for a pancreas transplant alone. While highly successful at restoring insulin independence, pancreas transplants carry the significant risks of major surgery and immunosuppression. Islet transplantation is a relatively minor procedure, usually performed for labile type 1 diabetes with severe hypoglycaemia. It is highly successful at resolving hypoglycaemia, but more than one pancreas donor may be required for insulin independence. Both pancreas and islet transplantation are limited in applicability by a paucity of deceased donors. Pigs provide one promising replenishable source of islets. Porcine islets can successfully reverse diabetes mellitus in non-human primates under the appropriate immunosuppressive conditions, with promise for eventually translating this success to a larger population of patients with diabetes mellitus in the future. Graphical abstract.

Signaling Molecules Regulating Pancreatic Endocrine Development from Pluripotent Stem Cell Differentiation.

Diabetes is one of the leading causes of death globally. Currently, the donor pancreas is the only source of human islets, placing extreme constraints on supply. Hence, it is imperative to develop renewable islets for diabetes research and treatment. To date, extensive efforts have been made to derive insulin-secreting cells from human pluripotent stem cells with substantial success. However, the in vitro generation of functional islet organoids remains a challenge due in part to our poor understanding of the signaling molecules indispensable for controlling differentiation pathways towards the self-assembly of functional islets from stem cells. Since this process relies on a variety of signaling molecules to guide the differentiation pathways, as well as the culture microenvironments that mimic in vivo physiological conditions, this review highlights extracellular matrix proteins, growth factors, signaling molecules, and microenvironments facilitating the generation of biologically functional pancreatic endocrine cells from human pluripotent stem cells. Signaling pathways involved in stepwise differentiation that guide the progression of stem cells into the endocrine lineage are also discussed. The development of protocols enabling the generation of islet organoids with hormone release capacities equivalent to native adult islets for clinical applications, disease modeling, and diabetes research are anticipated.

2132-P: Single-Cell RNA-Sequencing of Mouse Islets after Inflammatory Cytokine Exposure

Type 1 diabetes is an autoimmune disease caused by destruction of pancreatic β-cells. During disease progression, β-cells are exposed to proinflammatory cytokines like interleukin-1β (IL-1β) and interferon-γ (IFN-γ), stimulating them to express inducible nitric oxide synthase (iNOS) and produce micromolar levels of nitric oxide. While some cytokine-mediated changes in islet gene expression are known, we do not have a global view of the genes induced by cytokines nor do we know how non-β-cells respond to cytokines. Following 6-hr treatment with IL-1β and IFN-γ, adult C57BL/6 mouse islets were subjected to single-cell RNA-sequencing using the 10x Genomics Chromium platform. iNOS mRNA (Nos2) was induced in 73% of β-cells in response to both cytokines. Unexpectedly, 72% of Δ-cells and 44% of α-cells expressed Nos2 at levels similar to that found in β-cells. This is surprising since β-cells are thought to be the primary islet source of nitric oxide. Of the 241 genes enriched in Nos2-expressing β-cells, 70% were also enriched in Nos2-expressing Δ- and α-cells, suggesting that these cells initially respond to cytokine exposure by activating similar pathways. Among the enriched genes were those indicative of anti-viral defenses, suggesting induction of protective responses. Nos2 expression was negatively correlated with expression of genes encoding heat shock proteins, such as Hsp70, consistent with previous reports that heat shock inhibits cytokine-induced iNOS expression. Finally, we observed IL-1β-induced repression of β-cell identity genes, including Mafa, Pdx1, Slc2a2, and Ucn3. Repression of these identity genes was negatively correlated with Hsp70 expression, suggesting that cellular stress prevents this dedifferentiation. While these studies are ongoing, they suggest that (1) Δ- and α-cells respond to cytokines by upregulating Nos2 mRNA, (2) cytokines repress β-cell identity genes, and (3) both of these outcomes can be inhibited by induction of cellular stress. Disclosure J. Stancill: None. A. Khatun: None. M. Kasmani: None. W. Cui: None. J.A. Corbett: None.

An islet maturation media to improve the development of young porcine islets during in vitro culture

ABSTRACT Background The use of pancreata from pre-weaned piglets has the potential to serve as an unlimited alternative source of islets for clinical xenotransplantation. As pre-weaned porcine islets (PPIs) are immature and require prolonged culture, we developed an islet maturation media (IMM) and evaluated its effect on improving the quantity and quality of PPIs over 14 days of culture. Methods PPIs were isolated from the pancreata of pre-weaned Yorkshire piglets (8–15 days old). Each independent islet isolation was divided for culture in either control Ham’s F-10 media (n = 5) or IMM (n = 5) for 14 days. On day 3, 7 and 14 of culture, islets were assessed for islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of beta cells, and insulin secretion during glucose stimulation. Results In comparison to control islets, culturing PPIs in IMM significantly increased islet yield. PPIs cultured in IMM also maintained a stable isolation index and viability throughout 14 days of culture. The insulin content, endocrine cellular composition, and differentiation of beta cells were significantly improved in PPIs cultured in IMM, which subsequently augmented their insulin secretory capacity in response to glucose challenge compared to control islets. Conclusions Culturing PPIs in IMM increases islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of endocrine progenitor cells toward beta cells, and insulin secretion. Due to the improved islet quantity and quality after in vitro culture, the use of IMM in the culture of PPIs will assist to advance the outcomes of clinical islet xenotransplantation.

Glucosamine potentiates the differentiation of adipose-derived stem cells into glucose-responsive insulin-producing cells

BackgroundIslet transplantation might be a logical strategy to restore insulin secretion for the treatment of diabetes, however, the scarcity of donors poses an obstacle for such a treatment. As an alternative islet source, differentiation of stem cells into insulin-producing cells (IPCs) has been tried. Many protocols have been developed to improve the efficiency of differentiation of stem cells into IPCs. In this study, we investigated whether glucosamine supplementation during differentiation of human adipose-derived stem cells (hADSCs) into IPCs can improve the insulin secretory function.MethodsGlucosamine was added to the original differentiation medium at different stages of differentiation of hADSCs into IPCs for 12 days and insulin secretion was analyzed.ResultsAddition of glucosamine alone to the growth medium of hADSCs did not affect the differentiation of hADSCs to IPCs. Supplementation of the differentiation medium with glucosamine at a later stage (protocol G3) proved to have the greatest effect on IPC differentiation. Basal and glucose-stimulated insulin secretion (GSIS) was significantly increased and the expression of insulin and C-peptide was increased in differentiated IPCs as compared with that in differentiated IPCs using the conventional protocol (protocol C). In addition, the expression of beta-cell specific transcription factors such as pancreatic and duodenal homeobox1 (PDX1) and neurogenin 3 (NGN3) was also increased. Furthermore, the expression of genes related to insulin secretion, including synaptotagmin 4 (Syt4), glucokinase (Gck) and glucose transporter 2 (Glut2), was also increased.ConclusionsWe conclude that glucosamine supplementation potentiates the differentiation of hADSCs into IPCs.

Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function

ABSTRACTThe pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success. Blood samples were collected from 21 pancreas donors and 14 healthy volunteers. Pancreatic tissue samples were also collected from the corresponding donor during organ procurement. Six serum cytokines were measured by a fluorescent bead-based immunoassay, and the expression of fifteen inflammatory target genes was quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). There was no correlation between serum inflammatory cytokines and mRNA expression of the corresponding genes in peripheral blood mononuclear cells (PBMCs) or pancreatic tissue. The IL6 expression in pancreatic tissue correlated negatively with post-isolation islet yield. Islets isolated from donors highly expressing IFNG in PBMCs and MAC1 in pancreatic tissue functioned poorly in vivo when transplanted in diabetic NODscid mice. Furthermore, the increased MAC1 in pancreatic tissue was positively correlated with donor hospitalization time. Brain death duration positively correlated with higher expression of IL1B in PBMCs and TNF in both PBMCs and pancreatic tissue but failed to show a significant correlation with islet yield and in vivo function. The study indicates that the increased inflammatory genes in donor pancreatic tissues may be considered as biomarkers associated with poor islet isolation outcome.

Engineering Strategies to Improve Islet Transplantation for Type 1 Diabetes Therapy.

Type 1 diabetes is an autoimmune disease in which the immune system attacks insulin-producing beta cells of pancreatic islets. Type 1 diabetes can be treated with islet transplantation; however, patients must be administered immunosuppressants to prevent immune rejection of the transplanted islets if they are not autologous or not engineered with immune protection/isolation. To overcome biological barriers of islet transplantation, encapsulation strategies have been developed and robustly investigated. While islet encapsulation can prevent the need for immunosuppressants, these approaches have not shown much success in clinical trials due to a lack of long-term insulin production. Multiple engineering strategies have been used to improve encapsulation and post-transplantation islet survival. In addition, more efficient islet cryopreservation methods have been designed to facilitate the scaling-up of islet transplantation. Other islet sources have been identified including porcine islets and stem cell-derived islet-like aggregates. Overall, islet-laden capsule transplantation has greatly improved over the past 30 years and is moving towards becoming a clinically feasible treatment for type 1 diabetes.