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Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies. [ABSTRACT FROM AUTHOR]

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)—cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)—cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets. [ABSTRACT FROM AUTHOR]

Human organoid models potentially offer a physiologically relevant platform to replace traditional monolayer cultures and animal models. In particular, the rapid development of engineered strategies including microfluidics, hydrogel, 3D printing and others, which have enormous advantages in comparison to conventional methods, is expected to further advance organoid technology. Up to now, many studies have demonstrated the engineered organoid models with complex cell composition, controlled structure, enhanced maturation, reduced heterogeneity, and so on. These engineered organoids are high promising for studies in development, disease, tissue repair, precision medicine and drug screening. In this review, a comprehensive summary of the engineered organoid model systems is provided based on microfluidics, hydrogel, 3D printing and so on. Then, the biomedical applications of these models are highlighted, which have displayed the great power in organoid field. Finally, the key bottlenecks and future development of organoid models are discussed about from an engineering perspective. [ABSTRACT FROM AUTHOR]

BACKGROUND: In recent years, the development of liver organoids has made it a hot spot in the field of international liver disease research, but there is still no article on the bibliometric analysis of liver organoids. OBJECTIVE: To explore the hot trends in liver organoids in the last 20 years based on bibliometrics and visualization analysis. METHODS: We searched the articles about liver organoids in the Web of Science Core Collection from January 1, 2002 to November 12, 2022. Origin, Office, and CiteSpace software were used for bibliometrics and visualization analysis. We statistically analyzed the number of annually published articles, countries, institutions, authors, journals, and keywords of the articles by generating charts. RESULTS AND CONCLUSION: The number of articles, citation frequency, institutions and personnel involved in the research about liver organoids showed an overall upward trend in the last 20 years, indicating that the field was growing rapidly and attention was increasing. The USA had published the most papers and had the strongest influence in this field. Although it had invested a lot of time and energy, the number of papers published by a single research institution in the USA was not the highest among many research institutions. China was second only to the USA in the number of publications, with the Chinese Academy of Sciences and Fudan University leading the list. Utrecht University in the Netherlands was the institution with the most publications. Clevers H was the author with the highest number of articles. The article with the highest co-citation frequency was “Long-term culture of genome-stable bipotent stem cells from adult human liver”. The main fields of study for liver organoids were Molecular Science, Biology, and Immunology. The most frequently occurring keywords were stem cell, in vitro, and culture. The research hotspots in the liver organoids field were mainly focused on in vitro stem cell three-dimensional culture, differentiation and gene expression. [ABSTRACT FROM AUTHOR]

Background: Mammalian enabled (MENA) protein is a member of the enabled/vasodilator stimulated phosphoprotein (Ena/VASP) protein family, which regulates cytoplasmic actin network assembly. It plays a significant role in breast cancer invasion, migration, and resistance against targeted therapy and chemotherapy. However, its role in the efficacy of endocrine therapy for the hormone receptor-positive (HR+) breast cancer patients is not known. This study investigated the role of MENA in the resistance against tamoxifen therapy in patients with HR+ breast cancer and the underlying mechanisms. Methods: MENA expression levels in the clinical HR+ breast cancer samples (n = 119) were estimated using immunohistochemistry (IHC) to determine its association with the clinicopathological features, tamoxifen resistance, and survival outcomes. Western blotting (WB) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was performed to estimate the MENA protein and mRNA levels in the tamoxifen-sensitive and -resistant HR+ breast cancer cell lines. Furthermore, CCK8, colony formation, and the transwell invasion and migration assays were used to analyze the effects of MENA knockdown on the biological behavior and tamoxifen sensitivity of the HR+ breast cancer cell lines. Xenograft tumor experiments were performed in the nude mice to determine the tumor growth rates and tamoxifen sensitivity of the control and MENA knockdown HR+ breast cancer cells in the presence and absence of tamoxifen treatment. Furthermore, we estimated the growth rates of organoids derived from the HR+ breast cancer patients (n = 10) with high and low MENA expression levels when treated with tamoxifen. Results: HR+ breast cancer patients with low MENA expression demonstrated tamoxifen resistance and poorer prognosis compared to those with high MENA expression. Univariate and multivariate Cox regression analysis demonstrated that MENA expression was an independent predictor of tamoxifen resistance in patients with HR+ breast cancer. MENA knockdown HR+ breast cancer cells showed significantly reduced tamoxifen sensitivity in the in vitro experiments and the in vivo xenograft tumor mouse model compared with the corresponding controls. Furthermore, MENA knockdown increased the in vitro invasion and migration of the HR+ breast cancer cells. HR+ breast cancer organoids with low MENA expression demonstrated reduced tamoxifen sensitivity than those with higher MENA expression. Mechanistically, P-AKT levels were significantly upregulated in the MENA-knockdown HR + breast cancer cells treated with or without 4-OHT compared with the corresponding controls. Conclusions: This study demonstrated that downregulation of MENA promoted tamoxifen resistance in the HR+ breast cancer tissues and cells by enhancing the AKT signaling pathway. Therefore, MENA is a promising prediction biomarker for determining tamoxifen sensitivity in patients with HR+ breast cancer. [ABSTRACT FROM AUTHOR]

In this review, we explore the growing role of artificial intelligence (AI) in advancing the biomedical applications of human pluripotent stem cell (hPSC)-derived organoids. Stem cell-derived organoids, these miniature organ replicas, have become essential tools for disease modeling, drug discovery, and regenerative medicine. However, analyzing the vast and intricate datasets generated from these organoids can be inefficient and error-prone. AI techniques offer a promising solution to efficiently extract insights and make predictions from diverse data types generated from microscopy images, transcriptomics, metabolomics, and proteomics. This review offers a brief overview of organoid characterization and fundamental concepts in AI while focusing on a comprehensive exploration of AI applications in organoid-based disease modeling and drug evaluation. It provides insights into the future possibilities of AI in enhancing the quality control of organoid fabrication, label-free organoid recognition, and three-dimensional image reconstruction of complex organoid structures. This review presents the challenges and potential solutions in AI-organoid integration, focusing on the establishment of reliable AI model decision-making processes and the standardization of organoid research. [ABSTRACT FROM AUTHOR]

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host–pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air–liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae , and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models. [ABSTRACT FROM AUTHOR]

Metabolic diseases have become a major threat to human health worldwide as a result of changing lifestyles. The exploration of the underlying molecular mechanisms of metabolic diseases and the development of improved therapeutic methods have been hindered by the lack of appropriate human experimental models. Organoids are three‐dimensional in vitro models of self‐renewing cells that spontaneously self‐organize into structures similar to the corresponding in vivo tissues, recapitulating the original tissue function. Off‐body organoid technology has been successfully applied to disease modelling, developmental biology, regenerative medicine, and tumour precision medicine. This new generation of biological models has received widespread attention. This article focuses on the construction process and research progress with regard to organoids related to metabolic diseases in recent years, and looks forward to their prospective applications. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is a major cause of gynecological cancer mortality, necessitating enhanced research. Organoids, cellular clusters grown in 3D model, have emerged as a disruptive paradigm, transcending the limitations inherent to conventional models by faithfully recapitulating key morphological, histological, and genetic attributes. This review undertakes a comprehensive exploration of the potential in organoids derived from murine, healthy population, and patient origins, encompassing a spectrum that spans foundational principles to pioneering applications. Organoids serve as preclinical models, allowing us to predict how patients will respond to treatments and guiding the development of personalized therapies. In the context of evaluating new drugs, organoids act as versatile platforms, enabling thorough testing of innovative combinations and novel agents. Remarkably, organoids mimic the dynamic nature of OC progression, from its initial formation to the spread to other parts of the body, shedding light on intricate details that hold significant importance. By functioning at an individualized level, organoids uncover the complex mechanisms behind drug resistance, revealing strategic opportunities for effective treatments. [ABSTRACT FROM AUTHOR]

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Neoadjuvant chemotherapy (NAC) followed by surgery is a standard approach for management of locally advanced esophageal squamous cell carcinoma (ESCC). Patients who do not respond well to NAC have a poor prognosis. Despite extensive research, the mechanisms of chemoresistance in ESCC remain largely unknown. Here, we established paired tumor organoids—designated as PreNAC-O and PostNAC-O—from one ESCC patient before and after NAC, respectively. Although the two organoids did not exhibit significant differences in proliferation, morphology or drug sensitivity in vitro, the tumorigenicity of PostNAC-O in vivo was significantly higher than that of PreNAC-O. Xenografts from PreNAC-O tended to exhibit keratinization, while those from PostNAC-O displayed conspicuous necrotic areas. The tumorigenicity of PostNAC-O xenografts during the chemotherapy was comparable to that of PreNAC-O without treatment. Furthermore, the gene expression profiles of the xenografts suggested that expression of genes involved in the EMT and/or hypoxia response might be related to the tumorigenicity of PostNAC-O. Our data suggested that the tumorigenicity of residual cancer had been enhanced, outweighing the effects of chemotherapy, rather than being attributable to intrinsic chemoresistance. Further studies are required to clarify the extent to which residual cancers share a common mechanism similar to that revealed here. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to evaluate the trends in organoid culture research within the field of regenerative medicine from 2002 to 2022. Methods: The worldwide distribution of organoid research in regenerative medicine articles indexed in the Scopus database was analyzed. Result: A total of 840 documents were analyzed, averaging 42 publications annually. The USA (n = 296) led in publications, followed by China (n = 127), Japan (n = 91) and the UK (n = 75). Since 2011, research has surged, particularly in China, which emerged as a prominent center. Conclusion: The findings highlight significant growth in organoid research, promising future organ transplantation. Research trends integrate tissue engineering, gene modification and induced pluripotent stem cell technologies, reflecting a move toward personalized medicine. [ABSTRACT FROM AUTHOR]

IntroductionThere have been relatively few attempts to quantitatively assess if, and in which areas, the use of non-animal methods (NAMs) is increasing in biomedical research and importantly, how this compares to the use of live animals.MethodsWe conducted a bibliometric analysis of the relative publication of papers reporting the use of NAMs-only compared to those reporting the use of animals, even if they also reported the use of NAMs, over the period 2003 to 2022 across seven research areas (breast cancer, lung disease, blood cancer, heart disease, neurodegenerative diseases, diabetes and toxicology) and five regions (USA, China, France, Germany, United Kingdom).ResultsWe found that the relative number of publications of research using NAMs-only has been higher than animal-based research for the last 20 years for all research areas and is growing. Research areas differed in their relative publication of NAMs-only based work, with breast cancer and lung disease having consistently the highest ratio of NAMs-only to animal-based publications and heart disease, diabetes and toxicology showing the greatest change over the time period. A key period of change was 2016–18. By 2022 the UK had the highest NAMs-only to animal-based research ratio than any other country for five of the seven research areas and China the lowest for six, accounting for publication rate. Tissue and in silico-based methods were the most common of all NAMs-only publications; lab-on-a-chip and stem cell models are increasing in their use but at much lower levels and rate of increase.ConclusionWe found that proportionately the reliance on animals in these research areas is decreasing, which will be encouraging to those that support the replacement of animal experiments.

IntroductionOrganoid models enable three-dimensional representation of cellular systems, providing flexible and accessible research tools, and can highlight key biomolecules. Such models of the intestinal epithelium can provide significant knowledge for the study of celiac disease and provide an additional context for the nature of markers observed from patient biopsy data.MethodsUsing LC–MS/MS, the proteomes of the crypt and enterocyte-like states of a mouse mini-gut organoid model were measured. The data were further compared with published biopsy data by comparing the changes induced by gluten challenge after a gluten-free diet.Results and discussionThese analyses identified 4,850 protein groups and revealed how 400 putative biomarkers of dietary challenge were differentially expressed in the organoid model. In addition to the extensive changes within the differentiated cells, the data reiterated the disruption of the crypt–villus axis after gluten challenge. The mass spectrometry data are available via ProteomeXchange with the identifier PXD025690.

Amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, is characterized by progressive motor neuron degeneration, leading to widespread weakness and respiratory failure. While a variety of mechanisms have been proposed as causes of this disease, a full understanding remains elusive. Electrophysiological alterations, including increased motor axon excitability, likely play an important role in disease progression. There remains a critical need for non-animal disease models that can integrate electrophysiological tools to better understand underlying mechanisms, track disease progression, and evaluate potential therapeutic interventions. This review explores the integration of electrophysiological technologies with ALS disease models. It covers cellular and clinical electrophysiological tools and their applications in ALS research. Additionally, we examine conventional animal models and highlight advancements in humanized models and 3D organoid technologies. By bridging the gap between these models, we aim to enhance our understanding of ALS pathogenesis and facilitate the development of new therapeutic strategies.

Summary: Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic “livers in a dish.” We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Introduction: Endocrine disruptors are compounds of manmade origin able to interfere with the endocrine system and constitute an important environmental concern. Indeed, detrimental effects on thyroid physiology and functioning have been described. Differences exist in the susceptibility of human sexes to the incidence of thyroid disorders, like autoimmune diseases or cancer. Methods: To study how different hormonal environments impact the thyroid response to endocrine disruptors, we exposed human embryonic stem cell-derived thyroid organoids to physiological concentrations of sex hormones resembling the serum levels of human females post-ovulation or males of reproductive age for three days. Afterwards, we added 10 µM benzo[a]pyrene or PCB153 for 24 h and analyzed the transcriptome changes via single-cell RNA sequencing with differential gene expression and gene ontology analysis. Results: The sex hormones receptors genes AR, ESR1, ESR2 and PGR were expressed at low levels. Among the thyroid markers, only TG resulted downregulated by benzo[a]pyrene or benzo[a]pyrene with the “male” hormones mix. Both hormone mixtures and benzo[a]pyrene alone upregulated ribosomal genes and genes involved in oxidative phosphorylation, while their combination decreased the expression compared to benzo[a]pyrene alone. The “male” mix and benzo[a]pyrene, alone or in combination, upregulated genes involved in lipid transport and metabolism (APOA1, APOC3, APOA4, FABP1, FABP2, FABP6). The combination of “male” hormones and benzo[a]pyrene induced also genes involved in inflammation and NFkB targets. Benzo[a]pyrene upregulated CYP1A1, CYP1B1 and NQO1 irrespective of the hormonal context. The induction was stronger in the “female” mix. Benzo[a]pyrene alone upregulated genes involved in cell cycle regulation, response to reactive oxygen species and apoptosis. PCB153 had a modest effect in presence of “male” hormones, while we did not observe any changes with the “female” mix. Conclusion: This work shows how single cell transcriptomics can be applied to selectively study the in vitro effects of endocrine disrupters and their interaction with different hormonal contexts.

The endometrium undergoes a series of precise monthly changes under the regulation of dynamic levels of ovarian hormones that are characterized by repeated shedding and subsequent regeneration without scarring. This provides the potential for wound healing during endometrial injuries. Bioengineering materials highlight the faithful replication of constitutive cells and the extracellular matrix that simulates the physical and biomechanical properties of the endometrium to a larger extent. Significant progress has been made in this field, and functional endometrial tissue bioengineering allows an in-depth investigation of regulatory factors for endometrial and myometrial defects in vitro and provides highly therapeutic methods to alleviate obstetric and gynecological complications. However, much remains to be learned about the latest progress in the application of bioengineering technologies to the human endometrium. Here, we summarize the existing developments in biomaterials and bioengineering models for endometrial regeneration and improving the female reproductive potential.

Mitochondrial diseases are a group of severe pathologies that cause complex neurodegenerative disorders for which, in most cases, no therapy or treatment is available. These organelles are critical regulators of both neurogenesis and homeostasis of the neurological system. Consequently, mitochondrial damage or dysfunction can occur as a cause or consequence of neurodevelopmental or neurodegenerative diseases. As genetic knowledge of neurodevelopmental disorders advances, associations have been identified between genes that encode mitochondrial proteins and neurological symptoms, such as neuropathy, encephalomyopathy, ataxia, seizures, and developmental delays, among others. Understanding how mitochondrial dysfunction can alter these processes is essential in researching rare diseases. Three-dimensional (3D) cell cultures, which self-assemble to form specialized structures composed of different cell types, represent an accessible manner to model organogenesis and neurodevelopmental disorders. In particular, brain organoids are revolutionizing the study of mitochondrial-based neurological diseases since they are organ-specific and model-generated from a patient’s cell, thereby overcoming some of the limitations of traditional animal and cell models. In this review, we have collected which neurological structures and functions recapitulate in the different types of reported brain organoids, focusing on those generated as models of mitochondrial diseases. In addition to advancements in the generation of brain organoids, techniques, and approaches for studying neuronal structures and physiology, drug screening and drug repositioning studies performed in brain organoids with mitochondrial damage and neurodevelopmental disorders have also been reviewed. This scope review will summarize the evidence on limitations in studying the function and dynamics of mitochondria in brain organoids.

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated β-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.

Background & Aims: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is up-regulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown. Methods: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air–liquid interface cultures stimulated with interleukin (IL)13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse-transcription polymerase chain reaction, Western blot, histology, and functional analyses of barrier integrity. Results: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL13 in differentiated cells. LOX-overexpressing organoids showed suppressed basal and up-regulated differentiation markers. In addition, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified an enriched bone morphogenetic protein (BMP) signaling pathway compared with wild-type organoids. In particular, LOX overexpression increased BMP2 and decreased the BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells. Conclusions: Our data support a model whereby LOX exhibits noncanonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of the BMP pathway in the esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies.

Abstract Gastrointestinal (GI) cancers (gastric cancer, oesophageal cancer, liver cancer, colorectal cancer, etc.) are the most common cancers with the highest morbidity and mortality in the world. The therapy for most GI cancers is difficult and is associated with a poor prognosis. In China, upper GI cancers, mainly gastric cancer (GC) and oesophageal cancer (EC), are very common due to Chinese people’s characteristics, and more than half of patients are diagnosed with distant metastatic or locally advanced disease. Compared to other solid cancers, such as lung cancer and breast cancer, personalized therapies, especially targeted therapy and immunotherapy, in GC and EC are relatively lacking, leading to poor prognosis. For a long time, most studies were carried out by using in vitro cancer cell lines or in vivo cell line-derived xenograft models, which are unable to reproduce the characteristics of tumours derived from patients, leading to the possible misguidance of subsequent clinical validation. The patient-derived models represented by patient-derived organoid (PDO) and xenograft (PDX) models, known for their high preservation of patient tumour features, have emerged as a very popular platform that has been widely used in numerous studies, especially in the research and development of antitumour drugs and personalized medicine. Herein, based on some of the available published literature, we review the research and application status of PDO and PDX models in GC and EC, as well as detail their future challenges and prospects, to promote their use in basic and translational studies or personalized therapy.

Cardiovascular disease comprises a group of disorders affecting or originating within tissues and organs of the cardiovascular system; most, if not all, will eventually result in cardiomyocyte dysfunction or death, negatively impacting cardiac function. Effective models of cardiac disease are thus important for understanding crucial aspects of disease progression, while recent advancements in stem cell biology have allowed for the use of stem cell populations to derive such models. These include three-dimensional (3D) models such as stem cell-based models of embryos (SCME) as well as organoids, many of which are frequently derived from embryoid bodies (EB). Not only can they recapitulate 3D form and function, but the developmental programs governing the self-organization of cell populations into more complex tissues as well. Many different organoids and SCME constructs have been generated in recent years to recreate cardiac tissue and the complex developmental programs that give rise to its cellular composition and unique tissue morphology. It is thus the purpose of this narrative literature review to describe and summarize many of the recently derived cardiac organoid models as well as their use for the recapitulation of genetic and acquired disease. Owing to the cellular composition of the models examined, this review will focus on disease and tissue injury associated with embryonic/fetal tissues.

Human noroviruses (HuNoVs) are the main pathogens that cause acute gastroenteritis and lead to huge economic losses annually. Due to the lack of suitable culture systems, the pathogenesis of HuNoVs and the development of vaccines and drugs have progressed slowly. Although researchers have employed various methods to culture HuNoVs in vitro in the last century, problems relating to the irreducibility, low viral titer, and non-infectiousness of the progeny virus should not be ignored. In 2016, researchers achieved the cultivation and successive passaging of some HuNoV genotypes using human intestinal enteroids, initially demonstrating the potential use of organoids in overcoming this challenge. This paper reviews the efforts made in the last 20 years to culture HuNoVs in vitro and discusses the superiority and limitations of employing human intestinal enteroids/organoids as an in vitro culture model for HuNoVs.

Complex transcriptional gene regulation allows for multifaceted isoform production during retinogenesis, and novel isoforms transcribed from a single locus can have unlimited potential to code for diverse proteins with different functions. In this study, we explored the CTBP2/RIBEYE gene locus and its unique repertoire of transcripts that are conserved among vertebrates. We studied the transcriptional coregulator (CTBP2) and ribbon synapse-specific structural protein (RIBEYE) in the chicken retina by performing comprehensive histochemical and sequencing analyses to pinpoint cell and developmental stage-specific expression of CTBP2/RIBEYE in the developing chicken retina. We demonstrated that CTBP2 is widely expressed in retinal progenitors beginning in early retinogenesis but becomes limited to GABAergic amacrine cells in the mature retina. Inversely, RIBEYE is initially epigenetically silenced in progenitors and later expressed in photoreceptor and bipolar cells where they localize to ribbon synapses. Finally, we compared CTBP2/RIBEYE regulation in the developing human retina using a pluripotent stem cell derived retinal organoid culture system. These analyses demonstrate that similar regulation of the CTBP2/RIBEYE locus during chick and human retinal development is regulated by different members of the K50 homeodomain transcription factor family.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

Abstract Ruminants play a vital economic role as livestock, providing high-quality protein for humans. At present, 3D-cultured ruminant abomasum and intestinal organoids have been successfully established to study host and pathogen interaction. The rumen is a unique digestive organ of ruminants that occupies 70% of the volume of the digestive tract and its microbiota can decompose lignocellulose to support animal growth. Here we report a method for culturing rumen epithelial organoids. We found that single rumen epithelial cells form self-organized 3D structures representative of typical stratified squamous epithelium, which is similar to rumen epithelium. EGF, Noggin, Wnt3a, IGF-1, and FGF-10 significantly enhanced the seeding efficiency of organoids. Moreover, the inclusion of CHIR-99021, A83-01, SB202190, and Y-27632 is crucial for organoid formation and maintenance. Importantly, we demonstrate that rumen epithelial cells retain their ability to form organoids after passage, cryopreservation, and resuscitation. The rumen epithelial organoids express rumen cell type-specific genes, uptake fatty acids, and generate 2D cultures. In summary, our data demonstrate that it is feasible to establish organoids from single rumen epithelial cells, which is a novel in vitro system that may reduce the use of experimental animals.

Abstract Background Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and receive effective chemotherapy have the best chance for long‐term survival. Unfortunately, because of the heterogeneity of pancreatic cancer, it is difficult to find a personalized treatment strategy for patients. Organoids are ideal preclinical models for personalized medicine. Therefore, we explore the cultivation conditions and construction methods of PDAC organoid models to screen the individualized therapy strategy. Methods Fresh PDAC tissues from surgical resection were collected and digested with digestive enzymes; then the tumor cells were embedded in Matrigel with a suitable medium to establish the PDAC organoid models. The genetic stability of the organoids was analyzed using whole exon sequencing; hematoxylin and eosin staining and immunohistochemistry of organoids were performed to analyze their consistency with the pathological morphology of the patient's tumor tissue; After 2 days of organoid culture, we selected four commonly used clinical chemotherapy drugs for single or combined treatment to analyze drug sensitivity. Results Two cases of PDAC organoid models were successfully established, and the results of their pathological characteristics and exome sequencing were consistent with those of the patient's tumor tissue. Both PDAC organoids showed more sensitivity to gemcitabine and cisplatin, and the combined treatment was more effective than monotherapy. Conclusion Both organoids better retained the pathological characteristics, genomic stability, and heterogeneity with the original tumor. Individual PDAC organoids exhibited different sensitivities to the same drugs. Thus, this study provided ideal experimental models for screening individualized therapy strategy for patients with PDAC.

Abstract Background Harnessing hepatocytes for basic research and regenerative medicine demands a complete understanding of the genetic determinants underlying hepatocyte differentiation and maturation. Single-cell CRISPR screens in organoids could link genetic perturbations with parallel transcriptomic readout in single cells, providing a powerful method to delineate roles of cell fate regulators. However, a big challenge for identifying key regulators during data analysis is the low expression levels of transcription factors (TFs), which are difficult to accurately estimate due to noise and dropouts in single-cell sequencing. Also, it is often the changes in TF activities in the transcriptional cascade rather than the expression levels of TFs that are relevant to the cell fate transition. Results Here, we develop Organoid-based Single-cell CRISPR screening Analyzed with Regulons (OSCAR), a framework using regulon activities as readouts to dissect gene knockout effects in organoids. In adult-stem-cell-derived liver organoids, we map transcriptomes in 80,576 cells upon 246 perturbations associated with transcriptional regulation of hepatocyte formation. Using OSCAR, we identify known and novel positive and negative regulators, among which Fos and Ubr5 are the top-ranked ones. Further single-gene loss-of-function assays demonstrate that Fos depletion in mouse and human liver organoids promote hepatocyte differentiation by specific upregulation of liver metabolic genes and pathways, and conditional knockout of Ubr5 in mouse liver delays hepatocyte maturation. Conclusions Altogether, we provide a framework to explore lineage specifiers in a rapid and systematic manner, and identify hepatocyte determinators with potential clinical applications.

Abstract Immunotherapy is garnering increasing attention as a therapeutic strategy for breast cancer (BC); however, the application of precise immunotherapy in BC has not been fully studied. Further studies on BC immunotherapy have a growing demand for preclinical models that reliably recapitulate the composition and function of the tumor microenvironment (TME) of BC. However, the classic two-dimensional in vitro and animal in vivo models inadequately recapitulate the intricate TME of the original tumor. Organoid models which allow the regular culture of primitive human tumor tissue are increasingly reported that they can incorporate immune components. Therefore, organoid platforms can be used to replicate the BC–TME to achieve the immunotherapeutic reaction modeling and facilitate relevant preclinical trial. In this study, we have investigated different organoid culture methods for BC–TME modeling and their applications for precision immunotherapy in BC.

Abstract Background Dysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder. Methods Adult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs. Results AdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs. Conclusions We demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

Laboratory animals are the foundational conditions and indispensable technical support in life science research and biomedical industry development. The scientific development of animal models of diseases is of great significance to biomedical research and industrial development. In light of the booming development of multiple emerging in vitro modelling technologies over the past decade, in 2022, the U.S. Senate unanimously passed the bill FDA Modernization Act 2.0. This bill rescinded the requirement for animal testing in investigating the safety and effectiveness of a drug—a federal mandate since 1938, and highlighted the potential of various in vitro disease modeling approaches in future biomedical fields. This paper provides a comprehensive review of the latest advances and applications of in vitro disease modeling approaches in academia and industry followed by an interpretation of the FDA bill, namely cell culture, organoid, organ-on-a-chip, 3D bio-printing model and computer-based model. The paper next introduces the crossed applications of various disease models and discusses the advantages and disadvantages of each system, thereby providing insights into future trends in the use of animal disease models in China.

Sensory hair cells are responsible for detecting and transmitting sound in the inner ear, and damage to HCs leads to hearing loss. HCs do not regenerate spontaneously in adult mammals, which makes the hearing loss permanent. However, hair cells and supporting cells have the same precursors in the inner ear, and in newborn mice, the adjacent SCs can be activated by gene manipulation to differentiate into newly regenerated hair cells. Here, we demonstrate the role of the Ras association domain family member 2 (Rassf2) in supporting cell to hair cell trans-differentiation in the inner ear. Using the AAV vector (AAV-ie) to upregulate Rassf2 expression promoted supporting cell division and hair cell production in cultured cochlear organoids. Also, AAV-Rassf2 enhanced the regenerative ability of Lgr5+ SCs in the postnatal cochlea without impairing hearing, and this might due to the modulation of the Wnt, Hedgehog and Notch signaling pathways. Furthermore, AAV-Rassf2 enhances cochlear supporting cell division and hair cell production in the neomycin injury model. In summary, our results suggest that Rassf2 is a key component in HC regenerative repair, and gene modulation mediated by adeno-associated virus may be a promising gene therapy for hearing repair.

The human mammary gland is the major organ involved in lactation. In the mammary gland, alveoli secrete milk and myoepithelial cells contract to propel the milk through branched structures called ducts and eventually to the nipple. It is through this process of lactation that infants receive milk, which is essential for proper infant growth and development. The lactation process is comprised of sophisticated interactive networks at the cellular level that are not well understood. Whereas the majority of published mammary gland lactation studies have relied on mouse mammary glands, recent advancements in techniques to study mammary glands enable in vitro reproduction of lactation using human-representative frameworks. Currently, the 3D breast organoid is the state-of-the-art model in human mammary gland research, utilizing induced pluripotent stem cells (iPSCs) or processed patient-derived breast tissues embedded in a special matrix that are then able to grow into complex structures that recapitulate aspects of native human breast tissue. Gaining comprehensive biological insight into the process of lactation through these breast tissue-mimetic 3D models is essential for further studies on lactation-associated human mammary gland diseases, human milk composition, and potential solutions to challenges in maternal milk accessibility. In this short review, the benefits and potential utility of 3D breast organoids in understanding the underlying science of lactation and advancing further human mammary gland studies are discussed.

Abstract Background Systemic therapy is the primary treatment for advanced thymic malignancies. However, there is an urgent need to improve clinical outcome. Personalized treatment based on predictive biomarkers is a potential approach to address this requirement. In this study, we aimed to show the correlation between drug sensitivity tests on CTCs‐derived organoids and clinical response in patients with thymic malignancies. This approach carries the potential to create personalized cancer avatars and improve treatment outcome for patients. Methods We previously reported potential treatment outcome prediction with patient‐derived organoids (cancer avatars) in patients with pancreatic ductal adenocarcinoma. To further investigate the feasibility of this approach in advanced thymic malignancies, we conducted a study in which 12 patients were enrolled and 21 liquid biopsies were performed. Results Cancer avatars were successfully derived in 16 out of 21 samples (success rate 76.2%). We found a sensitivity of 1.0 and specificity of 0.6 for drug sensitivity tests on the cancer avatars, and a two‐tailed Fisher's exact test revealed a significant correlation between drug sensitivity tests and clinical responses (p = 0.0275). Conclusion This study supports the potential of circulating tumor cell‐derived organoids to inform personalized treatment for advanced thymic malignancies. Further validation of this proof of concept finding is ongoing.

The regeneration of hierarchical osteochondral units is challenging due to difficulties in inducing spatial, directional and controllable differentiation of mesenchymal stem cells (MSCs) into cartilage and bone compartments. Emerging organoid technology offers new opportunities for osteochondral regeneration. In this study, we developed gelatin-based microcryogels customized using hyaluronic acid (HA) and hydroxyapatite (HYP), respectively for inducing cartilage and bone regeneration (denoted as CH-Microcryogels and OS-Microcryogels) through in vivo self-assembly into osteochondral organoids. The customized microcryogels showed good cytocompatibility and induced chondrogenic and osteogenic differentiation of MSCs, while also demonstrating the ability to self-assemble into osteochondral organoids with no delamination in the biphasic cartilage-bone structure. Analysis by mRNA-seq showed that CH-Microcryogels promoted chondrogenic differentiation and inhibited inflammation, while OS-Microcryogels facilitated osteogenic differentiation and suppressed the immune response, by regulating specific signaling pathways. Finally, the in vivo engraftment of pre-differentiated customized microcryogels into canine osteochondral defects resulted in the spontaneous assembly of an osteochondral unit, inducing simultaneous regeneration of both articular cartilage and subchondral bone. In conclusion, this novel approach for generating self-assembling osteochondral organoids utilizing tailor-made microcryogels presents a highly promising avenue for advancing the field of tissue engineering.

Research on bidirectional communication between the heart and brain has often relied on studies involving nonhuman animals. Dependance on animal models offer limited applicability to humans and a lack of high-throughput screening. Recently, the field of 3D cell biology, specifically organoid technology, has rapidly emerged as a valuable tool for studying interactions across organ systems, i.e., gut-brain axis. The initial success of organoid models indicates the usefulness of 3D cultures for elucidating the intricate interactivity of the autonomic nervous system and overall health. This perspective aims to explore the potential of advancing in vitro modeling of the heart-brain axis by discussing the benefits, applications, and adaptability of organoid technologies. We closely examine the current state of brain organoids in conjunction with the advancements of cardiac organoids. Moreover, we explore the use of combined organoid systems to investigate pathophysiology and provide a platform for treatment discovery. Finally, we address the challenges that accompany the use of 3D models for studying the heart-brain axis with an emphasis on generating tailored engineering strategies for further refinement of dynamic organ system modeling in vitro.

The significant correlation between particulate matter with aerodynamic diameters of ≤ 2.5 µm (PM2.5) and the high morbidity and mortality of respiratory diseases has become the consensus of the research. Epidemiological studies have clearly pointed out that there is no safe concentration of PM2.5, and mechanism studies have also shown that exposure to PM2.5 will first cause pulmonary inflammation. Therefore, the purpose of this study is to explore the mechanism of early lung injury induced by low-level PM2.5 from the perspective of epigenetics. Based on the previous results of population samples, combined with an in vitro/vivo exposure model of PM2.5, it was found that low-level PM2.5 promoted the transport of circ_0092363 from intracellular to extracellular spaces.The decreased expression of intracellular circ_0092363 resulted in reduced absorption of miR-31-5p, leading to inhibition of Rho associated coiled-coil containing protein kinase 1 (ROCK1) and the subsequent abnormal expression of tight junction proteins such as Zonula occludens protein 1 (ZO-1) and Claudin-1, ultimately inducing the occurrence of early pulmonary injury. Furthermore, this study innovatively introduced organoid technology and conducted a preliminary exploration for a study of the relationship among environmental exposure genomics, epigenetics and disease genomics in organoids. The role of circ_0092363 in early pulmonary injury induced by low-level PM2.5 was elucidated, and its value as a potential diagnostic biomarker was confirmed.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or “mini-guts” has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.

Developing in vitro models that accurately mimic the microenvironment of biological structures or processes holds substantial promise for gaining insights into specific biological functions. In the field of tissue engineering and regenerative medicine, in vitro models able to capture the precise structural, topographical, and functional complexity of living tissues, prove to be valuable tools for comprehending disease mechanisms, assessing drug responses, and serving as alternatives or complements to animal testing. The choice of the right biomaterial and fabrication technique for the development of these in vitro models plays an important role in their functionality. In this sense, elastin-like recombinamers (ELRs) have emerged as an important tool for the fabrication of in vitro models overcoming the challenges encountered in natural and synthetic materials due to their intrinsic properties, such as phase transition behavior, tunable biological properties, viscoelasticity, and easy processability. In this review article, we will delve into the use of ELRs for molecular models of intrinsically disordered proteins (IDPs), as well as for the development of in vitro 3D models for regenerative medicine. The easy processability of the ELRs and their rational design has allowed their use for the development of spheroids and organoids, or bioinks for 3D bioprinting. Thus, incorporating ELRs into the toolkit of biomaterials used for the fabrication of in vitro models, represents a transformative step forward in improving the accuracy, efficiency, and functionality of these models, and opening up a wide range of possibilities in combination with advanced biofabrication techniques that remains to be explored.

ABSTRACTCongenital hearing loss is a common chronic condition affecting children in both developed and developing nations. Viruses correlated with congenital hearing loss include human cytomegalovirus (HCMV) and Zika virus (ZIKV), which causes congenital Zika syndrome. The mechanisms by which HCMV and ZIKV infections cause hearing loss are poorly understood. It is challenging to study human inner ear cells because they are encased in bone and also scarce as autopsy samples. Recent advances in culturing human stem cell-derived otic progenitor cells (OPCs) have allowed us herein to describe successful in vitro infection of OPCs with HCMV and ZIKV, and also to propose potential mechanisms by which each viral infection could affect hearing. We find that ZIKV infection rapidly and significantly induces the expression of type I interferon and interferon-stimulated genes, while OPC viability declines, at least in part, from apoptosis. In contrast, HCMV infection did not appear to upregulate interferons or cause a reduction in cell viability, and instead disrupted expression of key genes and pathways associated with inner ear development and function, including Cochlin, nerve growth factor receptor, SRY-box transcription factor 11, and transforming growth factor-beta signaling. These findings suggest that ZIKV and HCMV infections cause congenital hearing loss through distinct pathways, that is, by inducing progenitor cell death in the case of ZIKV infection, and by disruption of critical developmental pathways in the case of HCMV infection.IMPORTANCECongenital virus infections inflict substantial morbidity and devastating disease in neonates worldwide, and hearing loss is a common outcome. It has been difficult to study viral infections of the human hearing apparatus because it is embedded in the temporal bone of the skull. Recent technological advances permit the differentiation of otic progenitor cells (OPCs) from human-induced pluripotent stem cells. This paper is important for demonstrating that inner ear virus infections can be modeled in vitro using OPCs. We infected OPCs with two viruses associated with congenital hearing loss: human cytomegalovirus (HCMV), a DNA virus, or Zika virus (ZIKV), an RNA virus. An important result is that the gene expression and cytokine production profiles of HCMV/ZIKV-infected OPCs are markedly dissimilar, suggesting that mechanisms of hearing loss are also distinct. The specific molecular regulatory pathways identified in this work could suggest important targets for therapeutics.

High Throughput Screening (HTS) with 3D cell models is possible thanks to the recent progress and development in 3D cell culture technologies. Results from multiple studies have demonstrated different drug responses between 2D and 3D cell culture. It is now widely accepted that 3D cell models more accurately represent the physiologic conditions of tumors over 2D cell models. However, there is still a need for more accurate tests that are scalable and better imitate the complex conditions in living tissues. Here, we describe ultrahigh throughput 3D methods of drug response profiling in patient derived primary tumors including melanoma as well as renal cell carcinoma that were tested against the NCI oncologic set of FDA approved drugs. We also tested their autologous patient derived cancer associated fibroblasts, varied the in-vitro conditions using matrix vs matrix free methods and completed this in both 3D vs 2D rendered cancer cells. The result indicates a heterologous response to the drugs based on their genetic background, but not on their maintenance condition. Here, we present the methods and supporting results of the HTS efforts using these 3D of organoids derived from patients. This demonstrated the possibility of using patient derived 3D cells for HTS and expands on our screening capabilities for testing other types of cancer using clinically approved anti-cancer agents to find drugs for potential off label use.

Gallbladder cancer incidence has been increasing globally, and it remains challenging to expect long prognosis with the current systemic chemotherapy. We identified a novel nucleic acid-mediated therapeutic target against gallbladder cancer by using innovative organoid-based gallbladder cancer models generated from KrasLSL-G12D/+; Trp53f/f mice. Using comprehensive microRNA expression analyses and a bioinformatics approach, we identified significant microRNA-34a-5p downregulation in both murine gallbladder cancer organoids and resected human gallbladder cancer specimens. In three different human gallbladder cancer cell lines, forced microRNA-34a-5p expression inhibited cell proliferation and induced cell-cycle arrest at the G1 phase by suppressing direct target (CDK6) expression. Furthermore, comprehensive RNA sequencing revealed the significant enrichment of gene sets related to the cell-cycle regulators after microRNA-34a-5p expression in gallbladder cancer cells. In a murine xenograft model, locally injected microRNA-34a-5p mimics significantly inhibited gallbladder cancer progression and downregulated CDK6 expression. These results provide a rationale for promising therapeutics against gallbladder cancer by microRNA-34a-5p injection, as well as a strategy to explore therapeutic targets against cancers using organoid-based models, especially for those lacking useful genetically engineered murine models, such as gallbladder cancer.

Induced pluripotent stem cell (iPSC)-derived cardiac organoids offer a versatile platform for personalized cardiac toxicity assessment, drug screening, disease modeling, and regenerative therapies. While previous image-based contractility analysis techniques allowed the assessment of contractility of two-dimensional cardiac models, they face limitations, including encountering high noise levels when applied to three-dimensional organoid models and requiring expensive equipment. Additionally, they offer fewer functional parameters compared to commercial software. To address these challenges, we developed an open-source, particle image velocimetry-based software (PIV-MyoMonitor) and demonstrated its capacity for accurate contractility analysis in both two- and three-dimensional cardiac models using standard lab equipment. Comparisons with four other open-source software programs highlighted the capability of PIV-MyoMonitor for more comprehensive quantitative analysis, providing 22 functional parameters and enhanced video outputs. We showcased its applicability in drug screening by characterizing the response of cardiac organoids to a known isotropic drug, isoprenaline. In sum, PIV-MyoMonitor enables reliable contractility assessment across various cardiac models without costly equipment or software. We believe this software will benefit a broader scientific community.

Background: The intra‐ and inter‐tumoral heterogeneity of gliomas and the complex tumor microenvironment make accurate treatment of gliomas challenging. At present, research on gliomas mainly relies on cell lines, stem cell tumor spheres, and xenotransplantation models. The similarity between traditional tumor models and patients with glioma is very low. Aims: In this study, we aimed to address the limitations of traditional tumor models by generating patient‐derived glioma organoids using two methods that summarized the cell diversity, histological features, gene expression, and mutant profiles of their respective parent tumors and assess the feasibility of organoids for personalized treatment. Materials and Methods: We compared the organoids generated using two methods through growth analysis, immunohistological analysis, genetic testing, and the establishment of xenograft models. Results: Both types of organoids exhibited rapid infiltration when transplanted into the brains of adult immunodeficient mice. However, organoids formed using the microtumor method demonstrated more similar cellular characteristics and tissue structures to the parent tumors. Furthermore, the microtumor method allowed for faster culture times and more convenient operational procedures compared to the Matrigel method. Discussion: Patient‐derived glioma organoids, especially those generated through the microtumor method, present a promising avenue for personalized treatment strategies. Their capacity to faithfully mimic the cellular and molecular characteristics of gliomas provides a valuable platform for elucidating tumor biology and evaluating therapeutic modalities. Conclusion: The success rates of the Matrigel and microtumor methods were 45.5% and 60.5%, respectively. The microtumor method had a higher success rate, shorter establishment time, more convenient passage and cryopreservation methods, better simulation of the cellular and histological characteristics of the parent tumor, and a high genetic guarantee. [ABSTRACT FROM AUTHOR]