HumanIslets: An integrated platform for human islet data access and analysis.

Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.

Web-based multi-omics integration using the Analyst software suite.

The growing number of multi-omics studies demands clear conceptual workflows coupled with easy-to-use software tools to facilitate data analysis and interpretation. This protocol covers three key components involved in multi-omics analysis, including single-omics data analysis, knowledge-driven integration using biological networks and data-driven integration through joint dimensionality reduction. Using the dataset from a recent multi-omics study of human pancreatic islet tissue and plasma samples, the first section introduces how to perform transcriptomics/proteomics data analysis using ExpressAnalyst and lipidomics data analysis using MetaboAnalyst.

Metabolic switching, growth kinetics and cell yields in the scalable manufacture of stem cell-derived insulin-producing cells.

Background: Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy.

Human A2-CAR T Cells Reject HLA-A2 + Human Islets Transplanted Into Mice Without Inducing Graft-versus-host Disease.

Background: Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD).

Integrating genetics with single-cell multiomic measurements across disease states identifies mechanisms of beta cell dysfunction in type 2 diabetes.

Dysfunctional pancreatic islet beta cells are a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of the underlying mechanisms, including gene dysregulation, is lacking. Here we integrate information from measurements of chromatin accessibility, gene expression and function in single beta cells with genetic association data to nominate disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 nondiabetic, pre-T2D and T2D donors, we identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift during T2D progression.

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease.

Background: Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- -host disease (xGVHD).

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

Unlabelled: Few studies have examined the differentiation of human embryonic stem cell (hESC)-derived pancreatic endoderm cells (PECs) in different implantation sites. Here, we investigate the influence of implantation site and recipient sex on the differentiation of hESC-derived PECs in vivo. Male and female mice were implanted with 5 × 106 hESC-derived PECs under the kidney capsule, in the gonadal fat pad, or subcutaneously within macroencapsulation (TheraCyte) devices.

Integration of single-cell multiomic measurements across disease states with genetics identifies mechanisms of beta cell dysfunction in type 2 diabetes.

Altered function and gene regulation of pancreatic islet beta cells is a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of mechanisms driving T2D is still missing. Here we integrate information from measurements of chromatin activity, gene expression and function in single beta cells with genetic association data to identify disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 non-diabetic, pre-T2D and T2D donors, we robustly identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift in T2D.

Dynamic Ins2 Gene Activity Defines β-Cell Maturity States.

Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas. However, it is not clear whether β-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in β-cells from Ins2GFP knockin mice.

From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science.

Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function.

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance.

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1 knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1-containing littermate controls at multiple ages and on multiple diets.

Deletion of pancreas-specific miR-216a reduces beta-cell mass and inhibits pancreatic cancer progression in mice.

miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, β-cell mass, and insulin levels.

Role of myeloid cell leptin signaling in the regulation of glucose metabolism.

Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2CreLepr) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2CreLepr).

AAV GCG-EGFP, a new tool to identify glucagon-secreting α-cells.

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population.

Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues.

The relative contribution of peripheral and central leptin signalling to the regulation of metabolism and the mechanisms through which leptin affects glucose homeostasis have not been fully elucidated. We generated complementary lines of mice with either leptin receptor (Lepr) knockdown or reconstitution in adipose tissues using Cre-lox methodology. Lepr knockdown mice were modestly lighter and had lower plasma insulin concentrations following an oral glucose challenge compared to controls, despite similar insulin sensitivity.

Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice.

Aims/hypothesis: Islet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.

Methods: Human islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system.

Regenerative medicine and cell-based approaches to restore pancreatic function.

The pancreas is a complex organ with exocrine and endocrine components. Many pathologies impair exocrine function, including chronic pancreatitis, cystic fibrosis and pancreatic ductal adenocarcinoma. Conversely, when the endocrine pancreas fails to secrete sufficient insulin, patients develop diabetes mellitus.

Cotransplantation of Mesenchymal Stem Cells With Neonatal Porcine Islets Improve Graft Function in Diabetic Mice.

Mesenchymal stem cells (MSCs) possess immunoregulatory, anti-inflammatory, and proangiogenic properties and, therefore, have the potential to improve islet engraftment and survival. We assessed the effect human bone marrow-derived MSCs have on neonatal porcine islets (NPIs) in vitro and determined islet engraftment and metabolic outcomes when cotransplanted in a mouse model. NPIs cocultured with MSCs had greater cellular insulin content and increased glucose-stimulated insulin secretion.

Justifying clinical trials for porcine islet xenotransplantation.

The development of the Edmonton Protocol encouraged a great deal of optimism that a cell-based cure for type I diabetes could be achieved. However, donor organ shortages prevent islet transplantation from being a widespread solution as the supply cannot possibly equal the demand. Porcine islet xenotransplantation has the potential to address these shortages, and recent preclinical and clinical trials show promising scientific support.

Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application.

One challenge that must be overcome to allow transplantation of neonatal porcine islets (NPIs) to become a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our standard protocol, we routinely isolate NPIs from a maximum of four pancreases, requiring tissue culture in 16 Petri dishes (four per pancreas) in Ham's F10 and bovine serum albumin (BSA). We have now developed a scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes.

Development and Characterization of a Collagen-Based Matrix for Vascularization and Cell Delivery.

Since the development of the Edmonton protocol, islet transplantation is increasingly encouraging as a treatment for type 1 diabetes. Strategies to ameliorate problems with the intraportal site include macroencapsulating the islets in diverse biomaterials. Characterization of these biomaterials is important to optimally tune the properties to support islets and promote vascularization.

Identification and differentiation of PDX1 β-cell progenitors within the human pancreatic epithelium.

Aim: To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm that β-cell progenitors reside within the pancreatic epithelium.

Methods: Due to mesenchymal stem cell (MSC) expansion and overgrowth, progenitor cells within the pancreatic epithelium cannot be characterized in vitro, though β-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition (EMT) may generate β-cell progenitors. Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth (dexamethasone, epidermal growth factor, 3,5,3'-triiodo-l-thyronine, bovine brain extract).

Bioengineering a highly vascularized matrix for the ectopic transplantation of islets.

Islet transplantation is a promising treatment for Type 1 diabetes; however limitations of the intra-portal site and poor revascularization of islets must be overcome. We hypothesize that engineering a highly vascularized collagen-based construct will allow islet graft survival and function in alternative sites. In this study, we developed such a collagen-based biomaterial.

Collagen-chitosan-laminin hydrogels for the delivery of insulin-producing tissue.

Islet transplantation is an emerging strategy for treating patients with type 1 diabetes mellitus. Although the proof of concept for cellular replacement therapy in diabetes has been firmly established, vascularity of the transplant site and the long-term survival and function of transplanted islets remains suboptimal. In the present study, human circulating angiogenic cells (CACs) and porcine islet cells embedded in collagen-chitosan hydrogels, with and without laminin, were investigated as potential engineered biomaterials for the treatment of type 1 diabetes.