Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

Human Beta Cell Regenerative Drug Therapy for Diabetes: Past Achievements and Future Challenges

A quantitative deficiency of normally functioning insulin-producing pancreatic beta cells is a major contributor to all common forms of diabetes. This is the underlying premise for attempts to replace beta cells in people with diabetes by pancreas transplantation, pancreatic islet transplantation, and transplantation of beta cells or pancreatic islets derived from human stem cells. While progress is rapid and impressive in the beta cell replacement field, these approaches are expensive, and for transplant approaches, limited by donor organ availability. For these reasons, beta cell replacement will not likely become available to the hundreds of millions of people around the world with diabetes. Since the large majority of people with diabetes have some residual beta cells in their pancreata, an alternate approach to reversing diabetes would be developing pharmacologic approaches to induce these residual beta cells to regenerate and expand in a way that also permits normal function. Unfortunately, despite the broad availability of multiple classes of diabetes drugs in the current diabetes armamentarium, none has the ability to induce regeneration or expansion of human beta cells. Development of such drugs would be transformative for diabetes care around the world. This picture has begun to change. Over the past half-decade, a novel class of beta cell regenerative small molecules has emerged: the DYRK1A inhibitors. Their emergence has tremendous potential, but many areas of uncertainty and challenge remain. In this review, we summarize the accomplishments in the world of beta cell regenerative drug development and summarize areas in which most experts would agree. We also outline and summarize areas of disagreement or lack of unanimity, of controversy in the field, of obstacles to beta cell regeneration, and of challenges that will need to be overcome in order to establish human beta cell regenerative drug therapeutics as a clinically viable class of diabetes drugs.

Loss of MANF Causes Childhood Onset Syndromic Diabetes due to Increased Endoplasmic Reticulum Stress

MANF is an endoplasmic reticulum resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse beta cells, its precise role in human beta cell development and function is unknown. Herein, we show that lack of MANF in humans results in diabetes due to increased ER stress leading to impaired beta cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the <i>MANF</i> gene. To study the role of MANF in human beta cell development and function, we knocked out the <i>MANF </i>gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of <i>MANF</i> induced mild ER stress and impaired insulin processing capacity of beta cells <i>in vitro</i>. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in diabetic recipients. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human beta cell function and demonstrate the crucial role of MANF in this process.

Loss of MANF Causes Childhood Onset Syndromic Diabetes due to Increased Endoplasmic Reticulum Stress

MANF is an endoplasmic reticulum resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse beta cells, its precise role in human beta cell development and function is unknown. Herein, we show that lack of MANF in humans results in diabetes due to increased ER stress leading to impaired beta cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the <i>MANF</i> gene. To study the role of MANF in human beta cell development and function, we knocked out the <i>MANF </i>gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of <i>MANF</i> induced mild ER stress and impaired insulin processing capacity of beta cells <i>in vitro</i>. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in diabetic recipients. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human beta cell function and demonstrate the crucial role of MANF in this process.

Human Islets Contain a Beta Cell Type That Expresses Proinsulin But Not the Enzyme That Converts the Precursor to Insulin

In pancreatic beta cells, proinsulin (ProIN) undergoes folding in endoplasmic reticulum/Golgi system and is translocated to secretory vesicles for processing into insulin and C-peptide by the proprotein convertases (PC)1/3 and PC2, and carboxypeptidase E. Human beta cells show significant variation in the level of expression of PC1/3, the critical proconvertase involved in proinsulin processing. To ascertain whether this heterogeneity is correlated with the level of expression of the prohormone and mature hormone, the expression of proinsulin, insulin, and PC1/3 in human beta cells was examined. This analysis identified a human beta cell type that expressed proinsulin but lacked PC1/3 (ProIN+PC1/3−). This beta cell type is absent in rodent islets and is abundant in human islets of adults but scarce in islets from postnatal donors. Human islets also contained a beta cell type that expressed both proinsulin and variable levels of PC1/3 (ProIN+PC1/3+) and a less abundant cell type that lacked proinsulin but expressed the convertase (ProIN−PC1/3+). These cell phenotypes were altered by type 2 diabetes. These data suggest that these three cell types represent different stages of a dynamic process with proinsulin folding in ProIN+PC1/3− cells, proinsulin conversion into insulin in ProIN+PC1/3+cells, and replenishment of the proinsulin content in ProIN−PC1/3+ cells:

The Harmine and Exendin-4 Combination Markedly Expands Human Beta Cell Mass In Vivo: Quantification and Visualization By iDISCO+ 3D Imaging

Since all diabetes results from reductions in numbers of functional pancreatic beta cells, beta cell regenerative drugs are required for optimal and scalable future diabetes treatment. While many diabetes drugs are in clinical use, none increases human beta cell numbers. We have shown that a combination of the DYRK1A inhibitor, harmine, with the GLP1 receptor agonist, exendin-4, markedly increases human beta cell proliferation in vitro. However, technological limitations have prevented assessment of human beta cell mass in vivo. Here, we describe a novel method that combines iDISCO+ tissue clearing, insulin immunolabeling, light sheet microscopy, and volumetric quantification of human beta cells transplanted into immunodeficient mice. We demonstrate a striking seven-fold in vivo increase in human beta cell mass in response to three months of combined harmine-exendin-4 combination infusion, accompanied by lower blood glucose levels, increased plasma human insulin concentrations and enhanced beta cell proliferation. These studies unequivocally demonstrate for the first time that pharmacologic human beta cell expansion is a realistic and achievable path to diabetes therapy, and provide a rigorous, entirely novel and reproducible tool for quantifying human beta cell mass in vivo.