Identification of unique cell type responses in pancreatic islets to stress.

Diabetes involves the death or dysfunction of pancreatic ß-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response, we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types, we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ß-, α-, and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1, which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics.

Roadblocks confronting widespread dissemination and deployment of Organs on Chips.

Organ on Chip platforms hold significant promise as alternatives to animal models or traditional cell cultures, both of which poorly recapitulate human pathophysiology and human level responses. Within the last 15 years, we have witnessed seminal scientific developments from academic laboratories, a flurry of startups and investments, and a genuine interest from pharmaceutical industry as well as regulatory authorities to translate these platforms. This Perspective identifies several fundamental design and process features that may act as roadblocks that prevent widespread dissemination and deployment of these systems, and provides a roadmap to help position this technology in mainstream drug discovery.

Comparative and integrative single cell analysis reveals new insights into the transcriptional immaturity of stem cell-derived ß cells.

Diabetes cell replacement therapy has the potential to be transformed by human pluripotent stem cell-derived ß cells (SC-ß cells). However, the precise identity of SC-ß cells in relationship to primary fetal and adult ß-cells remains unclear. Here, we used single-cell sequencing datasets to characterize the transcriptional identity of islets from in vitro differentiation, fetal islets, and adult islets. Our analysis revealed that SC-ß cells share a core ß-cell transcriptional identity with human adult and fetal ß-cells, however SC-ß cells possess a unique transcriptional profile characterized by the persistent expression and activation of progenitor and neural-biased gene networks. These networks are present in SC-ß cells, irrespective of the derivation protocol used. Notably, fetal ß-cells also exhibit this neural signature at the transcriptional level. Our findings offer insights into the transcriptional identity of SC-ß cells and underscore the need for further investigation of the role of neural transcriptional networks in their development.

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.

Insulin-producing ß cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between ß cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes.

The generation of islet-like endocrine clusters from human pluripotent stem cells (hPSCs) has the potential to provide an unlimited source of insulin-producing ß cells for the treatment of diabetes. In order for this cell therapy to become widely adopted, highly functional and well-characterized stem cell-derived islets (SC-islets) need to be manufactured at scale. Furthermore, successful SC-islet replacement strategies should prevent significant cell loss immediately following transplantation and avoid long-term immune rejection. This review highlights the most recent advances in the generation and characterization of highly functional SC-islets as well as strategies to ensure graft viability and safety after transplantation.

Modular Microphysiological System for Modeling of Biologic Barrier Function.

Microphysiological systems, also known as organs-on-chips, are microfluidic devices designed to model human physiology in vitro. Polydimethylsiloxane (PDMS) is the most widely used material for organs-on-chips due to established microfabrication methods, and properties that make it suitable for biological applications such as low cytotoxicity, optical transparency, gas permeability. However, absorption of small molecules and leaching of uncrosslinked oligomers might hinder the adoption of PDMS-based organs-on-chips for drug discovery assays. Here, we have engineered a modular, PDMS-free microphysiological system that is capable of recapitulating biologic barrier functions commonly demonstrated in PDMS-based devices. Our microphysiological system is comprised of a microfluidic chip to house cell cultures and pneumatic microfluidic pumps to drive flow with programmable pressure and shear stress. The modular architecture and programmable pumps enabled us to model multiple in vivo microenvironments. First, we demonstrate the ability to generate cyclic strain on the culture membrane and establish a model of the alveolar air-liquid interface. Next, we utilized three-dimensional finite element analysis modeling to characterize the fluid dynamics within the device and develop a model of the pressure-driven filtration that occurs at the glomerular filtration barrier. Finally, we demonstrate that our model can be used to recapitulate sphingolipid induced kidney injury. Together, our results demonstrate that a multifunctional and modular microphysiological system can be deployed without the use of PDMS. Further, the bio-inert plastic used in our microfluidic device is amenable to various established, high-throughput manufacturing techniques, such as injection molding. As a result, the development plastic organs-on-chips provides an avenue to meet the increasing demand for organ-on-chip technology.

Integrated human pseudoislet system and microfluidic platform demonstrate differences in GPCR signaling in islet cells.

Pancreatic islets secrete insulin from ß cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated ß and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human ß and α cells.

Microelectrode Array based Functional Testing of Pancreatic Islet Cells.

Electrophysiological techniques to characterize the functionality of islets of Langerhans have been limited to short-term, one-time recordings such as a patch clamp recording. We describe the use of microelectrode arrays (MEAs) to better understand the electrophysiology of dissociated islet cells in response to glucose in a real-time, non-invasive method over prolonged culture periods. Human islets were dissociated into singular cells and seeded onto MEA, which were cultured for up to 7 days. Immunofluorescent imaging revealed that several cellular subtypes of islets; ß, δ, and γ cells were present after dissociation. At days 1, 3, 5, and 7 of culture, MEA recordings captured higher electrical activities of islet cells under 16.7 mM glucose (high glucose) than 1.1 mM glucose (low glucose) conditions. The fraction of the plateau phase (FOPP), which is the fraction of time with spiking activity recorded using the MEA, consistently showed distinguishably greater percentages of spiking activity with high glucose compared to the low glucose for all culture days. In parallel, glucose stimulated insulin secretion was measured revealing a diminished insulin response after day 3 of culture. Additionally, MEA spiking profiles were similar to the time course of insulin response when glucose concentration is switched from 1.1 to 16.7 mM. Our analyses suggest that extracellular recordings of dissociated islet cells using MEA is an effective approach to rapidly assess islet functionality, and could supplement standard assays such as glucose stimulate insulin response.

Evaluating Vascularization of Heterotopic Islet Constructs for Type 1 Diabetes Using an In Vitro Platform.

Type 1 diabetes (T1D) results from the autoimmune destruction of ß-cells within the pancreatic islets of Langerhans. Clinical islet transplantation from healthy donors is proposed to ameliorate symptoms, improve quality of life, and enhance the life span of afflicted T1D patients. However, post-transplant outcomes are dependent on the survival of the transplanted islets, which relies on the engraftment of the islets with the recipient's vasculature among other factors. Treatment strategies to improve engraftment include combining islets with supporting cells including endothelial cells (EC) and mesenchymal stem cells (MSC), dynamic cells capable of robust immunomodulatory and vasculogenic effects. In this study, we developed an in vitro model of transplantation to investigate the cellular mechanisms that enhance rapid vascularization of heterotopic islet constructs. Self-assembled vascular beds of fluorescently stained EC served as reproducible in vitro transplantation sites. Heterotopic islet constructs composed of islets, EC, and MSC were transferred to vascular beds for modeling transplantation. Time-lapsed imaging was performed for analysis of the vascular bed remodeling for parameters of neo-vascularization. Moreover, sampling of media following modeled transplantation showed secretory profiles that were correlated with imaging analyses as well as with islet function using glucose-stimulated insulin secretion. Together, evidence revealed that heterotopic constructs consisting of islets, EC, and MSC exhibited the most rapid recruitment and robust branching of cells from the vascular beds suggesting enhanced neo-vascularization compared to islets alone and control constructs. Together, this evidence supports a promising cell transplantation strategy for T1D and also demonstrates a valuable tool for rapidly investigating candidate cellular therapies for transplantation.

Comparison of temperature change and resulting ablation size induced by a 902-928 MHz and a 2450 MHz microwave ablation system in in-vivo porcine kidneys.

INTRODUCTION: Microwave ablation (MWA) uses heat to ablate undesired tissue. Development of pre-planning algorithms for MWA of small renal masses requires understanding of microwave-tissue interactions at different operating parameters. The objective of this study was to compare the performance of two MWA systems in in-vivo porcine kidneys. METHODS: Five ablations were performed using a 902-928 MHz system (24 W, 5 min) and a 2450 MHz system (180 W, 2 min). Nonlinear regression analysis of temperature changes measured 5 mm from the antenna axis was completed for the initial 10 s of ablation using the power equation ΔT=atb and after the inflection point using an exponential equation. Thermal damage was calculated using the Arrhenius equation. Long and short axis ablation diameters were measured. RESULTS: The average 'a' varied significantly between systems (902-928 MHz: 0.0299 ± 0.027, 2450 MHz: 0.1598 ± 0.158), indicating proportionality to the heat source, but 'b' did not (902-928 MHz: 1.910 ± 0.372, 2450 MHz: 2.039 ± 0.366), signifying tissue type dependence. Past the inflection point, average steady-state temperature increases were similar between systems but reached more quickly with the 2450 MHz system. Complete damage was reached at 5 mm for both systems. The 2450 MHz system produced significantly larger short axis ablations (902-928 MHz: 2.40 ± 0.54 cm, 2450 MHz: 3.32 ± 0.41cm). CONCLUSION: The 2450 MHz system achieved similar steady state temperature increases compared to the 902-928 MHz system, but more quickly due to higher output power. Further investigations using various treatment parameters and precise thermal sensor placement are warranted to refine equation parameters for the development of an ablation model.

Engineered Microenvironments for Maturation of Stem Cell Derived Cardiac Myocytes.

Through the use of stem cell-derived cardiac myocytes, tissue-engineered human myocardial constructs are poised for modeling normal and diseased physiology of the heart, as well as discovery of novel drugs and therapeutic targets in a human relevant manner. This review highlights the recent bioengineering efforts to recapitulate microenvironmental cues to further the maturation state of newly differentiated cardiac myocytes. These techniques include long-term culture, co-culture, exposure to mechanical stimuli, 3D culture, cell-matrix interactions, and electrical stimulation. Each of these methods has produced various degrees of maturation; however, a standardized measure for cardiomyocyte maturation is not yet widely accepted by the scientific community.