HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes.

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3, HAAO, HNF1A, MAP3K11) and previously unidentified (ABCD3, CDKN2AIP, USH1C, VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1, GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation.

Hepatocyte-intrinsic SMN deficiency drives metabolic dysfunction and liver steatosis in spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is typically characterized as a motor neuron disease, but extra-neuronal phenotypes are present in almost every organ in severely affected patients and animal models. Extra-neuronal phenotypes were previously underappreciated as patients with severe SMA phenotypes usually died in infancy; however, with current treatments for motor neurons increasing patient lifespan, impaired function of peripheral organs may develop into significant future comorbidities and lead to new treatment-modified phenotypes. Fatty liver is seen in SMA animal models , but generalizability to patients and whether this is due to hepatocyte-intrinsic Survival Motor Neuron (SMN) protein deficiency and/or subsequent to skeletal muscle denervation is unknown. If liver pathology in SMA is SMN-dependent and hepatocyte-intrinsic, this suggests SMN repleting therapies must target extra-neuronal tissues and motor neurons for optimal patient outcome. Here we showed that fatty liver is present in SMA and that SMA patient-specific iHeps were susceptible to steatosis. Using proteomics, functional studies and CRISPR/Cas9 gene editing, we confirmed that fatty liver in SMA is a primary SMN-dependent hepatocyte-intrinsic liver defect associated with mitochondrial and other hepatic metabolism implications. These pathologies require monitoring and indicate need for systematic clinical surveillance and additional and/or combinatorial therapies to ensure continued SMA patient health.

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.

Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility.

Cell culture technology has evolved, moving from single-cell and monolayer methods to 3D models like reaggregates, spheroids, and organoids, improved with bioengineering like microfabrication and bioprinting. These advancements, termed microphysiological systems (MPSs), closely replicate tissue environments and human physiology, enhancing research and biomedical uses. However, MPS complexity introduces standardization challenges, impacting reproducibility and trust. We offer guidelines for quality management and control criteria specific to MPSs, facilitating reliable outcomes without stifling innovation. Our fit-for-purpose recommendations provide actionable advice for achieving consistent MPS performance.

Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells.

Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

PAX4 loss of function increases diabetes risk by altering human pancreatic endocrine cell development.

The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-ßH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk.

FGFR-mediated ERK1/2 signaling contributes to mesendoderm and definitive endoderm formation in vitro.

The differentiation of human pluripotent stem cells into the SOX17+ definitive endoderm (DE) germ layer is important for generating tissues for regenerative medicine. Multiple developmental and stem cell studies have demonstrated that Activin/Nodal signaling is the primary driver of definitive endoderm formation. Here, we uncover that the FGF2-FGFR-ERK1/2 signaling contributes to mesendoderm and SOX17+ DE formation. Without ERK1/2 signaling, the Activin/Nodal signaling is insufficient to drive mesendoderm and DE formation. Besides FGF2-FGFR-mediated signaling, IGF1R signaling possibly contributes to the ERK1/2 signaling for DE formation. We identified a temporal relationship between Activin/Nodal-SMAD2 and FGF2-FGFR-ERK1/2 signaling in which Activin/Nodal-SMAD2 participates in the initiation of mesendoderm and DE specification that is followed by increasing activity of FGF2-FGFR-ERK1/2 to facilitate and permit the successful generation of SOX17+ DE. Overall, besides the role of Activin/Nodal signaling for DE formation, our findings shed light on the contribution of ERK1/2 signaling for mesendoderm and DE formation.

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.

Prioritization of genes associated with type 2 diabetes mellitus for functional studies.

Existing therapies for type 2 diabetes mellitus (T2DM) show limited efficacy or have adverse effects. Numerous genetic variants associated with T2DM have been identified, but progress in translating these findings into potential drug targets has been limited. Here, we describe the tools and platforms available to identify effector genes from T2DM-associated coding and non-coding variants and prioritize them for functional studies. We discuss QSER1 and SLC12A8 as examples of genes that have been identified as possible T2DM candidate genes using these tools and platforms. We suggest further approaches, including the use of sequencing data with increased sample size and ethnic diversity, single-cell omics data for analyses, glycaemic trait associations to predict gene function and, potentially, human induced pluripotent stem cell 'village' cultures, to strengthen current gene functionalization workflows. Effective prioritization of T2DM-associated genes for experimental validation could expedite our understanding of the genetic mechanisms responsible for T2DM to facilitate the use of precision medicine in its treatment.

HNF1A binds and regulates the expression of SLC51B to facilitate the uptake of estrone sulfate in human renal proximal tubule epithelial cells.

Renal defects in maturity onset diabetes of the young 3 (MODY3) patients and Hnf1a-/- mice suggest an involvement of HNF1A in kidney development and/or its function. Although numerous studies have leveraged on Hnf1α-/- mice to infer some transcriptional targets and function of HNF1A in mouse kidneys, species-specific differences obviate a straightforward extrapolation of findings to the human kidney. Additionally, genome-wide targets of HNF1A in human kidney cells have yet to be identified. Here, we leveraged on human in vitro kidney cell models to characterize the expression profile of HNF1A during renal differentiation and in adult kidney cells. We found HNF1A to be increasingly expressed during renal differentiation, with peak expression on day 28 in the proximal tubule cells. HNF1A ChIP-Sequencing (ChIP-Seq) performed on human pluripotent stem cell (hPSC)-derived kidney organoids identified its genome-wide putative targets. Together with a qPCR screen, we found HNF1A to activate the expression of SLC51B, CD24, and RNF186 genes. Importantly, HNF1A-depleted human renal proximal tubule epithelial cells (RPTECs) and MODY3 human induced pluripotent stem cell (hiPSC)-derived kidney organoids expressed lower levels of SLC51B. SLC51B-mediated estrone sulfate (E1S) uptake in proximal tubule cells was abrogated in these HNF1A-deficient cells. MODY3 patients also exhibit significantly higher excretion of urinary E1S. Overall, we report that SLC51B is a target of HNF1A responsible for E1S uptake in human proximal tubule cells. As E1S serves as the main storage form of nephroprotective estradiol in the human body, lowered E1S uptake and increased E1S excretion may reduce the availability of nephroprotective estradiol in the kidneys, contributing to the development of renal disease in MODY3 patients.

Core-Shell Particles: From Fabrication Methods to Diverse Manipulation Techniques.

Core-shell particles are micro- or nanoparticles with solid, liquid, or gas cores encapsulated by protective solid shells. The unique composition of core and shell materials imparts smart properties on the particles. Core-shell particles are gaining increasing attention as tuneable and versatile carriers for pharmaceutical and biomedical applications including targeted drug delivery, controlled drug release, and biosensing. This review provides an overview of fabrication methods for core-shell particles followed by a brief discussion of their application and a detailed analysis of their manipulation including assembly, sorting, and triggered release. We compile current methodologies employed for manipulation of core-shell particles and demonstrate how existing methods of assembly and sorting micro/nanospheres can be adopted or modified for core-shell particles. Various triggered release approaches for diagnostics and drug delivery are also discussed in detail.

Tissue engineering of decellularized pancreas scaffolds for regenerative medicine in diabetes.

Diabetes mellitus is a global disease requiring long-term treatment and monitoring. At present, pancreas or islet transplantation is the only reliable treatment for achieving stable euglycemia in Type I diabetes patients. However, the shortage of viable pancreata for transplantation limits the use of this therapy for the majority of patients. Organ decellularization and recellularization is emerging as a promising solution to overcome the shortage of viable organs for transplantation by providing a potential alternative source of donor organs. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have been performed, and show promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we provide an overview of the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with an outlook on the remaining work that needs to be done in order to realize the goal of using this technology to create bioengineered pancreata for transplantation in diabetes patients. STATEMENT OF SIGNIFICANCE: Pancreas or islet transplantation is a means of providing insulin-independence in diabetes patients. However, due to the shortage of viable pancreata, whole-organ decellularization and recellularization is emerging as a promising solution to overcome organ shortage for transplantation. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have shown promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we highlight the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with future work that needs to be done in order to realize clinical translation of bioengineered pancreata for transplantation in diabetes patients.

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.

Trans-interaction of risk loci 6p24.1 and 10q11.21 is associated with endothelial damage in coronary artery disease.

BACKGROUND AND AIMS: Single nucleotide polymorphism rs6903956 has been identified as one of the genetic risk factors for coronary artery disease (CAD). However, rs6903956 lies in a non-coding locus on chromosome 6p24.1. We aim to interrogate the molecular basis of 6p24.1 containing rs6903956 risk alleles in endothelial disease biology. METHODS AND RESULTS: We generated induced pluripotent stem cells (iPSCs) from CAD patients (AA risk genotype at rs6903956) and non-CAD subjects (GG non-risk genotype at rs6903956). CRISPR-Cas9-based deletions (Δ63-89bp) on 6p24.1, including both rs6903956 and a short tandem repeat variant rs140361069 in linkage disequilibrium, were performed to generate isogenic iPSC-derived endothelial cells. Edited CAD endothelial cells, with removal of 'A' risk alleles, exhibited a global transcriptional downregulation of pathways relating to abnormal vascular physiology and activated endothelial processes. A CXC chemokine ligand on chromosome 10q11.21, CXCL12, was uncovered as a potential effector gene in CAD endothelial cells. Underlying this effect was the preferential inter-chromosomal interaction of 6p24.1 risk locus to a weak promoter of CXCL12, confirmed by chromatin conformation capture assays on our iPSC-derived endothelial cells. Functionally, risk genotypes AA/AG at rs6903956 were associated significantly with elevated levels of circulating damaged endothelial cells in CAD patients. Circulating endothelial cells isolated from patients with risk genotypes AA/AG were also found to have 10 folds higher CXCL12 transcript copies/cell than those with non-risk genotype GG. CONCLUSIONS: Our study reveals the trans-acting impact of 6p24.1 with another CAD locus on 10q11.21 and is associated with intensified endothelial injury.

Generating pancreatic beta-like cells from human pluripotent stem cells.

Diabetes is a major healthcare burden globally, affecting over 463 million people today, according to the International Diabetes Federation. The most common types of diabetes are Type I diabetes (T1D) and Type II diabetes (T2D), characterized by hyperglycemia due to autoimmune destruction of ß cells (T1D) and ß cell dysfunction, usually on a background of insulin resistance (T2D). There is currently no cure for diabetes, and patients with T1D require lifelong insulin therapy. Additionally, while most cases of T2D can be managed by lifestyle and diet modifications, with or without antidiabetic drugs, severe cases of T2D may also require insulin therapy. The only means to restore stable euglycemia in these patients is now via whole pancreas or islet transplantation. However, this is limited by the scarcity of donors. In recent years, advances in human pluripotent stem cell (hPSC) technologies and pancreatic ß cell differentiation protocols have opened up new potential avenues for cell replacement therapies for diabetes. These advances have also created opportunities to use hPSC-derived ß-like cells for studies of disease mechanisms and drug discovery, which in turn have the potential to lead to better therapies for diabetes patients. Here, we describe the protocol used in our laboratory to generate ß-like cells from hPSCs to study the mechanisms underlying various types of diabetes.

Quality criteria for in vitro human pluripotent stem cell-derived models of tissue-based cells.

The advent of the technology to isolate or generate human pluripotent stem cells provided the potential to develop a wide range of human models that could enhance understanding of mechanisms underlying human development and disease. These systems are now beginning to mature and provide the basis for the development of in vitro assays suitable to understand the biological processes involved in the multi-organ systems of the human body, and will improve strategies for diagnosis, prevention, therapies and precision medicine. Induced pluripotent stem cell lines are prone to phenotypic and genotypic changes and donor/clone dependent variability, which means that it is important to identify the most appropriate characterization markers and quality control measures when sourcing new cell lines and assessing differentiated cell and tissue culture preparations for experimental work. This paper considers those core quality control measures for human pluripotent stem cell lines and evaluates the state of play in the development of key functional markers for their differentiated cell derivatives to promote assurance of reproducibility of scientific data derived from pluripotent stem cell-based systems.

Chromatin Immunoprecipitation in Human Pluripotent Stem Cell-Derived 3D Organoids to Analyze DNA-Protein Interactions.

Chromatin immunoprecipitation (ChIP) is a technique that has been widely used to interrogate DNA-protein interactions in cells. In recent years, human pluripotent stem cell (hPSC)-derived 3D organoids have emerged as a powerful model to understand human development and diseases. Performing ChIP in hPSC-derived 3D organoids is a useful approach to dissect the roles of transcription factors or co-factors and to understand the epigenetic landscape in human development and diseases. However, performing ChIP in 3D organoids is more challenging than monolayer cultures, and an optimized protocol is needed for interpretable data. Hence, in this chapter, we describe in detail a protocol for performing ChIP in hPSC-derived islet-like cells as an example, from organoid harvest to ChIP-qPCR data analysis. This chapter also highlights potential pitfalls and provides recommendations for troubleshooting.

Multidisciplinary Effort to Drive Precision-Medicine for the Future.

In the past one or two decades, countries across the world have successively implemented different precision medicine (PM) programs, and also cooperated to implement international PM programs. We are now in the era of PM. Singapore's National Precision Medicine (NPM) program, initiated in 2017, is now entering its second phase to generate a large genomic database for Asians. The National University of Singapore (NUS) also launched its own PM translational research program (TRP) in 2021, aimed at consolidating multidisciplinary expertise within the Yong Loo Lin School of Medicine to develop collaborative projects that can help to identify and validate novel therapeutic targets for the realization of PM. To achieve this, appropriate data collection, data processing, and results interpretation must be taken into consideration. There may be some difficulties during these processes, but with the improvement of relevant rules and the continuous development of omics-based technologies, we will be able to solve these problems, eventually achieving precise prediction, diagnosis, treatment, or even prevention of diseases.

Manufacturing clinical-grade human induced pluripotent stem cell-derived beta cells for diabetes treatment.

The unlimited proliferative capacity of human pluripotent stem cells (hPSCs) fortifies it as one of the most attractive sources for cell therapy application in diabetes. In the past two decades, vast research efforts have been invested in developing strategies to differentiate hPSCs into clinically suitable insulin-producing endocrine cells or functional beta cells (ß cells). With the end goal being clinical translation, it is critical for hPSCs and insulin-producing ß cells to be derived, handled, stored, maintained and expanded with clinical compliance. This review focuses on the key processes and guidelines for clinical translation of human induced pluripotent stem cell (hiPSC)-derived ß cells for diabetes cell therapy. Here, we discuss the (1) key considerations of manufacturing clinical-grade hiPSCs, (2) scale-up and differentiation of clinical-grade hiPSCs into ß cells in clinically compliant conditions and (3) mandatory quality control and product release criteria necessitated by various regulatory bodies to approve the use of the cell-based products.