Harnessing Human Pluripotent Stem Cell-Derived Pancreatic In Vitro Models for High-Throughput Toxicity Testing and Diabetes Drug Discovery.

The long-standing goals in diabetes research are to improve ß-cell survival, functionality and increase ß-cell mass. Current strategies to manage diabetes progression are still not ideal for sustained maintenance of normoglycemia, thereby increasing demand for the development of novel drugs. Available pancreatic cell lines, cadaveric islets, and their culture methods and formats, either 2D or 3D, allow for multiple avenues of experimental design to address diverse aims in the research setting. More specifically, these pancreatic cells have been employed in toxicity testing, diabetes drug screens, and with careful curation, can be optimized for use in efficient high-throughput screenings (HTS). This has since spearheaded the understanding of disease progression and related mechanisms, as well as the discovery of potential drug candidates which could be the cornerstone for diabetes treatment. This book chapter will touch on the pros and cons of the most widely used pancreatic cells, including the more recent human pluripotent stem cell-derived pancreatic cells, and HTS strategies (cell models, design, readouts) that can be used for the purpose of toxicity testing and diabetes drug discovery.

New insights into human beta cell biology using human pluripotent stem cells.

Pancreatic ß-cells are responsible for maintaining glucose homeostasis. Therefore, their dysregulation leads to diabetes. Pancreas or islet transplants can be used to treat diabetes but these human tissues remain in short supply. Significant progress has now been made in differentiating human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into pancreatic ß-like cells for potential cell replacement therapy. Additionally, these hPSC-derived ß-like cells represent a new invaluable model for studying diabetes disease mechanisms. Here, we review the use of hPSC-derived ß-like cells as a platform to model various types of defects in human ß-cells in diabetes, comparing them against existing animal models, ex vivo human islets and human ß-cell line. We also discuss how hPSC-derived ß-like cells are being used as a platform for screening novel therapeutic compounds. Last but not least, we evaluate the strengths and limitations of this human cell-based platform as an avenue to study and reveal new insights into human ß-cell biology.

A new perspective of probe development for imaging pancreatic beta cell in vivo.

Beta cells assume a fundamental role in maintaining blood glucose homeostasis through the secretion of insulin, which is contingent on both beta cell mass and function, in response to elevated blood glucose levels or secretagogues. For this reason, evaluating beta cell mass and function, as well as scrutinizing how they change over time in a diabetic state, are essential prerequisites in elucidating diabetes pathophysiology. Current clinical methods to measure human beta cell mass and/or function are largely lacking, indirect and sub-optimal, highlighting the continued need for noninvasive in vivo beta cell imaging technologies such as optical imaging techniques. While numerous probes have been developed and evaluated for their specificity to beta cells, most of them are more suited to visualize beta cell mass rather than function. In this review, we highlight the distinction between beta cell mass and function, and the importance of developing more probes to measure beta cell function. Additionally, we also explore various existing probes that can be employed to measure beta cell mass and function in vivo, as well as the caveats in probe development for in vivo beta cell imaging.

Defective insulin receptor signaling in hPSCs skews pluripotency and negatively perturbs neural differentiation.

Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry-based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1+PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.

Fabrication and characterization of core-shell microparticles containing an aqueous core.

Core-shell microparticles containing an aqueous core have demonstrated their value for microencapsulation and drug delivery systems. The most important step in generating these uniquely structured microparticles is the formation of droplets and double emulsion. The droplet generator must meet the performance and reliability requirements, including accurate size control with tunability and monodispersity. Herein, we present a facile technique to generate surfactant-free core-shell droplets with an aqueous core in a microfluidic device. We demonstrate that the geometry of the core-shell droplets can be precisely adjusted by the flow rates of the droplet components. As the shell is polymerized after the formation of the core-shell droplets, the resulting solid microparticles ensure the encapsulation of the aqueous core and prevent undesired release. We then study experimentally and theoretically the behaviour of resultant microparticles under heating and compression. The microparticles demonstrate excellent stability under both thermal and mechanical loads. We show that the rupture force can be quantitatively predicted from the shell thickness relative to the outer shell radius. Experimental results and theoretical predictions confirm that the rupture force scales directly with the shell thickness.

Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction.

AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. METHODS: We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. RESULTS: The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. CONCLUSIONS/INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Replicates in stem cell models-How complicated!

Current complexities in human pluripotent stem cell (hPSC)-based studies. hPSC studies begin with the recruitment of patients harboring disease-associated gene variant(s) that may increase susceptibility to disease development. Somatic reprogramming is then performed to derive patient-specific human induced pluripotent stem cells (hiPSCs), followed by step-wise directed differentiation to a relevant cell type before qualitative/quantitative assays are performed to assess for phenotypic or gene expression differences between the healthy and diseased hiPSCs.

Dynamic proteome profiling of human pluripotent stem cell-derived pancreatic progenitors.

A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation.

Multimodal Imaging Probe Development for Pancreatic ß Cells: From Fluorescence to PET.

Pancreatic ß cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic ß cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

On-Demand Droplet Merging with an AC Electric Field for Multiple-Volume Droplet Generation.

We introduce a unique system to achieve on-demand droplet merging and splitting using a perpendicular AC electric field. The working mechanism involves a micropillar to split droplets, followed by electrocoalescence using an AC electric field. Adjusting the parameters of the AC signal and conductivity of the fluid result in different merging regimes. We observed a minimum threshold voltage and a strong influence of the surfactant. We hypothesize that the merging process is caused by dipole-dipole coalescence between the daughter droplets. At the same time, adjustment of the conductivity reveals a shift in the merging regimes and can be explained with an electric circuit diagram. Size-based sorting using this merging phenomenon is subsequently demonstrated, where alternate, single, double, and triple droplets sorting were achieved. The concept presented in this paper is potentially useful for drug dispensing or multivolume digital polymerase chain reaction, as droplets of multiple sizes can be generated simultaneously.

Fundamentals of Differential Particle Inertial Focusing in Symmetric Sinusoidal Microchannels.

Focusing and separation of particles such as cells at high throughput is extremely attractive for biomedical applications. Particle manipulation based on inertial effects requires a high flow speed and thus is well-suited to high-throughput applications. Recently, inertial focusing and separation using curvilinear microchannels has been attracting a great amount of interest because of the linear structure for parallelization, small device footprint, superior particle-focusing performance, and easy implementation of particle separation. However, the curvature directions of these microchannels alternate, leading to variations in both the magnitude and direction of the induced secondary flow. Accumulation of this variation along the channel causes unpredictable behaviors of particles. This paper systematically investigates the inertial-focusing phenomenon in low-aspect-ratio symmetric sinusoidal channels. First, we comprehensively studied the effects of parameters such as viscosity, flow conditions, particle size, and geometric dimensions of the microchannel on differential particle focusing. We found that particle inertial focusing is generally independent of fluid kinematic viscosity but highly dependent on particle size, flow conditions, and channel dimensions. Next, we derived an explicit scaling factor and included all four dimensionless parameters (particle-blockage ratio, curvature ratio, Dean number, and channel aspect ratio) in a single operational map to illustrate the particle-focusing patterns. Finally, we proposed a rational guideline to intuitively instruct the design of channel dimensions for separation of a given particle mixture.

Influence of Interfacial Gas Enrichment on Controlled Coalescence of Oil Droplets in Water in Microfluidics.

Interfacial gas enrichment (IGE) of dissolved gases in water is shown to govern the strong attraction between solid hydrophobic surfaces of an atomic force microscopy (AFM) colloidal probe and solid substrate. However, the role of IGE in controlling the attraction between fluid-fluid interfaces of foam films and emulsion films is difficult to establish by AFM techniques because of the extremely fast coalescence. Here, we applied droplet-based microfluidics to capture the fast coalescence event under the creeping flow condition and quantify the effect of IGE on the drainage and stability of water films between coalescing oil droplets. The amount of dissolved gases is controlled by partially degassing the oil phase. When the amount of dissolved gases (oxygen) in oil decreases (from 7.89 to 4.59 mg/L), the average drainage time of coalescence significantly increases (from 19 to 50 ms). Our theoretical quantification of the coalescence by incorporating IGE into the multilayer van der Waals attraction theory confirms the acceleration of film drainage dynamics by the van der Waals attractive force generated by IGE. The thickness of the IGE layer decreases from 5.5 to 4.9 nm when the amount of dissolved gas decreases from 7.89 to 4.59 mg/L. All these results establish the universal role of dissolved gases in governing the strong attraction between particulate hydrophobic interfaces.

Pluripotency factors regulate definitive endoderm specification through eomesodermin.

Understanding the molecular mechanisms controlling early cell fate decisions in mammals is a major objective toward the development of robust methods for the differentiation of human pluripotent stem cells into clinically relevant cell types. Here, we used human embryonic stem cells and mouse epiblast stem cells to study specification of definitive endoderm in vitro. Using a combination of whole-genome expression and chromatin immunoprecipitation (ChIP) deep sequencing (ChIP-seq) analyses, we established an hierarchy of transcription factors regulating endoderm specification. Importantly, the pluripotency factors NANOG, OCT4, and SOX2 have an essential function in this network by actively directing differentiation. Indeed, these transcription factors control the expression of EOMESODERMIN (EOMES), which marks the onset of endoderm specification. In turn, EOMES interacts with SMAD2/3 to initiate the transcriptional network governing endoderm formation. Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development.

The molecular functions of hepatocyte nuclear factors - In and beyond the liver.

The hepatocyte nuclear factors (HNFs) namely HNF1α/ß, FOXA1/2/3, HNF4α/γ and ONECUT1/2 are expressed in a variety of tissues and organs, including the liver, pancreas and kidney. The spatial and temporal manner of HNF expression regulates embryonic development and subsequently the development of multiple tissues during adulthood. Though the HNFs were initially identified individually based on their roles in the liver, numerous studies have now revealed that the HNFs cross-regulate one another and exhibit synergistic relationships in the regulation of tissue development and function. The complex HNF transcriptional regulatory networks have largely been elucidated in rodent models, but less so in human biological systems. Several heterozygous mutations in these HNFs were found to cause diseases in humans but not in rodents, suggesting clear species-specific differences in mutational mechanisms that remain to be uncovered. In this review, we compare and contrast the expression patterns of the HNFs, the HNF cross-regulatory networks and how these liver-enriched transcription factors serve multiple functions in the liver and beyond, extending our focus to the pancreas and kidney. We also summarise the insights gained from both human and rodent studies of mutations in several HNFs that are known to lead to different disease conditions.

An arduous journey from human pluripotent stem cells to functional pancreatic ß cells.

Type 1 and type 2 diabetes are caused by a destruction and decrease in the number of functional insulin-producing ß cells, respectively; therefore, the generation of functional ß cells from human embryonic stem cells and human induced pluripotent stem cells, collectively known as human pluripotent stem cells (hPSCs), for potential cell replacement therapy and disease modelling is an intensely investigated area. Recent scientific breakthroughs enabled derivation of large quantities of human pancreatic ß-like cells in vitro, although with varied glucose-stimulated insulin secretion kinetics. In the present review, we comprehensively summarize, compare and critically analyze the intricacies of these developing technologies, including differentiation platforms, robustness of protocols, and methodologies used to characterize hPSC-derived ß-like cells. We also discuss experimental issues that need to be resolved before these ß-like cells can be used clinically.

Negative Pressure Induced Droplet Generation in a Microfluidic Flow-Focusing Device.

We introduce an effective method to actively induce droplet generation using negative pressure. Droplets can be generated on demand using a series of periodic negative pressure pulses. Fluidic network models were developed using the analogy to electric networks to relate the pressure conditions for different flow regimes. Experimental results show that the droplet volume is correlated to the pressure ratio with a power law of 1.3. Using a pulsed negative pressure at the outlet, we are able to produce droplets in demand and with a volume proportional to the pulse width.

Setting sail for glucose homeostasis with the AKAP150-PP2B-anchor.

Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in ß cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.

Large retroperitoneal isolated fibrous cyst in absence of preceding trauma or acute pancreatitis.

BACKGROUND: Isolated retroperitoneal cystic masses are uncommon with an estimated incidence of 1/5750 to 1/250,000. The majority present with size related symptoms, complications, or a mass. Approximately a third of patients are asymptomatic and are diagnosed incidentally. Aetiologies of retroperitoneal cystic masses (RPC) include mesenteric, omental, splenic and enteric duplication cysts. Neoplastic RPCs can be divided into epithelial (mucinous or serous cystadenoma), mesothelial (mesothelioma), germ cell (cystic teratoma) and cystic changes in a solid neoplasm (paraganglioma, neurilemmoma, sarcoma). CASE PRESENTATION: A 53 year-old man presented to us with abdominal pain related to a large mass in his left upper quadrant with associated anorexia and weight loss. He gave no history of previous trauma and denied having symptoms or a history of pancreatitis. He said he had felt this mass increasing in size over the course of several years. Clinical examination of his abdomen revealed a large firm left sided mass extending to his left upper quadrant. Imaging with computed tomography (CT) and magnetic resonance imaging cholangio-pancreatogram (MRCP) revealed a 13.7 cm × 12.2 cm × 10.9 cm cystic lesion in the retroperitoneum which was separate from the kidney, pancreas, spleen and bowel. At laparotomy, this mass was easily dissected from the surrounding viscera and was excised completely intact. Histopathological assessment found the mass to be a large fibrous pseudocyst with no epithelial lining. CONCLUSION: We present a rare case of an isolated large retroperitoneal fibrous pseudocyst unrelated to previous pancreatitis which was successfully managed with surgery.

Derivation of human induced pluripotent stem cells from patients with maturity onset diabetes of the young.

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human "stem cell cassette" containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.

Innovations in bio-engineering and cell-based approaches to address immunological challenges in islet transplantation.

Human allogeneic pancreatic islet transplantation is a life-changing treatment for patients with severe Type 1 Diabetes (T1D) who suffer from hypoglycemia unawareness and high risk of severe hypoglycemia. However, intensive immunosuppression is required to prevent immune rejection of the graft, that may in turn lead to undesirable side effects such as toxicity to the islet cells, kidney toxicity, occurrence of opportunistic infections, and malignancies. The shortage of cadaveric human islet donors further limits islet transplantation as a treatment option for widespread adoption. Alternatively, porcine islets have been considered as another source of insulin-secreting cells for transplantation in T1D patients, though xeno-transplants raise concerns over the risk of endogenous retrovirus transmission and immunological incompatibility. As a result, technological advancements have been made to protect transplanted islets from immune rejection and inflammation, ideally in the absence of chronic immunosuppression, to improve the outcomes and accessibility of allogeneic islet cell replacement therapies. These include the use of microencapsulation or macroencapsulation devices designed to provide an immunoprotective environment using a cell-impermeable layer, preventing immune cell attack of the transplanted cells. Other up and coming advancements are based on the use of stem cells as the starting source material for generating islet cells 'on-demand'. These starting stem cell sources include human induced pluripotent stem cells (hiPSCs) that have been genetically engineered to avoid the host immune response, curated HLA-selected donor hiPSCs that can be matched with recipients within a given population, and multipotent stem cells with natural immune privilege properties. These strategies are developed to provide an immune-evasive cell resource for allogeneic cell therapy. This review will summarize the immunological challenges facing islet transplantation and highlight recent bio-engineering and cell-based approaches aimed at avoiding immune rejection, to improve the accessibility of islet cell therapy and enhance treatment outcomes. Better understanding of the different approaches and their limitations can guide future research endeavors towards developing more comprehensive and targeted strategies for creating a more tolerogenic microenvironment, and improve the effectiveness and sustainability of islet transplantation to benefit more patients.