Human System Model Research Articles

Abstract 2796: Colibactin mutation signatures are associated with younger age of onset in colorectal cancer

Abstract Escherichia coli (E. coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin associated with pathogenesis of colorectal cancer (CRC). Colibactin binds directly to DNA, causing alkylating damage leading to double stranded DNA breaks. Colibactin mutagenesis results in mutations in hexameric A-T rich DNA motifs that can be identified by whole genome sequencing (WGS) as mutation signatures SBS28 and SBS88 (SBS-pks). Since WGS is not routinely used clinically, we developed an approach to specifically identify SBS-pks using a clinical targeted exon capture assay, MSK-IMPACT. In an institutional pan-cancer clinical cohort of 78,905 tumors we identified 15,967 samples with > 20 mutations detected by MSK-IMPACT. We identified 150/1,884 samples with microsatellite stable CRC with >10% mutations attributable to SBS-pks. Examining clinicopathologic and genomic features of SBS-pks+ compared to SBS-pks- CRC tumors, we found that SBS-pks+ signatures are significantly enriched in younger age of onset compared to SBS-pks- CRC patients (median age 55 vs median age 59, p= 0.0028). SBS-pks+ CRC exhibit similar driver mutations and tumor mutational burden (TMB) compared to SBS-pks- CRC but are associated with increased fraction of genome altered (FGA). To mechanistically investigate colibactin-associated damage we developed a novel long-term human colon organoid-microbe co-culture model system that takes advantage of reversal of polarity in suspension culture. WGS of human CRC organoids co-cultured for three months with pks+ and delta-pks E. coli NC101 confirmed induction of pks+ specific SBS-pks signatures, validating the model. Leveraging this model and a biobank of normal colon organoids from donors aged </> 50 years, we are investigating the molecular mechanisms underlying the association of SBS-pks with younger age of onset CRC. Citation Format: Stefanie Gerstberger, Melissa Lumish, Saskia Hartner, Farheen Shah, Seongmin Choi, Anisha Luthra, Qingwen Jiang, Hyung Jun Woo, Ahmed Mahmoud, Henry Walch, Simran Asawa, Mark Donoghue, Andrea Cercek, Rona Yaeger, Andrew McPherson, Francisco Sanchez-Vega, Karuna Ganesh. Colibactin mutation signatures are associated with younger age of onset in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2796.

Abstract 136: Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk RMS has not increased above 20% in the last three decades. A deeper understanding of the basic biology driving RMS tumorigenesis is needed to discover novel therapeutic targets. RMS can be divided into two major histologic subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS accounts for 20% of pediatric cases and has poorer survival. The majority of ARMS tumors harbor t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. ARMS tumors that lack oncofusions more closely resemble ERMS. Classifying on fusion status more accurately describes the disease and highlights the significance of P3/7F. ARMS tumor genomes only contain an average of 6.4 somatic mutations suggesting the oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. RMS is commonly thought to arise from skeletal muscle progenitor cells that fail to terminally differentiate. However, RMS occurs throughout the body in sites devoid of skeletal muscle, underscoring the potential for alternative cells of origin. Our lab established that endothelial progenitors are indeed a cell of origin for ARMS by generating a genetically engineered mouse model with endothelial cell specific P3F expression coupled with Cdkn2a loss. We further generated a human model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which we direct differentiation to endothelial cell fate and then force P3F expression. P3F expression during endothelial cell directed differentiation blocked endothelial maturation and instead reprogrammed cells shifting cell fate to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the kinetics and specific genomic contributions facilitated by P3F during RMS transformation. To investigate the genomic changes occurring during iARMS transformation I utilized CUT&RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These results define the fundamental genomic changes needed for transformation into FP-RMS by P3F and uncovers the key determinants of ARMS cell identity. The iARMS system provides a unique platform to uncover how P3F establishes and maintains RMS cell state and to dissect the specific dependencies required to maintain the ARMS cell state. Understanding the fundamental mechanism of P3F-driven tumorigenesis could provide greater resolution into the key disease determinants to focus pre-clinical efforts on pivotal targets. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Brian J. Abraham, Mark E. Hatley. Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 136.

Abstract 6779: Modality agnostic tumor models of cancer which can more accurately define drug efficacy and mechanism to help develop more effective therapeutics

Abstract Introduction: In the path to clinic, researchers require robust tools to effectively evaluate the capacity of novel therapeutic modalities to generate anti-tumor responses. Using human-relevant, translational ex-vivo models is widely considered to reduce attrition during drug discovery and development for a more effective prediction of clinical outcomes during preclinical testing of drug candidates. Methods: The three levels of our patient derived tumoroid model platform, COMPASS (Custom Organoid Modeling Platform for Accurate SolutionS) was built to be versatile modality-agnostic human on human model system for pre-clinical testing. COMPASS 1 has stroma with epithelium; COMPASS 2 has stroma, epithelium, and immune components (from PBMCs) and COMPASS 3 has stroma, epithelium, immune components, and oxygenation. For widespread adaptation and utilization of our COMPASS platform we encourage extensive characterization by articulating the context of use to help build confidence in cases where limited knowledge and only few real-life cases exist. Results: Our proof-of-concept examples articulating the context of use demonstrate the modality-agnostic adaptability our COMPASS platform brings to the field of preclinical testing. We encourage a collaborative approach with researchers to support their testing needs to build confidence in our model to enable transition from existing screens, assays, and animal models. We envision preclinical use of COMPASS to help stratify patients for clinical trials, identify subpopulations that need dose modification and predict the response of patients and/or their risk of adverse effects. Conclusion: Due to its greater physiological relevance and ability to encompass downstream processes in a time and cost-efficient way, industry and academia should deliberate the impact platform such as COMPASS would have on their workflow and consider adapting to using these models routinely. Citation Format: Preethi Samuel, Damieanus Odour Ochola, Huckie Del Mundo, Bhuvanesh Dave. Modality agnostic tumor models of cancer which can more accurately define drug efficacy and mechanism to help develop more effective therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6779.

Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

Cross-species modeling of muscular dystrophy in Caenorhabditis elegans using patient-derived extracellular vesicles.

Reliable disease models are critical for medicine advancement. Here, we established a versatile human disease model system using patient-derived extracellular vesicles (EVs), which transfer a pathology-inducing cargo from a patient to a recipient naïve model organism. As a proof of principle, we applied EVs from the serum of patients with muscular dystrophy to Caenorhabditis elegans and demonstrated their capability to induce a spectrum of muscle pathologies, including lifespan shortening and robust impairment of muscle organization and function. This demonstrates that patient-derived EVs can deliver disease-relevant pathologies between species and can be exploited for establishing novel and personalized models of human disease. Such models can potentially be used for disease diagnosis, prognosis, analyzing treatment responses, drug screening and identification of the disease-transmitting cargo of patient-derived EVs and their cellular targets. This system complements traditional genetic disease models and enables modeling of multifactorial diseases and of those not yet associated with specific genetic mutations.

Lessons from inducible pluripotent stem cell models on neuronal senescence in aging and neurodegeneration.

Age remains the central risk factor for many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Although the mechanisms of aging are complex, the age-related accumulation of senescent cells in neurodegeneration is well documented and their clearance can alleviate disease-related features in preclinical models. Senescence-like characteristics are observed in both neuronal and glial lineages, but their relative contribution to aging and neurodegeneration remains unclear. Human pluripotent stem cell-derived neurons provide an experimental model system to induce neuronal senescence. However, the extensive heterogeneity in the profile of senescent neurons and the methods to assess senescence remain major challenges. Here, we review the evidence of cellular senescence in neuronal aging and disease, discuss human pluripotent stem cell-based model systems used to investigate neuronal senescence and propose a panel of cellular and molecular hallmarks to characterize senescent neurons. Understanding the role of neuronal senescence may yield novel therapeutic opportunities in neurodegenerative disease.

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

Comparison of permeable cell culture inserts for use in culture of a human invitro air-liquid interface model system.

In this study, we compared 12 mm cell culture inserts with permeable polyester membranes (0.4 μm pores) from two different manufacturers: CELLTREAT® and Corning®. Physical dimensions and masses of the inserts were found to be very similar between the two brands, with CELLTREAT® inserts having a slightly smaller diameter and growth area (11.91 mm; 1.11 cm2 ) compared to Corning® Transwells® (12 mm; 1.13 cm2 ). We compared cell differentiation outcomes of human nasal epithelial cells (HNECs) at air-liquid interface grown on inserts from the two different manufacturers, including trans-epithelial electrical resistance, ciliary beat frequency, ciliated area, and gene expression. HNECs from three male donors were used for all endpoints. No statistically significant differences were observed between paired cultures grown on different brands of insert. In conclusion, these inserts are comparable for use with airway epithelial cell model systems and likely do not impact cellular differentiation or cell culture quality.

Human Hepatic Spheroid Coculture Model for the Assessment of Drug-Induced Liver Injury.

Accurate evaluation of potential drug risks such as drug-induced liver injury (DILI) continues to be a challenge faced by pharmaceutical industry and regulatory agencies. Preclinical testing has served as a foundation for the evaluation of the potential risks and effectiveness of investigational new drug (IND) products in humans. However, current two-dimensional (2D) in vitro human primary hepatocyte (HPH) culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, while animal studies present inherited species-specific differences and low throughput scales. Thus, there is a continued demand to establish new approaches that can better characterize DILI during drug discovery and development. Among others, the three-dimensional (3D) hepatic spheroid model comprising self-aggregated primary human hepatocytes cocultured with non-parenchymal cells (NPCs) appears to be a more accurate representation of the natural hepatic microenvironment with intercellular interactions between hepatocytes, stellate cells, Kupffer cells, liver sinusoidal endothelial cells (LSECs), and other cell types. This model holds the potential to improve the ability for long-term functional and toxicological studies. Here, we provide methodological details for this human hepatic spheroid coculture model system.

Limitations in the field of vision of young operators of utility all-terrain vehicles

Problem: All-Terrain Vehicles (ATVs) cause a distressing number of fatalities and injuries among farm youth in the United States. The number of severe injuries caused by ATV crashes on farms, which stood at 25 children in 2019, is expected to rise due to increased ATV sales during the COVID-19 pandemic. Existing research into children's injuries has explored physical capabilities like anthropometry, strength, and visual acuity. Yet, studies assessing youths' physical ability to operate ATVs remain limited. This study tests the hypothesis that diminished vision field in young ATV operators raises crash risks. Methods: This study used SAMMIECAD digital human modeling system to simulate and compare the field of vision of youth and adult ATV operators. The simulations incorporated 3-D mockups of ATVs, humans, sprayer tanks, windshields, and obstacles. Ten utility ATVs and children of both genders across nine ages (8–16) and three height percentiles (5th, 50th, 95th) were evaluated. Results: Simulations showed that young ATV operators generally have a more restricted field of vision than adults, particularly those aged 6–11 years. Discussion: Visual limitations hinder riders' ability to see ATV controls, potentially leading to vehicle control loss. Moreover, they impair environmental perception during riding, compromising youths' ATV operation abilities and increasing crash risks. Practical Applications: These findings offer crucial data in support of modifying ATV safety guidelines. Additionally, they can guide youth occupational health professionals in preventing ATV-related incidents in agricultural settings.

High‐throughput CRISPRi screening to investigate mechanisms underlying Alzheimer’s risk loci

AbstractBackgroundAlzheimer’s disease (AD) is a highly (60‐80%) heritable disease with 75 known genetic risk loci. Interestingly, preliminary data from human tissues suggests that these risk loci predominantly act through myeloid cells, implicating microglia, the brain’s resident immune cell, as playing a critical role in AD risk. However, whether the mechanisms underlying these risk loci can be studied in induced pluripotent stem cell (iPSC)‐derived systems, which are currently the only physiologically relevant human model system, remains unknown.MethodWe generated neural progenitors, excitatory neurons, astrocytes and microglia from two human iPSC lines, and performed RNA‐seq to identify all expressed genes and ChIP‐seq for H3K27ac to identify all active gene promoters and enhancers. We compared these data to ChIP‐seq data from primary uncultured (ex vivo) adult human microglia to compare the enrichment of AD risk loci polymorphisms in active promoters and enhancers, as well as a larger panel of neuropsychiatric and neurodegenerative conditions.ResultiPSC‐derived microglia‐like cells (iMGLs) recapitulated an enrichment of AD risk loci polymorphisms in microglial promoters and enhancers previously seen in ex vivo primary human microglia. Furthermore, the enrichments of polymorphisms conferring risk for other neurological and neurodegenerative conditions were similar in iMGLs and ex vivo microglia, suggesting that the epigenome of iMGLs is similar to that of in vivo human microglia.ConclusioniMGLs will serve as a useful cell‐based model for studying AD risk loci, and we are currently developing high‐throughput CRISPR‐based genome and epigenome editing systems to dissect the molecular and cellular mechanisms at these loci, which will nominate novel genes and pathways for therapeutic development.

Stage-Specific Erythropoietic Regulation By Long Non-Coding RNAs (lncRNAs) in Patients with Monge's Disease

Background: Approximately 20% of individuals living in Andean mountains suffer from Chronic Mountain Sickness, CMS, or Monge's disease. They manifest the disease in adulthood and often die because of excessive erythropoiesis (EE) (Hematocrit of >65%, averaging 72%, mostly occurring in males), an extreme phenotype that can often lead to myocardial infarction or stroke. The uniqueness of this Andean population is even more significant when we realize that there are individuals who live side by side at the same altitude as those with CMS but do not suffer from the same extreme phenotype (non-CMS). Methods: To understand the mechanistic basis of EE in these patients, we performed RNA seq of cells from both CMS and non-CMS populations. We further utilized an in-vitro human iPS-derived model system to study the functional role of genes at various erythroid stages. Results: Interestingly, we discovered a unique non-overlapping profile of differentially expressed long non-coding RNA (lncRNAs) between CMS and non-CMS cells. Among these lncRNAs, LINC00431 was significantly downregulated in non-CMS cells as compared to CMS in hypoxia (mimicking high altitude). We further validated the expression of LINC00431 and found it to be significantly downregulated under hypoxia in the non-CMS cells as determined by the qPCR (P<0.01). We hypothesized that this lncRNA plays a role in regulating EE response at high altitude. Knock-down of LINC00431in the CMS cells did not affect the early stages of erythroid proliferation (such BFU-E) but there was a large effect in the later erythroid stages such as the CFU-E and reticulocyte stages (CD71+, CD235a+ cells). Particularly, KD of LINC00431 resulted in a major reduction (from ~60% to ~7%, p<0.0001) of CD235a+ erythroid cells (Reticulocyte stage-a later erythroid stage). In contrast, we have also shown that the knock-down of LINC02228 has a large impact at the early erythropoietic stage BFU-E of the CMS cell reducing by 50% when we KD LINC00228. Conclusion: These results show a stage-specific erythropoietic regulation by lncRNAs in the CMS and non-CMS patients. LINC02228 plays a critical role at the BFU stage and LINC00431 plays an important role particularly at later erythroid stages (CD71+, CD235+ Reticulocyte stage). This stage specificity also suggests distinct mechanisms for each lncRNAs and their downstream targets.

Development of a human model system for investigating pancreatic ducts

Development of a human model system for investigating pancreatic ducts

Human cellular model systems of β-thalassemia enable in-depth analysis of disease phenotype

β-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for β-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in β-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.

Augmented reality and indoor positioning based mobile production monitoring system to support workers with human-in-the-loop

With the increasing demand for product customization, the exponential increase in large-scale and small-batch production orders has yielded new challenges for the capacity of traditional production lines. Due to the information redundancy and complex production process on the production site of the factory, the traditional monitoring system is incapable of meeting the information interaction requirements between the factory management level and the executive level. Meanwhile, the traditional augmented reality (AR) based on image recognition is not suitable in the complex industrial environments. To address the gap, we propose a novel mobile production monitoring system (AIMPMs) with human-in-the-loop control by leveraging the cutting-edge AR and indoor positioning technique. In the proposed system, Ultra-wideband (UWB) and Inertial Measurement Unit (IMU) fusion indoor positioning technology is proposed, which provides accurate indoor positioning information for the production factors in the factory. Subsequently, we build the lightweight indoor map for positioning that can serve as the location reference and path planning, and a nearest-neighbor decision algorithm with double rejection decision (NN-DRD) is proposed to match the positioning features to trigger virtual monitoring information. Finally, the AIMPMs is applied in a hydraulic cylinder factory to verify its enforceability and effectiveness, and the human factors evaluation model and index system are constructed to evaluate two systems, (1) the mobile monitoring system based on positioning information, and (2) the mobile monitoring system based on image recognition, respectively. The experimental results indicate that after the application of AIMPMs, the physiological and mental fatigue of the production personnel is immensely decreased. Therefore, the system realizes a smarter and highly humanized human-machine interaction mode with human-in-the-loop control.

Biofabrication of an in-vitro bone model for Gaucher disease

Gaucher disease (GD), the most prevalent lysosomal disorder, is caused by GBA1 gene mutations, leading to deficiency of glucocerebrosidase, and accumulation of glycosphingolipids in cells of the mononuclear phagocyte system. While skeletal diseases are the leading cause of morbidity and reduced quality of life in GD, the pathophysiology of bone involvement is not yet fully understood, partly due to lack of relevant human model systems. In this work, we present the first 3D human model of GD using aspiration-assisted freeform bioprinting, which enables a platform tool with a potential for decoding the cellular basis of the developmental bone abnormalities in GD. In this regard, human bone marrow-derived mesenchymal stem cells (obtained commercially) and peripheral blood mononuclear cells derived from a cohort of GD patients, at different severities, were co-cultured to form spheroids and differentiated into osteoblast and osteoclast lineages, respectively. Co-differentiated spheroids were then 3D bioprinted into rectangular tissue patches as a bone tissue model for GD. The results revealed positive alkaline phosphatase (ALP) and tartrate-resistant ALP activities, with multi-nucleated cells demonstrating the efficacy of the model, corroborating with gene expression studies. There were no significant changes in differentiation to osteogenic cells but pronounced morphological deformities in spheroid formation, more evident in the ‘severe’ cohort, were observed. Overall, the presented GD model has the potential to be adapted to personalized medicine not only for understanding the GD pathophysiology but also for personalized drug screening and development.

IL-6 trans-signaling in a humanized mouse model of scleroderma

Fibrosis is regulated by interactions between immune and mesenchymal cells. However, the capacity of cell types to modulate human fibrosis pathology is poorly understood due to lack of a fully humanized model system. MISTRG6 mice were engineered by homologous mouse/human gene replacement to develop an immune system like humans when engrafted with human hematopoietic stem cells (HSCs). We utilized MISTRG6 mice to model scleroderma by transplantation of healthy or scleroderma skin from a patient with pansclerotic morphea to humanized mice engrafted with unmatched allogeneic HSC. We identified that scleroderma skin grafts contained both skin and bone marrow-derived human CD4 and CD8 T cells along with human endothelial cells and pericytes. Unlike healthy skin, fibroblasts in scleroderma skin were depleted and replaced by mouse fibroblasts. Furthermore, HSC engraftment alleviated multiple signatures of fibrosis, including expression of collagen and interferon genes, and proliferation and activation of human T cells. Fibrosis improvement correlated with reduced markers of T cell activation and expression of human IL-6 by mesenchymal cells. Mechanistic studies supported a model whereby IL-6 trans-signaling driven by CD4 T cell-derived soluble IL-6 receptor complexed with fibroblast-derived IL-6 promoted excess extracellular matrix gene expression. Thus, MISTRG6 mice transplanted with scleroderma skin demonstrated multiple fibrotic responses centered around human IL-6 signaling, which was improved by the presence of healthy bone marrow-derived immune cells. Our results highlight the importance of IL-6 trans-signaling in pathogenesis of scleroderma and the ability of healthy bone marrow-derived immune cells to mitigate disease.

관절 데이터 기반 사람 동작 분류 모델 및 자세 위험성 평가 시스템

관절 데이터 기반 사람 동작 분류 모델 및 자세 위험성 평가 시스템

What is a cell type?

A next step for cell atlases should be to chart perturbations in human model systems.

SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells

Aims/hypothesisIncreased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood.MethodsWe generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised.ResultsβS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking.Conclusions/interpretationTaken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway.Data availabilityRNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).Graphical