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BACKGROUND: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. METHODS: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC). RESULTS: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. CONCLUSION: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.

Temporal development of neural electrophysiology follows genetic programming, similar to cellular maturation and organization during development. The emergent properties of this electrophysiological development, namely neural oscillations, can be used to characterize brain development. Recently, we utilized the innate programming encoded in the human genome to generate functionally mature cortical organoids. In brief, stem cells are suspended in culture via continuous shaking and naturally aggregate into embryoid bodies before being exposed to media formulations for neural induction, differentiation and maturation. The specific culture format, media composition and duration of exposure to these media distinguish organoid protocols and determine whether a protocol is guided or unguided toward specific neural fate. The 'semi-guided' protocol presented here has shorter induction and differentiation steps with less-specific patterning molecules than most guided protocols but maintains the use of neurotrophic factors such as brain-derived growth factor and neurotrophin-3, unlike unguided approaches. This approach yields the cell type diversity of unguided approaches while maintaining reproducibility for disease modeling. Importantly, we characterized the electrophysiology of these organoids and found that they recapitulate the maturation of neural oscillations observed in the developing human brain, a feature not shown with other approaches. This protocol represents the potential first steps toward bridging molecular and cellular biology to human cognition, and it has already been used to discover underlying features of human brain development, evolution and neurological conditions. Experienced cell culture technicians can expect the protocol to take 1 month, with extended maturation, electrophysiology recording, and adeno-associated virus transduction procedure options.

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.

In an earlier essay, I advocated that translational bioethics uses the public's values, determined through social science, in its analysis of translational science technologies. It may be unclear what those values might be, and whether such a translational ethics would necessarily conclude that cutting edge technologies should not be developed. In this essay, I show the public's values relevant to human brain organoids and argue that a translational bioethics analysis using these values would support continued organoid research.

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.

Cell atlases serve as vital references for automating cell labeling in new samples, yet existing classification algorithms struggle with accuracy. Here we introduce SIMS (scalable, interpretable machine learning for single cell), a low-code data-efficient pipeline for single-cell RNA classification. We benchmark SIMS against datasets from different tissues and species. We demonstrate SIMS's efficacy in classifying cells in the brain, achieving high accuracy even with small training sets (

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by neurofibromatosis type 1 syndrome. cNFs are benign lesions: they do not undergo malignant transformation or metastasize. Nevertheless, they can cover a significant proportion of the body, with some individuals developing hundreds to thousands of lesions. cNFs can cause pain, itching, and disfigurement resulting in substantial socio-emotional repercussions. Currently, surgery and laser desiccation are the sole treatment options but may result in scarring and potential regrowth from incomplete removal. To identify effective systemic therapies, we introduce an approach to establish and screen cNF organoids. We optimized conditions to support the ex vivo growth of genomically diverse cNFs. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors as measured by immunohistopathology, methylation, RNA sequencing, and flow cytometry. Our cNF organoid platform enables rapid screening of hundreds of compounds in a patient- and tumor-specific manner.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Three Prime Repair Exonuclease 1 (TREX1) gene mutations have been associated with Aicardi-Goutières Syndrome (AGS) - a rare, severe pediatric autoimmune disorder that primarily affects the brain and has a poorly understood etiology. Microglia are brain-resident macrophages indispensable for brain development and implicated in multiple neuroinflammatory diseases. However, the role of TREX1 - a DNase that cleaves cytosolic nucleic acids, preventing viral- and autoimmune-related inflammatory responses - in microglia biology remains to be elucidated. Here, we leverage a model of human embryonic stem cell (hESC)-derived engineered microglia-like cells, bulk, and single-cell transcriptomics, optical and transmission electron microscopy, and three-month-old assembloids composed of microglia and oligodendrocyte-containing organoids to interrogate TREX1 functions in human microglia. Our analyses suggest that TREX1 influences cholesterol metabolism, leading to an active microglial morphology with increased phagocytosis in the absence of TREX1. Notably, regulating cholesterol metabolism with an HMG-CoA reductase inhibitor, FDA-approved atorvastatin, rescues these microglial phenotypes. Functionally, TREX1 in microglia is necessary for the transition from gliogenic intermediate progenitors known as pre-oligodendrocyte precursor cells (pre-OPCs) to precursors of the oligodendrocyte lineage known as OPCs, impairing oligodendrogenesis in favor of astrogliogenesis in human assembloids. Together, these results suggest routes for therapeutic intervention in pathologies such as AGS based on microglia-specific molecular and cellular mechanisms.

The use of patient-derived organoids (PDOs) to characterize therapeutic sensitivity and resistance is a promising precision medicine approach, and its potential to inform clinical decisions is now being tested in several large multiinstitutional clinical trials. PDOs are cultivated in the extracellular matrix from basement membrane extracts (BMEs) that are most commonly acquired commercially. Each clinical site utilizes distinct BME lots and may be restricted due to the availability of commercial BME sources. However, the effect of different sources of BMEs on organoid drug response is unknown. Here, we tested the effect of BME source on proliferation, drug response, and gene expression in mouse and human pancreatic ductal adenocarcinoma (PDA) organoids. Both human and mouse organoids displayed increased proliferation in Matrigel compared with Cultrex and UltiMatrix. However, we observed no substantial effect on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources, and PDOs maintained their classical or basal-like designation. Overall, we found that the BME source (Matrigel, Cultrex, UltiMatrix) does not shift PDO dose-response curves or drug testing results, indicating that PDO pharmacotyping is a robust approach for precision medicine.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organs epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

BackgroundSocial affective and communication symptoms are central to autism spectrum disorder (ASD), yet their severity differs across toddlers: Some toddlers with ASD display improving abilities across early ages and develop good social and language skills, while others with "profound" autism have persistently low social, language and cognitive skills and require lifelong care. The biological origins of these opposite ASD social severity subtypes and developmental trajectories are not known.MethodsBecause ASD involves early brain overgrowth and excess neurons, we measured size and growth in 4910 embryonic-stage brain cortical organoids (BCOs) from a total of 10 toddlers with ASD and 6 controls (averaging 196 individual BCOs measured/subject). In a 2021 batch, we measured BCOs from 10 ASD and 5 controls. In a 2022 batch, we  tested replicability of BCO size and growth effects by generating and measuring an independent batch of BCOs from 6 ASD and 4 control subjects. BCO size was analyzed within the context of our large, one-of-a-kind social symptom, social attention, social brain and social and language psychometric normative datasets ranging from N = 266 to N = 1902 toddlers. BCO growth rates were examined by measuring size changes between 1- and 2-months of organoid development. Neurogenesis markers at 2-months were examined at the cellular level. At the molecular level, we measured activity and expression of Ndel1; Ndel1 is a prime target for cell cycle-activated kinases; known to regulate cell cycle, proliferation, neurogenesis, and growth; and known to be involved in neuropsychiatric conditions.ResultsAt the BCO level, analyses showed BCO size was significantly enlarged by 39% and 41% in ASD in the 2021 and 2022 batches. The larger the embryonic BCO size, the more severe the ASD social symptoms. Correlations between BCO size and social symptoms were r = 0.719 in the 2021 batch and r = 0. 873 in the replication 2022 batch. ASD BCOs grew at an accelerated rate nearly 3 times faster than controls. At the cell level, the two largest ASD BCOs had accelerated neurogenesis. At the molecular level, Ndel1 activity was highly correlated with the growth rate and size of BCOs. Two BCO subtypes were found in ASD toddlers: Those in one subtype had very enlarged BCO size with accelerated rate of growth and neurogenesis; a profound autism clinical phenotype displaying severe social symptoms, reduced social attention, reduced cognitive, very low language and social IQ; and substantially altered growth in specific cortical social, language and sensory regions. Those in a second subtype had milder BCO enlargement and milder social, attention, cognitive, language and cortical differences.LimitationsLarger samples of ASD toddler-derived BCO and clinical phenotypes may reveal additional ASD embryonic subtypes.ConclusionsBy embryogenesis, the biological bases of two subtypes of ASD social and brain development-profound autism and mild autism-are already present and measurable and involve dysregulated cell proliferation and accelerated neurogenesis and growth. The larger the embryonic BCO size in ASD, the more severe the toddler's social symptoms and the more reduced the social attention, language ability, and IQ, and the more atypical the growth of social and language brain regions.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO) account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Precise modulation of brain activity is fundamental for the proper establishment and maturation of the cerebral cortex. To this end, cortical organoids are promising tools to study circuit formation and the underpinnings of neurodevelopmental disease. However, the ability to manipulate neuronal activity with high temporal resolution in brain organoids remains limited. To overcome this challenge, we introduce a bioelectronic approach to control cortical organoid activity with the selective delivery of ions and neurotransmitters. Using this approach, we sequentially increased and decreased neuronal activity in brain organoids with the bioelectronic delivery of potassium ions (K+) and γ-aminobutyric acid (GABA), respectively, while simultaneously monitoring network activity. This works highlights bioelectronic ion pumps as tools for high-resolution temporal control of brain organoid activity toward precise pharmacological studies that can improve our understanding of neuronal function.

Biliary complications are still a major cause for morbidity and mortality after liver transplantation (LT). Ischemia/reperfusion injury (IRI) leads to disruption of the biliary epithelium. We introduce a novel model to study the effect of IRI on human cholangiocytes using extrahepatic cholangiocyte organoids (ECOs). Extrahepatic bile duct tissue was collected during LT at static cold storage and after reperfusion (n = 15); gallbladder tissue was used for controls (n = 5). ECOs (n = 9) were cultured from extrahepatic biliary tissue, with IRI induced in an atmosphere of 95% air (nitrogen), 1% O2 and 5% CO2for 48 h, followed by 24 h of reoxygenation. Qualitative and quantitative histology and qRT-PCR were performed to discern phenotype, markers of hypoxia, programmed cell death and proliferation. ECOs self-organized into circular structures resembling biliary architecture containing cholangiocytes that expressed EpCAM, CK19, LGR5 and SOX-9. After hypoxia, ECOs showed increased expression of VEGF A (p < 0.0001), SLC2A1 (p < 0.0001) and ACSL4 (p < 0.0001) to indicate response to hypoxic damage and subsequent programmed cell death. Increase in cyclin D1 (p < 0.0001) after reoxygenation indicated proliferative activity in ECOs. Therefore, ECO structure and response to IRI are comparable to that found in-vivo, providing a suitable model to study IRI of the bile duct in-vitro. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) pose a substantial burden on global healthcare systems. When unraveling the complex pathophysiology of UTIs, bladder models are used to understand complex and multifaceted interactions between different components within the system. This review aimed to bridge the gap between in vitro and in vivo experimental bladder models towards UTI research. We reviewed clinical, animal, and analytical studies and patents from 1959 to the end of 2023. Both in vivo and in vitro models offer unique benefits and drawbacks in understanding UTIs. In vitro models provide controlled environments for studying specific aspects of UTI biology and testing potential treatments, while in vivo models offer insights into how UTIs manifest and progress within living organisms. Thus, both types of models are leading to the development of more effective diagnostic tools and therapeutic interventions against UTIs. Moreover, advanced methodologies involving three-dimensional bladder organoids have also been used to study bladder biology, model bladder-related disorders, and explore new treatments for bladder cancers, UTIs, and urinary incontinence. Narrowing the distance between fundamental scientific research and practical medical applications, these pioneering models hold the key to unlocking new avenues for the development of personalized diagnostics, precision medicine, and ultimately, the alleviation of UTI-related morbidity worldwide. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) has been demonstrated to be dysregulated and crucial for malignant progression in gastric cancer (GC), but the mechanism is not well understood. Here, that discoidin domain receptor 1 (DDR1), a principal ECM receptor, is recognized as a key driver of GC progression is reported. Mechanistically, DDR1 directly interacts with the PAS domain of hypoxia‐inducible factor‐1α (HIF‐1α), suppresses its ubiquitination and subsequently strengthens its transcriptional regulation of angiogenesis. Additionally, DDR1 upregulation in GC cells promotes actin cytoskeleton reorganization by activating HIF‐1α/ Ras Homolog Family Member A (RhoA)/Rho‐associated protein kinase 1 (ROCK1) signaling, which in turn enhances the metastatic capacity. Pharmacological inhibition of DDR1 suppresses GC progression and angiogenesis in patient‐derived xenograft (PDX) and organoid models. Taken together, this work first indicates the effects of the DDR1‐HIF‐1α axis on GC progression and reveals the related mechanisms, providing experimental evidence for DDR1 as a therapeutic target for GC. [ABSTRACT FROM AUTHOR]

Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development, metabolism, and reproduction. In mammals, THRα has two major splicing isoforms, THRα1 and THRα2. The better-characterized isoform, THRα1, is a transcriptional stimulator of genes involved in cell metabolism and growth. The less-well-characterized isoform, THRα2, lacks the ligand-binding domain (LBD) and is thought to act as an inhibitor of THRα1 activity. The ratio of THRα1 to THRα2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However, the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies, we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3′-end. In all datasets, we found a strong predominance of THRA2 transcripts at all examined stages of human brain development and in the central nervous system of healthy human adults. [ABSTRACT FROM AUTHOR]

Introduction: Cervical cancer represents one of the main causes of female, cancer-related mortality worldwide. The majority of cancers are caused by human papillomaviruses such as HPV16 and HPV18. As chemotherapeutic resistance to first-line platinum treatment is still a predominant clinical challenge in advanced cervical cancer, novel treatment options including combinatorial therapies are urgently required to overcome chemotherapeutic resistance. Inhibition of A Disintegrin And Metalloproteinase (ADAM)-family members, heavily involved in tumour progression of a vast range of solid tumours, strongly improved response to chemotherapeutic treatment in other tumour entities including ovarian cancer. Methods: We established two- and three-dimensional models derived from three traditional cervical cancer cell lines and ectocervical cancer-derived organoids. Following characterisation, these models were used to investigate their response to cisplatin treatment in the absence and presence of ADAM inhibitors using viability assays and automated live cell imaging. Results: The pivotal role of the metalloprotease ADAM17 driving chemotherapy resistance was detectable in all ectocervical cultures irrespective of the model system used, whereas ADAM10 inhibition was predominantly effective only in loosely aggregated spheroids. We showed prominent differences regarding treatment responses between 2D monolayers compared to 3D spheroid and 3D organoid model systems. Particularly, the organoid system, regarded as the closest representation of primary tumours, exhibited reliably the combinatorial effect of ADAM17 inhibition and cisplatin in all three individual donors. Discussion: As two- and three-dimensional models of the same cell lines differ in their responses to chemotherapy it is essential to validate treatment strategies in more advanced model systems representing the patient situation more realistically. Ectocervical organoids showed reliable results regarding treatment responses closely mimicking the primary tumours and could therefore serve as an important tool for personalized medicine in cervical cancer. These findings strengthen the role of ADAM17 as a potential novel target for combinatorial treatments to overcome chemoresistance in cervical cancer. [ABSTRACT FROM AUTHOR]

Aggregated a-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD. [ABSTRACT FROM AUTHOR]

Organoids, characterized by their high physiological attributes, effectively preserve the genetic characteristics, physiological structure, and function of the simulated organs. Since the inception of small intestine organoids, other organoids for organs including the liver, lungs, stomach, and pancreas have subsequently been developed. However, a comprehensive summary and discussion of research findings on gastrointestinal tract (GIT) organoids as disease models and drug screening platforms is currently lacking. Herein, in this review, we address diseases related to GIT organoid simulation and highlight the notable advancements that have been made in drug screening and pharmacokinetics, as well as in disease research and treatment using GIT organoids. Organoids of GIT diseases, including inflammatory bowel disease, irritable bowel syndrome, necrotizing enterocolitis, and Helicobacter pylori infection, have been successfully constructed. These models have facilitated the study of the mechanisms and effects of various drugs, such as metformin, Schisandrin C, and prednisolone, in these diseases. Furthermore, GIT organoids have been used to investigate viruses that elicit GIT reactions, including Norovirus, SARS-CoV-2, and rotavirus. Previous studies by using GIT organoids have shown that dasabuvir, gemcitabine, and imatinib possess the capability to inhibit viral replication. Notably, GIT organoids can mimic GIT responses to therapeutic drugs at the onset of disease. The GIT toxicities of compounds like gefitinib, doxorubicin, and sunset yellow have also been evaluated. Additionally, these organoids are instrumental for the study of immune regulation, post-radiation intestinal epithelial repair, treatment for cystic fibrosis and diabetes, the development of novel drug delivery systems, and research into the GIT microbiome. The recent use of conditioned media as a culture method for replacing recombinant hepatocyte growth factor has significantly reduced the cost associated with human GIT organoid culture. This advancement paves the way for large-scale culture and compound screening of GIT organoids. Despite the ongoing challenges in GIT organoid development (e.g., their inability to exist in pairs, limited cell types, and singular drug exposure mode), these organoids hold considerable potential for drug screening. The use of GIT organoids in this context holds great promises to enhance the precision of medical treatments for patients living with GIT diseases. [ABSTRACT FROM AUTHOR]

AbstractBetter in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance. [ABSTRACT FROM AUTHOR]

Organoids are in vitro tissue models derived from human or animal primary tissues or stem cells that allow for studying three-dimensional (3D) tissue biology, toxicity testing, biomarker evaluation, and assessment of compound efficacy, supplementing or potentially minimizing use of animal models. Organoids are typically cultured in a 3D format within an extracellular matrix and, at the end of an experiment, can be further processed for various cellular or molecular readouts. Analysis often relies on whole mount immunolabeling for markers of interest, which consumes the entire sample/well, thereby limiting sample availability for downstream assays. In addition, 3D cultures become more friable after fixation and are susceptible to sample loss during washing steps. In contrast, by fixing and processing organoids to a paraffin block, dozens or hundreds of unstained slides can be generated, enabling robust characterization via multiple assays, including histologic evaluation and (immuno)histochemical stains, thus maximizing the yield of these time- and labor-intensive cultures. Here we describe three methods to process 3D Matrigel cultures into paraffin blocks using Histogel as an embedding agent. The three techniques all yield high-quality sections but vary in complexity of implementation at different steps, and their application for different use cases is discussed. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cellular assemblies, such as cancer spheroids and organoids, are increasingly valued for their physiological relevance, and versatility in biological applications. Nanopatterns that mimic the extracellular matrix provide crucial topological cues, creating a physiologically relevant cellular environment and guiding cellular behaviors. However, the high cost and complex, time-consuming nature of the nanofabrication process have limited the widespread adoption of nanopatterns in diverse biological applications. In this study, we present a straightforward and cost-effective elastomer replica molding method utilizing commercially available optical discs to generate various nanopatterns, such as nanogroove/ridge, nanoposts, and nanopits, varying in spacing and heights. Using the nanopatterned well chips (NW-Chips), we demonstrated the efficient formation of 3D multicellular self-assemblies of three different types of cancer cells. Our findings highlight the accessibility and affordability of optical discs as tools for nanopattern generation, offering promising avenues for modulating cell behaviors and advancing diverse biological applications. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common malignant tumor in women. Due to the high heterogeneity of breast cancer cells, traditional in vitro research models still have major limitations. Therefore, it is urgent to establish an experimental model that can accurately simulate the characteristics of human breast cancer. Breast cancer organoid technology emerged as the times required, that is, to construct tissue analogs with organ characteristics by using a patient's tumor tissue through 3D culture in vitro. Since the breast cancer organoid can fully preserve the histology and genetic characteristics of the original tumor, it provides a reliable model for preclinical drug screening, establishment of breast cancer organoid biobanks, research into the mechanisms of tumor development, and determination of cancer targets. It has promoted personalized treatment for clinical breast cancer patients. This article mainly focuses on recent research progress and applications of organoid technology in breast cancer, discussing the current limitations and prospects of breast cancer organoid technology. [ABSTRACT FROM AUTHOR]

Background: Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer's disease (AD), contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153), a key epigenetic regulator of NSC stemness, characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression. Methods: Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment. Results: Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism, nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration, mitochondrial dysfunction, proteasomal processing and RNA degradation. Furthermore, recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors, marked by BrdU/DCX and BrdU/PSANCAM positivity and, later, the integration of differentiating neurons in the cell granule layer (BrdU/NeuN+ cells) compared with GFP-injected AD mice. Consistently, Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation. Conclusions: Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target. [ABSTRACT FROM AUTHOR]

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media. [ABSTRACT FROM AUTHOR]

Simple Summary: Primary hepatocytes constitute the majority of liver cells and are responsible for many liver-related diseases, including cancer. However, the lack of robust methods for engineering primary hepatocytes ex vivo has hindered their versatile application in disease modeling. Here, we worked to enable genome manipulation in primary mouse hepatocytes in both monolayer culture and organoids, and achieved up to 80% and 20% single gene knockout efficiency, respectively. Using genome engineering of human pluripotent stem cell-derived hepatocytes, we developed a spectrum of isogenic cellular models of hepatocarcinoma with various single or combined oncogenic alterations, and demonstrated that the introduction of these alterations initiated malignant transformation, accelerated cancer growth, and altered transcriptomic profiles and pathway activities. These results reveal that oncogenic alterations dominate cancer characteristics and highlight a powerful genome-engineering-based platform for cancer mechanism studies. Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wild-type cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here, we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming, highlighting the critical roles of these frequently mutated genes in driving liver neoplasia. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models of hepatocarcinoma, providing insights into early tumorigenesis mechanisms. [ABSTRACT FROM AUTHOR]

Objective To construct and identify a patient-derived organoid model, and to investigate the sensitivity of chemotherapy drugs using this model. Methods Pancreatic cancer cells were obtained from the surgical specimens of two female patients with a confirmed diagnosis of pancreatic cancer after tumor tissue digestion, and then the cells were inoculated into a culture dish using matrigel for three-dimensional culture. Paraffin sections were prepared for HE staining and immunohistochemical staining and were compared with the parent tumor tissue to determine whether the histopathological features of the tumor in vivo were preserved. The pancreatic cancer organoids were treated with seven chemotherapy drugs at different concentrations； Cell TiterGlo®3D reagent was used to measure cell viability, and the results of drug sensitivity were analyzed. Results Two patient-derived pancreatic cancer organoids were successfully constructed, and HE staining and immunohistochemical staining showed that the pancreatic cancer organoids had consistent histopathological features with the tumors of the corresponding patient. Both pancreatic cancer organoids were more sensitive to gemcitabine monotherapy and the combination of oxaliplatin+SN38+fluorouracil, and patient 1 was more sensitive than patient 2. There were individual differences in the response to drugs between the organoids from different patients. Conclusion The pancreatic cancer organoid model successfully constructed in this study can reflect the histological classification of parent pancreatic tumors and can be used for in vitro chemotherapy drug sensitivity test, which is expected to provide a reference for clinical medication. [ABSTRACT FROM AUTHOR]

Although the advent of organoids has opened unprecedented perspectives for basic and translational research, immune system-related organoids remain largely underdeveloped. Here, we established organoids from the thymus, the lymphoid organ responsible for T-cell development. We identified conditions enabling mouse thymic epithelial progenitor cell proliferation and development into organoids with diverse cell populations and transcriptional profiles resembling in vivo thymic epithelial cells (TECs) more closely than traditional TEC cultures. In contrast to these two-dimensional cultures, thymic epithelial organoids maintained thymus functionality in vitro and mediated physiological T-cell development upon reaggregation with T-cell progenitors. The reaggregates showed in vivo-like epithelial diversity and the ability to attract T-cell progenitors. Thymic epithelial organoids are the first organoids originating from the stromal compartment of a lymphoid organ. They provide new opportunities to study TEC biology and T-cell development in vitro, paving the way for future thymic regeneration strategies in ageing or acute injuries. [ABSTRACT FROM AUTHOR]

To support complex cognition, neuronal circuits must integrate information across multiple temporal scales, ranging from milliseconds to decades. Neuronal timescales describe the duration over which activity within a network persists, posing a putative explanatory mechanism for how information might be integrated over multiple temporal scales. Little is known about how timescales develop in human neural circuits or other model systems, limiting insight into how the functional dynamics necessary for cognition emerge. In our work, we show that neuronal timescales develop in a nonlinear fashion in human cortical organoids, which is partially replicated in dissociated rat hippocampus cultures. We use spectral parameterization of spiking activity to extract an estimate of neuronal timescale that is unbiased by coevolving oscillations. Cortical organoid timescales begin to increase around month 6 postdifferentiation. In rodent hippocampal dissociated cultures, we see that timescales decrease from in vitro days 13–23 before stabilizing. We speculate that cortical organoid development over the duration studied here reflects an earlier stage of a generalized developmental timeline in contrast to the rodent hippocampal cultures, potentially accounting for differences in timescale developmental trajectories. The fluctuation of timescales might be an important developmental feature that reflects the changing complexity and information capacity in developing neuronal circuits. NEW & NOTEWORTHY: Neuronal timescales describe the persistence of activity within a network of neurons. Timescales were found to fluctuate with development in two model systems. In cortical organoids timescales increased, peaked, and then decreased throughout development; in rat hippocampal dissociated cultures timescales decreased over development. These distinct developmental models overlap to highlight a critical window in which timescales lengthen and contract, potentially indexing changes in the information capacity of neuronal systems. [ABSTRACT FROM AUTHOR]

Purpose: To investigate whether the transcriptome profile differs between progesterone-treated infertile and fertile endometrial organoids. Methods: Endometrial biopsies were obtained from 14 infertile and seven fertile women, after which organoids were generated from isolated epithelial cells. To mimic the secretory phase, organoids were sequentially treated with 17β-estradiol (E2) and progesterone (P4) and subjected to RNA sequencing. Differentially expressed genes (DEGs) were identified using DESeq2 (lfcThreshold = 0, log2 Fold Change ≥ 1.0 or ≤ −1.0), and a principal component analysis (PCA) plot was generated. Functional enrichment analysis was performed by overrepresentation analysis and Gene Set Enrichment Analysis (GSEA). To functionally assess proliferation, OrganoSeg surface measurements were performed before (T0) and after (T1) differentiation of organoids, and T1/T0 ratios were calculated to determine the proliferation rate. Results: Although the PCA plot did not show clear clustering of the fertile and infertile samples, 363 significant DEGs (129 upregulated and 234 downregulated) were detected in infertile compared to fertile organoids. Mainly cell cycle processes were highly enriched in infertile organoids. Thus, we hypothesised that proliferative activity during differentiation may be higher in infertile organoids compared to fertile organoids. However, this could not be validated by cell surface measurements. Conclusions: This study revealed that cell cycle processes were enriched in E2/P4-treated infertile endometrial organoids as compared to fertile organoids. This could reflect persistently higher proliferative activity of the endometrial epithelial cells in differentiated infertile organoids compared to fertile organoids. To confirm this hypothesis, further studies are warranted. [ABSTRACT FROM AUTHOR]

Pancreatic cancer, as a common malignant tumor of the digestive system, has a very low survival rate. In recent years, pancreatic cancer has made great progress in diagnostic methods, radiation therapy techniques, and systemic chemotherapy, but its therapeutic effect has not been considerably improved. As a new type of tumor research platform, organoids have made research progress in many fields. Constructing pancreatic cancer organoids is of great research value to guide the individualized treatment of pancreatic cancer. This article reviews the research and clinical application prospect of organoid model in radiotherapy of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Brain tumors display remarkable cellular and molecular diversity, significantly impacting the progression and outcomes of the disease. The utilization of tumor tissue acquired through surgical handheld devices for tumor characterization raises important questions regarding translational research. This study seeks to evaluate the integrity of tissue resected using a microdebrider (MD) in the context of establishing tumor organoids from glioblastomas (GBM). Tumor samples were collected from patients with GBM using both tumor forceps (en bloc) and a MD. The time required to protocol completion and cell viability of paired samples was measured. H&E staining was performed to examine histologic morphology. Ten paired samples were obtained from GBM patients using tumor forceps and the MD. Samples collected with the MD demonstrated significantly shorter processing times compared to those obtained through en bloc resection, with overall means of 31.7 ± 2.4 mins and 38.8±3 mins, respectively (P < 0.001). Cell viability measured at the end of protocol completion was comparable between tissues obtained using both the MD and en bloc, with mean viabilities of 80.2 ± 12.4% and 79.1 ± 12.5%, respectively (P = 0.848). H&E examination of tissues revealed no significant differences in the cellular and histologic characteristics of paired samples obtained using both methods across GBM tumors, nor in the corresponding established organoids. Tumor tissues obtained using the MD and en bloc methods demonstrate a high success rate in establishing GBM organoids, with the MD offering the advantage of significantly reduced processing time. Both methods display comparable cell viability and maintain consistent histologic characteristics in the resected tissue and the corresponding organoids. [ABSTRACT FROM AUTHOR]

Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours. [ABSTRACT FROM AUTHOR]

There are several in vitro systems that enable evaluation of the absorption direction, but there are few quantitative systems that enable easy evaluation of the excretion direction. Enteroids, organoids derived from intestine, have been frozen and passaged for various research. But it is not clear how the freezing and passaging affect the expression and function of transporters. We investigated the effects of passage and cryopreservation of enteroids. We focused on P-gp (P-glycoprotein) and compared the transfer rates of rhodamine 123 (Rh123) into the lumen of enteroids with and without a P-gp inhibitor. mRNA expression levels did not change significantly before and after passage and cryopreservation. Accumulation of Rh123 in the lumen of enteroids was observed. With some P-gp inhibitors, excretion of Rh123 into the lumen of enteroids was inhibited and the nonexcreted Rh123 accumulated in enteroids epithelial cells. The transfer rate of Rh123 into the lumen of enteroids with a P-gp inhibitor was significantly decreased compared to that of without a P-gp inhibitor. Before and after passage and cryopreservation, the transfer rate was almost the same as that of primary cultured enteroids. We succeeded in easily evaluating whether a component is a substrate of P-gp using enteroids. [Display omitted] • mRNA expression level of efflux intestinal transporter was not changed before and after passage and number of culture days. • This method allows semi-quantitative evaluation of efflux transporters function regardless of the shapes of enteroids. • Our method enables to easily evaluate whether a component is a substrate of P-gp using enteroids. [ABSTRACT FROM AUTHOR]

Antisense technology demonstrates significant potential for addressing inherited brain diseases, with over a dozen products already available and numerous others in the development pipeline. The versatility of differentiating induced pluripotent stem cells (iPSCs) into nearly all neural cell types proves invaluable for comprehending the mechanisms behind neurological diseases, replicating cellular phenotypes, and advancing the testing and development of new therapies, including antisense oligonucleotide therapeutics. While delivering antisense oligonucleotides (ASOs) to human iPSC-based neuronal models has posed challenges, this study explores various delivery methods, including lipid-based transfection, gymnotic uptake, Ca(2+)-enhanced medium (CEM)-based delivery, and electroporation, in 2D and 3D hiPSC-derived neuronal models. This study reveals that CEM-based delivery exhibits efficiency and low toxicity in both 2D neuronal cultures and 3D brain organoids. Furthermore, the findings indicate that CEM is slightly more effective in neurons than in astrocytes, suggesting promising avenues for further exploration and optimization of preclinical ASO strategies in the treatment of neurological disorders. [ABSTRACT FROM AUTHOR]

Studying the behavior of electroactive cells, such as firing dynamics and chemical secretion, is crucial for developing human disease models and therapeutics. Following the recent advances in cell culture technology, traditional monolayers are optimized to resemble more 3D, organ‐like structures. The biological and electrochemical complexity of these structures requires devices with adaptive shapes and novel features, such as precise electrophysiological mapping and stimulation in the case of brain‐ and heart‐derived tissues. However, conventional organ‐on‐chip platforms often fall short, as they do not recreate the native environment of the cells and lack the functional interfaces necessary for long‐term monitoring. Origami‐on‐a‐chip platforms offer a solution for this problem, as they can flexibly adapt to the structure of the desired biological sample and can be integrated with functional components enabled by chosen materials. In this review, the evolution of origami‐on‐a‐chip biointerfaces is discussed, emphasizing folding stimuli, materials, and critical findings. In the prospects, microfluidic integration, functional tissue engineering scaffolds, and multi‐organoid networks are included, allowing patient‐specific diagnoses and therapies through computational and in vitro disease modeling. [ABSTRACT FROM AUTHOR]

Recent advancements in canine intestinal organoid research have paved the way for the development of enhanced in vitro models, crucial for exploring intestinal physiology and diseases. Despite these strides, there is a notable gap in creating specific in vitro models that focus on intestinal inflammation. Our study aims to bridge this gap by investigating the impact of proinflammatory cytokines on canine intestinal epithelial cells (IECs) within the context of organoid models. Canine intestinal organoids were treated with proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. The expression of stem cell markers Lgr5, Sox9, Hopx, and Olfm4 was evaluated through RT-qPCR, while membrane integrity was assessed using immunofluorescence staining for tight junction proteins and transport assays for permeability. IFN-γ significantly decreased Lgr5 expression, a key intestinal stem cell marker, at both 24 and 48 h post-treatment (p=0.030 and p=0.002, respectively). Conversely, TNF-α increased Olfm4 expression during the same intervals (p=0.018 and p=0.011, respectively). A reduction in EdU-positive cells, indicative of decreased cell proliferation, was observed following IFN-γ treatment. Additionally, a decrease in tight junction proteins E-cadherin and ZO-1 (p<0.001 and p=0.003, respectively) and increased permeability in IECs (p=0.012) were noted, particularly following treatment with IFN-γ. The study highlights the profound impact of proinflammatory cytokines on canine IECs, influencing both stem cell dynamics and membrane integrity. These insights shed light on the intricate cellular processes underlying inflammation in the gut and open avenues for more in-depth research into the long-term effects of inflammation on intestinal health. [ABSTRACT FROM AUTHOR]

Ensuring optimal infant nutrition is crucial for the health and development of children. Many infants aged 0–6 months are fed with infant formula rather than breast milk. Research on cancer cell lines and animal models is limited to examining the nutrition effects of formula and breast milk, as it does not comprehensively consider absorption, metabolism, and the health and social determinants of the infant and its physiology. Our study utilized small intestine organoids induced from human embryo stem cell (ESC) to compare the nutritional effects of breast milk from five donors during their postpartum lactation period of 1–6 months and three types of Stage 1 infant formulae from regular retail stores. Using transcriptomics and untargeted metabolomics approaches, we focused on the differences such as cell growth and development, cell junctions, and extracellular matrix. We also analyzed the roles of pathways including AMPK, Hippo, and Wnt, and identified key genes such as ALPI, SMAD3, TJP1, and WWTR1 for small intestine development. Through observational and in-vitro analysis, our study demonstrates ESC-derived organoids might be a promising model for exploring nutritional effects and underlying mechanisms. [ABSTRACT FROM AUTHOR]

SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression‐free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4− mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFβ signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Gastrulation is a key milestone in the development of an organism. It is a period of cell proliferation and coordinated cellular rearrangement, that creates an outline of the body plan. Our current understanding of mammalian gastrulation has been improved by embryo culture, but there are still many open questions that are difficult to address because of the intrauterine development of the embryos and the low number of specimens. In the case of humans, there are additional difficulties associated with technical and ethical challenges. Over the last few years, pluripotent stem cell models are being developed that have the potential to become useful tools to understand the mammalian gastrulation. Here we review these models with a special emphasis on gastruloids and provide a survey of the methods to produce them robustly, their uses, relationship to embryos, and their prospects as well as their limitations. [ABSTRACT FROM AUTHOR]

Despite advancements in cancer research, epithelial ovarian cancer remains a leading threat to women’s health with a low five-year survival rate of 48%. Prognosis for advanced cases, especially International Federation of Gynecology and Obstetrics (FIGO) III-IV, is poor. Standard care includes surgical resection and platinum-based chemo, but 70-80% face recurrence and chemoresistance. In recent years, three- dimensional (3D) cancer models, especially patients-derived organoids (PDOs), have revolutionized cancer research for personalized treatment. By transcending the constraints of conventional models, organoids accurately recapitulate crucial morphological, histological, and genetic characteristics of diseases, particularly in the context of ovarian cancer. The extensive potential of ovarian cancer organoids is explored, spanning from foundational theories to cutting-edge applications. As potent preclinical models, organoids offer invaluable tools for predicting patient treatment responses and guiding the development of personalized therapeutic strategies. Furthermore, in the arena of drug evaluation, organoids demonstrate their unique versatility as platforms, enabling comprehensive testing of innovative drug combinations and novel candidates, thereby pioneering new avenues in pharmaceutical research. Notably, organoids mimic the dynamic progression of ovarian cancer, from inception to systemic dissemination, shedding light on intricate and subtle disease mechanisms, and providing crucial insights. Operating at an individualized level, organoids also unravel the complex mechanisms underlying drug resistance, presenting strategic opportunities for the development of effective treatment strategies. This review summarizes the emerging role of ovarian cancer organoids, meticulously cultivated cellular clusters within threedimensional models, as a groundbreaking paradigm in research. [ABSTRACT FROM AUTHOR]