Helicobacter pylori VacA-induced mitochondrial damage in the gastric pit cells of the antrum and therapeutic rescue.

Exploring host cell specificity, pathogenicity, and molecular mechanisms of the vacuolating cytotoxin A (VacA), secreted by Helicobacter pylori (Hp) is crucial for developing novel treatment strategies. VacA affects subcellular events, particularly mitochondria, at a cell-type-specific level. However, the lack of reliable models that mimic VacA-induced subcellular damages and enable novel drug screening linked to the human stomach clinically limits our understanding of the mitochondrial networks in vivo. Here, human antrum gastric organoids (hAGOs) and tissue samples from Hp-infected patients were used to show the toxic effects of VacA-induced mitochondrial damage mainly in mucus-producing gastric pit cells by employing transcriptional, translational, and functional analyses. In VacA-intoxicated or Hp-infected hAGOs, robust mitochondrial fragmentation in gastric pit cells reduced ATP production during respiration, and loss of mucosal barrier integrity was first demonstrated experimentally. Using hAGOs, clinically relevant small molecules were screened for efficacy, and MLN8054, an Aurora kinase A inhibitor, reversed VacA-induced mitochondrial damage and loss of gastric epithelium integrity. MLN8054 was effective in VacA-treated and Hp-infected hAGOs and mice, highlighting hAGOs as a promising drug-screening model. These findings suggest that mitochondrial quality control may serve as a promising therapeutic target for Hp VacA-mediated toxicity and disease progression.

Standardization and quality assessment for human intestinal organoids.

To enhance the practical application of intestinal organoids, it is imperative to establish standardized guidelines. This proposed standardization outlines a comprehensive framework to ensure consistency and reliability in the development, characterization, and application of intestinal organoids. The recommended guidelines encompass crucial parameters, including culture conditions, critical quality attributes, quality control measures, and functional assessments, aimed at fostering a standardized approach across diverse research initiatives. The implementation of these guidelines is anticipated to significantly contribute to the reproducibility and comparability of results in the burgeoning field of intestinal organoid research.

Epigenetic alterations of TP53INP1 by EHMT2 regulate the cell cycle in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a type of cancer with high incidence and mortality rates. Although various chemical interventions are being developed to treat gastric cancer, there is a constant demand for research into new GC treatment targets and modes of action (MOAs) because of the low effectiveness and side effects of current treatments. METHODS: Using the TCGA data portal, we identified EHMT2 overexpression in GC samples. Using RNA-seq and EHMT2-specific siRNA, we investigated the role of EHMT2 in GC cell proliferation and validated its function with two EHMT2-specific inhibitors. Through the application of 3D spheroid culture, patient-derived gastric cancer organoids (PDOs), and an in vivo model, we confirmed the role of EHMT2 in GC cell proliferation. RESULTS: In this study, we found that EHMT2, a histone 3 lysine 9 (H3K9) methyltransferase, is significantly overexpressed in GC patients compared with healthy individuals. Knockdown of EHMT2 with siRNA induced G1 cell cycle arrest and attenuated GC cell proliferation. Furthermore, we confirmed that TP53INP1 induction by EHMT2 knockdown induced cell cycle arrest and inhibited GC cell proliferation. Moreover, specific EHMT2 inhibitors, BIX01294 and UNC0638, induced cell cycle arrest in GC cell lines through TP53INP1 upregulation. The efficacy of EHMT2 inhibition was further confirmed in a 3D spheroid culture system, PDOs, and a xenograft model. CONCLUSIONS: Our findings suggest that EHMT2 is an attractive therapeutic target for GC treatment.

Guidelines for Manufacturing and Application of Organoids: Kidney.

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

Maternal nanoplastic ingestion induces an increase in offspring body weight through altered lipid species and microbiota.

The rapidly increasing prevalence of obesity and overweight, especially in children and adolescents, has become a serious societal issue. Although various genetic and environmental risk factors for pediatric obesity and overweight have been identified, the problem has not been solved. In this study, we examined whether environmental nanoplastic (NP) pollutants can act as environmental obesogens using mouse models exposed to NPs derived from polystyrene and polypropylene, which are abundant in the environment. We found abnormal weight gain in the progeny until 6 weeks of age following the oral administration of NPs to the mother during gestation and lactation. Through a series of experiments involving multi-omic analyses, we have demonstrated that NP-induced weight gain is caused by alterations in the lipid composition (lysophosphatidylcholine/phosphatidylcholine ratio) of maternal breast milk and he gut microbiota distribution of the progeny. These data indicate that environmental NPs can act as obesogens in childhood.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Generation of a human fibroblast-derived induced pluripotent stem cell line KRIBBi009-A from a patient with breast cancer.

Patient-derived human induced pluripotent stem cells (iPSCs) provide a potentially useful resource for studying disease pathology and therapeutics. In this study, we generated the breast cancer patient-derived KRIBBi009-A-iPSC line from normal fibroblasts using the Sendai virus, which expressed pluripotent markers and exhibited differentiation capacity across 3 germ layers through in vitro differentiation and in vivo teratoma assay. A normal karyotype and the absence of cross-contamination of the cell lines were confirmed. Consequently, the developed iPSC line has been confirmed to be suitable for use in various studies.

GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. METHODS: Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8's involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. RESULTS: GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. CONCLUSIONS: Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes.

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line.

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.

FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia.

BACKGROUND: Spastin significantly influences microtubule regulation in neurons and is implicated in the pathogenesis of hereditary spastic paraplegia (HSP). However, post-translational regulation of the spastin protein remains nebulous. The association between E3 ubiquitin ligase and spastin provides a potential therapeutic strategy. RESULTS: As evidenced by protein chip analysis, FBXL17 inversely correlated with SPAST-M1 at the protein level in vitro and, also in vivo during embryonic developmental stage. SPAST-M1 protein interacted with FBXL17 specifically via the BTB domain at the N-terminus of SPAST-M1. The SCFFBXL17 E3 ubiquitin ligase complex degraded SPAST-M1 protein in the nuclear fraction in a proteasome-dependent manner. SPAST phosphorylation occurred only in the cytoplasmic fraction by CK2 and was involved in poly-ubiquitination. Inhibition of SCFFBXL17 E3 ubiquitin ligase by small chemical and FBXL17 shRNA decreased proteasome-dependent degradation of SPAST-M1 and induced axonal extension. The SPAST Y52C mutant, harboring abnormality in BTB domain could not interact with FBXL17, thereby escaping protein regulation by the SCFFBXL17 E3 ubiquitin ligase complex, resulting in loss of functionality with aberrant quantity. Although this mutant showed shortening of axonal outgrowth, low rate proliferation, and poor differentiation capacity in a 3D model, this phenotype was rescued by inhibiting SCFFBXL17 E3 ubiquitin ligase. CONCLUSIONS: We discovered that a novel pathway, FBXL17-SPAST was involved in pathogenicity of HSP by the loss of function and the quantitative regulation. This result suggested that targeting FBXL17 could provide new insight into HSP therapeutics.

The proliferative and multipotent epidermal progenitor cells for human skin reconstruction in vitro and in vivo.

OBJECTIVES: The skin exhibits tremendous regenerative potential, as different types of progenitor and stem cells regulate skin homeostasis and damage. However, in vitro primary keratinocytes present with several drawbacks, such as high donor variability, short lifespan, and limited donor tissue availability. Therefore, more stable primary keratinocytes are needed to generate multiple uniform in vitro and in vivo skin models. RESULTS: We identified epidermal progenitor cells from primary keratinocytes using Integrin beta 1 (ITGB1) an epidermal stem cell marker markedly decreased after senescence in vitro. Epidermal progenitor cells exhibited unlimited proliferation and the potential for multipotent differentiation capacity. Moreover, they could completely differentiate to form an organotypic skin model including conversed mesenchymal cells in the dermis and could mimic the morphologic and biochemical processes of human epidermis. We also discovered that proliferation and the multipotent differentiation capacity of these cells relied on ITGB1 expression. Eventually, we examined the in vitro and in vivo wound healing capacity of these epidermal progenitor cells. CONCLUSIONS: Overall, the findings suggest that these stable and reproducible cells can differentiate into multiple lineages, including human skin models. They are a potentially powerful tool for studying skin regeneration, skin diseases, and are an alternative for in vivo experiments.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer.

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

Next-Generation Intestinal Toxicity Model of Human Embryonic Stem Cell-Derived Enterocyte-Like Cells.

The gastrointestinal tract is the most common exposure route of xenobiotics, and intestinal toxicity can result in systemic toxicity in most cases. It is important to develop intestinal toxicity assays mimicking the human system; thus, stem cells are rapidly being developed as new paradigms of toxicity assessment. In this study, we established human embryonic stem cell (hESC)-derived enterocyte-like cells (ELCs) and compared them to existing in vivo and in vitro models. We found that hESC-ELCs and the in vivo model showed transcriptomically similar expression patterns of a total of 10,020 genes than the commercialized cell lines. Besides, we treated the hESC-ELCs, in vivo rats, Caco-2 cells, and Hutu-80 cells with quarter log units of lethal dose 50 or lethal concentration 50 of eight drugs-chloramphenicol, cycloheximide, cytarabine, diclofenac, fluorouracil, indomethacin, methotrexate, and oxytetracycline-and then subsequently analyzed the biomolecular markers and morphological changes. While the four models showed similar tendencies in general toxicological reaction, hESC-ELCs showed a stronger correlation with the in vivo model than the immortalized cell lines. These results indicate that hESC-ELCs can serve as a next-generation intestinal toxicity model.

EHMT1 knockdown induces apoptosis and cell cycle arrest in lung cancer cells by increasing CDKN1A expression.

Dozens of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases such as cancer remain largely unclear. Here, we demonstrate the involvement of EHMT1, a histone lysine methyltransferase, in lung cancer. Immunohistochemical analysis indicated that the expression levels of EHMT1 are significantly elevated in human lung carcinomas compared with non-neoplastic lung tissues. Through gene ontology analysis of RNA-seq results, we showed that EHMT1 is clearly associated with apoptosis and the cell cycle process. Moreover, FACS analysis and cell growth assays showed that knockdown of EHMT1 induced apoptosis and G1 cell cycle arrest via upregulation of CDKN1A in A549 and H1299 cell lines. Finally, in 3D spheroid culture, compared to control cells, EHMT1 knockdown cells exhibited reduced aggregation of 3D spheroids and clear upregulation of CDKN1A and downregulation of E-cadherin. Therefore, the results of the present study suggest that EHMT1 plays a critical role in the regulation of cancer cell apoptosis and the cell cycle by modulating CDKN1A expression. Further functional analyses of EHMT1 in the context of human tumorigenesis may aid in the development of novel therapeutic strategies for cancer.

Development of a quantitative prediction algorithm for target organ-specific similarity of human pluripotent stem cell-derived organoids and cells.

Human pluripotent stem cell (hPSC)-derived organoids and cells have similar characteristics to human organs and tissues. Thus, in vitro human organoids and cells serve as a superior alternative to conventional cell lines and animal models in drug development and regenerative medicine. For a simple and reproducible analysis of the quality of organoids and cells to compensate for the shortcomings of existing experimental validation studies, a quantitative evaluation method should be developed. Here, using the GTEx database, we construct a quantitative calculation system to assess similarity to the human organs. To evaluate our system, we generate hPSC-derived organoids and cells, and detected organ similarity. To facilitate the access of our system by researchers, we develop a web-based user interface presenting similarity to the appropriate organs as percentages. Thus, this program could provide valuable information for the generation of high-quality organoids and cells and a strategy to guide proper lineage-oriented differentiation.

The development of a functional human small intestinal epithelium model for drug absorption.

Advanced technologies are required for generating human intestinal epithelial cells (hIECs) harboring cellular diversity and functionalities to predict oral drug absorption in humans and study normal intestinal epithelial physiology. We developed a reproducible two-step protocol to induce human pluripotent stem cells to differentiate into highly expandable hIEC progenitors and a functional hIEC monolayer exhibiting intestinal molecular features, cell type diversity, and high activities of intestinal transporters and metabolic enzymes such as cytochrome P450 3A4 (CYP3A4). Functional hIECs are more suitable for predicting compounds metabolized by CYP3A4 and absorbed in the intestine than Caco-2 cells. This system is a step toward the transition from three-dimensional (3D) intestinal organoids to 2D hIEC monolayers without compromising cellular diversity and function. A physiologically relevant hIEC model offers a novel platform for creating patient-specific assays and support translational applications, thereby bridging the gap between 3D and 2D culture models of the intestine.

Impedance Measurement System for Assessing the Barrier Integrity of Three-Dimensional Human Intestinal Organoids.

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) hold unprecedented promise for basic biology and translational applications. However, developing a quantitative method to evaluate the epithelial cell membrane integrity of HIOs as an in vitro intestinal barrier model is a major challenge because of their complex three-dimensional (3D) structure. In this study, we developed an impedance system to measure the change in electrical resistance of 3D HIOs depending on the integrity of the intestinal epithelial cell membrane, which can reflect functionality and maturity. The expression of intestinal maturation- and tight junction-related markers was significantly higher in HIOs matured in vitro by treatment with IL-2 than in control HIOs. Analysis of gap junction size indicated that mature HIOs have greater integrity, with approximately 30% more compact gaps than immature HIOs. We designed a multi-microchannel system controlled by the inhalation pressure where the HIO is loaded, which enhances the stability and sensitivity of the impedance signal. We demonstrated the applicability of the impedance system by showing the difference in resistance between control and mature HIOs, reflecting the expression of tight junction proteins and their maturation status. We also validated the impedance system by monitoring its resistance in real time during junctional damage to HIOs induced by a digestive agent. In summary, we suggest a quantitative method to directly quantify the physiological changes in complex 3D organoid structures based on impedance spectroscopy, which can be applied to noninvasively monitor live cells and therefore enable their use in subsequent experiments.

Coiled-coil domain containing 50-V2 protein positively regulates neurite outgrowth.

The coiled-coil domain containing 50 (CCDC50) protein is a phosphotyrosine-dependent signalling protein stimulated by epidermal growth factor. It is highly expressed in neuronal cells in the central nervous system; however, the roles of CCDC50 in neuronal development are largely unknown. In this study, we showed that the depletion of CCDC50-V2 impeded the neuronal development process, including arbor formation, spine density development, and axonal outgrowth, in primary neurons. Mechanistic studies revealed that CCDC50-V2 positively regulated the nerve growth factor receptor, while it downregulated the epidermal growth factor receptor pathway. Importantly, JNK/c-Jun activation was found to be induced by the CCDC50-V2 overexpression, in which the interaction between CCDC50-V2 and JNK2 was also observed. Overall, the present study demonstrates a novel mechanism of CCDC50 function in neuronal development and provides new insight into the link between CCDC50 function and the aetiology of neurological disorders.

Fabrication of a Polycaprolactone/Alginate Bipartite Hybrid Scaffold for Osteochondral Tissue Using a Three-Dimensional Bioprinting System.

Osteochondral defects, including damage to both the articular cartilage and the subchondral bone, are challenging to repair. Although many technological advancements have been made in recent years, there are technical difficulties in the engineering of cartilage and bone layers, simultaneously. Moreover, there is a great need for a valuable in vitro platform enabling the assessment of osteochondral tissues to reduce pre-operative risk. Three-dimensional (3D) bioprinting systems may be a promising approach for fabricating human tissues and organs. Here, we aimed to develop a polycaprolactone (PCL)/alginate bipartite hybrid scaffold using a multihead 3D bioprinting system. The hybrid scaffold was composed of PCL, which could improve the mechanical properties of the construct, and alginate, encapsulating progenitor cells that could differentiate into cartilage and bone. To differentiate the bipartite hybrid scaffold into osteochondral tissue, a polydimethylsiloxane coculture system for osteochondral tissue (PCSOT) was designed and developed. Based on evaluation of the biological performance of the novel hybrid scaffold, the PCL/alginate bipartite scaffold was successfully fabricated; importantly, our findings suggest that this PCSOT system may be applicable as an in vitro platform for osteochondral tissue engineering.

A new experimental model to study human drug responses.

Accurate prediction of pharmacokinetic (PK) and pharmacodynamic (PD) characteristics is critical for drug development. Oral drugs are particularly difficult because they are absorbed by the intestine and metabolized in the liver before systemic metabolism in vivo; this is called the first-pass effect and is a critical factor for predicting oral bioavailability (BA). Here, we fabricated a new networking and circulating cell culture system (NCCS), mimicking the circulatory system and interaction of organs for studying the pharmacokinetic and pharmacodynamics of oral drugs in vitro. NCCS consisted of a micro-pump for circulating fluids, two types of multi-insert culture dishes for culturing different cell types, and an orbital shaker for mixing; flow rate and shaking-speed were controlled by weight-sensors and drivers. A first-pass effect test was performed using functionally differentiated HepaRG and Caco-2 cell lines, using a new modified spheroid forming unit (SFU) protocol. To verify the similarity of PK (first-pass effect) data of NCCS with the data from the human body, 15 reference drugs were chosen and their associated data were obtained by liquid chromatography-mass spectrometry analysis. NCCS generated absorption and metabolism data showed >70% similarity to human data respectively. NCCS can also be used to demonstrate species differences. Animal models are the primary basis for drug discovery, development, and testing. However, the weak correlation between humans and animals, particularly regarding absorption and metabolism, is a substantial limitation for the use of animal models. Here we compare human and mouse acetaminophen (APAP) metabolism using NCCS, and its application can be extended to assess cellular responses, such as efficacy and toxicity, simultaneously.