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.

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.

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.

Development of "LvL UP 1.0": a smartphone-based, conversational agent-delivered holistic lifestyle intervention for the prevention of non-communicable diseases and common mental disorders.

Background: Non-communicable diseases (NCDs) and common mental disorders (CMDs) are the leading causes of death and disability worldwide. Lifestyle interventions via mobile apps and conversational agents present themselves as low-cost, scalable solutions to prevent these conditions. This paper describes the rationale for, and development of, "LvL UP 1.0″, a smartphone-based lifestyle intervention aimed at preventing NCDs and CMDs. Materials and Methods: A multidisciplinary team led the intervention design process of LvL UP 1.0, involving four phases: (i) preliminary research (stakeholder consultations, systematic market reviews), (ii) selecting intervention components and developing the conceptual model, (iii) whiteboarding and prototype design, and (iv) testing and refinement. The Multiphase Optimization Strategy and the UK Medical Research Council framework for developing and evaluating complex interventions were used to guide the intervention development. Results: Preliminary research highlighted the importance of targeting holistic wellbeing (i.e., both physical and mental health). Accordingly, the first version of LvL UP features a scalable, smartphone-based, and conversational agent-delivered holistic lifestyle intervention built around three pillars: Move More (physical activity), Eat Well (nutrition and healthy eating), and Stress Less (emotional regulation and wellbeing). Intervention components include health literacy and psychoeducational coaching sessions, daily "Life Hacks" (healthy activity suggestions), breathing exercises, and journaling. In addition to the intervention components, formative research also stressed the need to introduce engagement-specific components to maximise uptake and long-term use. LvL UP includes a motivational interviewing and storytelling approach to deliver the coaching sessions, as well as progress feedback and gamification. Offline materials are also offered to allow users access to essential intervention content without needing a mobile device. Conclusions: The development process of LvL UP 1.0 led to an evidence-based and user-informed smartphone-based intervention aimed at preventing NCDs and CMDs. LvL UP is designed to be a scalable, engaging, prevention-oriented, holistic intervention for adults at risk of NCDs and CMDs. A feasibility study, and subsequent optimisation and randomised-controlled trials are planned to further refine the intervention and establish effectiveness. The development process described here may prove helpful to other intervention developers.

Women's Usage Behavior and Perceived Usefulness with Using a Mobile Health Application for Gestational Diabetes Mellitus: Mixed-Methods Study.

The prevalence of gestational diabetes mellitus (GDM) is increasing, and only a few mobile health (mHealth) applications are specifically designed to manage GDM. In this mixed-methods study, a follow-up study of a randomized controlled trial (RCT) analyzed a largely automated mHealth application-based lifestyle coaching program to (a) measure the application's usage behavior and (b) explore users' perceptions of its usefulness in GDM management. Quantitative data were collected from the 170 application users who had participated in the intervention arm of the RCT. Semi-structured interviews (n = 14) captured users' experiences when using the application. Data were collected from June 2019 to January 2020. Quantitative data were analyzed descriptively, and interviews were analyzed thematically. Only 57/170 users (34%) logged at least one meal, and only 35 meals on average were logged for eight weeks because of the incorrectly worded food items and limited food database. On the contrary, an average of 1.85 (SD = 1.60) weight values were logged per week since the weight tracking component was easy to use. Many users (6/14 (43%)) mentioned that the automatic coach messages created an immediate sense of self-awareness in food choices and motivated behavior. The findings suggest that for GDM management, a largely automated mHealth application has the potential to promote self-awareness of healthy lifestyle choices, reducing the need for intensive human resources. Additionally, several gaps in the application's design were identified which need to be addressed in future works.

Decreased GLUT2 and glucose uptake contribute to insulin secretion defects in MODY3/HNF1A hiPSC-derived mutant ß cells.

Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A+/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived ß-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients.

A Randomized Controlled Trial to Evaluate the Effects of a Smartphone Application-Based Lifestyle Coaching Program on Gestational Weight Gain, Glycemic Control, and Maternal and Neonatal Outcomes in Women With Gestational Diabetes Mellitus: The SMART-GDM Study.

OBJECTIVE: SMART-GDM examined whether Habits-GDM, a smartphone application (app) coaching program, can prevent excessive gestational weight gain (EGWG) and improve glycemic control and maternal and neonatal outcomes in gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS: In this randomized controlled trial, women diagnosed with GDM between 12 and 30 weeks were randomly assigned to usual care (control) or to additional support from Habits-GDM that integrated dietary, physical activity, weight, and glucose monitoring (intervention). The primary outcome was the proportion of participants with EGWG. Secondary outcomes included absolute gestational weight gain (GWG), glycemic control, and maternal, delivery, and neonatal outcomes. RESULTS: In total, 340 women were randomized (170 intervention, 170 control; mean ± SD age 32.0 ± 4.2 years; mean BMI 25.6 ± 5.6 kg/m2). There were no statistically significant differences in the proportions of women with EGWG, absolute GWG, or maternal and delivery outcomes between experimental groups. Average glucose readings were lower in the intervention group (mean difference -0.15 mmol/L [95% CI -0.26; -0.03], P = 0.011) as were the proportions of glucose above targets (premeal: 17.9% vs. 23.3%, odds ratio 0.68 [95% CI 0.53; 0.87], P = 0.003; 2-h postmeal: 19.9% vs. 50%, 0.54 [0.42; 0.70], P < 0.001). When regarded as a composite (although not prespecified), the overall neonatal complications (including birth trauma, neonatal hypoglycemia, hyperbilirubinemia, respiratory distress, neonatal intensive care unit admission, and perinatal death) were significantly lower in the intervention group (38.1% vs. 53.7%, 0.53 [0.34; 0.84], P = 0.006). CONCLUSIONS: When added to usual care, Habits-GDM resulted in better maternal glycemic control and composite neonatal outcomes (nonprespecified) but did not reduce EGWG among women with GDM.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

HNF4A Haploinsufficiency in MODY1 Abrogates Liver and Pancreas Differentiation from Patient-Derived Induced Pluripotent Stem Cells.

Maturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes condition caused by heterozygous HNF4A mutations. We investigate how HNF4A haploinsufficiency from a MODY1/HNF4A mutation influences the development of foregut-derived liver and pancreatic cells through differentiation of human induced pluripotent stem cells from a MODY1 family down the foregut lineage. In MODY1-derived hepatopancreatic progenitors, which expressed reduced HNF4A levels and mislocalized HNF4A, foregut genes were downregulated, whereas hindgut-specifying HOX genes were upregulated. MODY1-derived hepatocyte-like cells were found to exhibit altered morphology. Hepatic and ß cell gene signatures were also perturbed in MODY1-derived hepatocyte-like and ß-like cells, respectively. As mutant HNF4A (p.Ile271fs) did not undergo complete nonsense-mediated decay or exert dominant negativity, HNF4A-mediated loss of function is likely due to impaired transcriptional activation of target genes. Our results suggest that in MODY1, liver and pancreas development is perturbed early on, contributing to altered hepatic proteins and ß cell defects in patients.

Single-cell analyses of human islet cells reveal de-differentiation signatures.

Human pancreatic islets containing insulin-secreting ß-cells are notoriously heterogeneous in cell composition. Since ß-cell failure is the root cause of diabetes, understanding this heterogeneity is of paramount importance. Recent reports have cataloged human islet transcriptome but not compared single ß-cells in detail. Here, we scrutinized ex vivo human islet cells from healthy donors and show that they exhibit de-differentiation signatures. Using single-cell gene expression and immunostaining analyses, we found healthy islet cells to contain polyhormonal transcripts, and INS+ cells to express decreased levels of ß-cell genes but high levels of progenitor markers. Rare cells that are doubly positive for progenitor markers/exocrine signatures, and endocrine/exocrine hormones were also present. We conclude that ex vivo human islet cells are plastic and can possibly de-/trans-differentiate across pancreatic cell fates, partly accounting for ß-cell functional decline once isolated. Therefore, stabilizing ß-cell identity upon isolation may improve its functionality.