Development of a Coaxial 3D Printing Platform for Biofabrication of Implantable Islet-Containing Constructs

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes

Desmoglein-2 is Important for Islet Function and β-Cell Survival

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg

Bioprinting an Artificial Pancreas for Type 1 Diabetes

Purpose of Review: Pancreatic islet cell transplantation is currently the only curative cell therapy for type 1 diabetes mellitus. However, its potential to treat many more patients is limited by several challenges. The emergence of 3D bioprinting technology from recent advances in 3D printing, biomaterials, and cell biology has provided the means to overcome these challenges. Recent Findings: 3D bioprinting allows for the precise fabrication of complex 3D architectures containing spatially distributed cells, biomaterials (bioink), and bioactive factors. Different strategies to capitalize on this ability have been investigated for the 3D bioprinting of pancreatic islets. In particular, with co-axial bioprinting technology, the co-printability of islets with supporting cells such as endothelial progenitor cells and regulatory T cells, which have been shown to accelerate revascularization of islets and improve the outcome of various transplantations, respectively, has been achieved. Summary: 3D bioprinting of islets for generation of an artificial pancreas is a newly emerging field of study with a vast potential to improve islet transplantation

Dog colour patterns explained by modular promoters of ancient canid origin

Distinctive colour patterns in dogs are an integral component of canine diversity. Colour pattern differences are thought to have arisen from mutation and artificial selection during and after domestication from wolves but important gaps remain in understanding how these patterns evolved and are genetically controlled. In other mammals, variation at the ASIP gene controls both the temporal and spatial distribution of yellow and black pigments. Here, we identify independent regulatory modules for ventral and hair cycle ASIP expression, and we characterize their action and evolutionary origin. Structural variants define multiple alleles for each regulatory module and are combined in different ways to explain five distinctive dog colour patterns. Phylogenetic analysis reveals that the haplotype combination for one of these patterns is shared with Arctic white wolves and that its hair cycle-specific module probably originated from an extinct canid that diverged from grey wolves more than 2 million years ago. Natural selection for a lighter coat during the Pleistocene provided the genetic framework for widespread colour variation in dogs and wolves. Dogs exhibit remarkable variation in colour patterns. Here, the authors identify structural variants of independent regulatory modules for ventral and hair cycle expression of the ASIP gene that explain five distinctive dog colour patterns and trace back the origin of one colour pattern to an extinct canid.Peer reviewe

Encapsulation of Human Natural and Induced Regulatory T-Cells in IL-2 and CCL1 Supplemented Alginate-GelMA Hydrogel for 3D Bioprinting

2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulatory T-cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time-consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate-gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg-specific bioactive factors interleukin-2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8+ T-cells enriched with anti-inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co-axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co-culture with human peripheral blood mononuclear cells. This establishes the co-encapsulation of Tregs by co-axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets

Encapsulation of Human Natural and Induced Regulatory T-Cells in IL-2 and CCL1 Supplemented Alginate-GelMA Hydrogel for 3D Bioprinting

2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulatory T-cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time-consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate-gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg-specific bioactive factors interleukin-2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8+ T-cells enriched with anti-inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co-axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co-culture with human peripheral blood mononuclear cells. This establishes the co-encapsulation of Tregs by co-axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response

Desmoglein-2 is important for islet function and β-cell survival

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes

Phenoxodiol, an experimental anticancer drug, shows potent antiangiogenic properties in addition to its antitumour effects

Phenoxodiol (2H-1-benzopyran-7-0,1, 3-[4-hydroxyphenyl], PXD) is a synthetic analogue of the naturally-occurring plant isoflavone and anticancer agent, genistein. PXD directly induces mitotic arrest and apoptosis in most cancer cells and is currently undergoing clinical trials, as a chemotherapeutic in ovarian and prostate cancers. We show here that PXD also exhibits potent antiangiogenic properties. Thus, it inhibited endothelial cell proliferation, migration and capillary tube formation and inhibited expression of the matrix metalloproteinase MMP-2, a major matrix degrading enzyme. Importantly, we demonstrate that PXD is functional in vivo since it inhibited the extent of capillary tube invasion in an in vivo model of angiogenesis. We show that phenoxodiol inhibits hallmarks of endothelial cell activation, namely TNF or IL-1 induced E-selectin and VCAM-1 expression and IL-8 secretion. However, PXD had no effect on unstimulated endothelial cells. We also describe that PXD inhibits the lipid kinase sphingosine kinase, which recently has been implicated in endothelial cell activation and angiogenesis as well as oncogenesis. Thus, our results suggest that PXD may be an effective anticancer drug targeting the two drivers of tumour growth--the proliferation of the tumour cells themselves and the angiogenic and inflammatory stimulation of the vasculature.Gamble, Jennifer R. ; Xia, Pu ; Hahn, Christopher N. ; Drew, Jenny J. ; Drogemuller, Christopher J. ; Brown, David ; Vadas, Mathew A