Protocol for controlled cortical impact in human cerebral organoids to model traumatic brain injury.

Modeling traumatic brain injury (TBI) has been a challenge. Rodent and cellular models have provided relevant contributions despite their limitations. Here, we present a protocol for a TBI model based on the controlled cortical impact (CCI) performed on human cerebral organoids (COs), self-assembled 3D cultures that recapitulate features of the human brain. Here, we generate COs from iPSCs obtained from reprogrammed fibroblasts. For complete details on the use and execution of this protocol, please refer to Ramirez et al. (2021).

The Evolving Role of Induced Pluripotent Stem Cells and Cerebral Organoids in Treating and Modeling Neurosurgical Diseases.

Over the past decade, the use of induced pluripotent stem cells (IPSCs), as both direct therapeutics and building blocks for 3D in vitro models, has exhibited exciting potential in both helping to elucidate pathogenic mechanisms and treating diseases relevant to neurosurgery. Transplantation of IPSCs is being studied in neurological injuries and diseases, such as spinal cord injury and Parkinson's disease, whose clinical manifestations stem from underlying neuronal and/or axonal degeneration. Both animal models and clinical trials have shown that IPSCs have the ability to regenerate damaged neural tissue. Such evidence makes IPSCs a potentially promising therapeutic modality for patients who suffer from these neurological injuries/diseases. In addition, the cerebral organoid, a 3D assembly of IPSC aggregates that develops heterogeneous brain regions, has become the first in vitro model to closely recapitulate the complexity of the brain extracellular matrix, a 3-dimensional network of molecules that structurally and biochemically support neighboring cells. Cerebral organoids have become an exciting prospect for modeling and testing drug susceptibility of brain tumors, such as glioblastoma and metastatic brain cancer. As patient-derived organoid models are becoming more faithful to the brain, they are becoming an increasingly accurate substitute for patient clinical trials; such patient-less trials would protect the patient from potentially ineffective drugs, and speed up trial results and optimize cost. In this review, we aim to describe the role of IPSCs and cerebral organoids in treating and modeling diseases that are relevant to neurosurgery.

Anti-inflammatory and tight junction protective activity of the herbal preparation STW 5-II on mouse intestinal organoids.

BACKGROUND: Irritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS. AIM: This study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II. METHODS: Intestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1ß, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II. RESULTS: An organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS. CONCLUSION: STW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.

Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut.

Small intestinal organoids, or enteroids, represent a valuable model to study host-pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4+ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host-pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.

State of the art on lung organoids in mammals.

The number and severity of diseases affecting lung development and adult respiratory function have stimulated great interest in developing new in vitro models to study lung in different species. Recent breakthroughs in 3-dimensional (3D) organoid cultures have led to new physiological in vitro models that better mimic the lung than conventional 2D cultures. Lung organoids simulate multiple aspects of the real organ, making them promising and useful models for studying organ development, function and disease (infection, cancer, genetic disease). Due to their dynamics in culture, they can serve as a sustainable source of functional cells (biobanking) and be manipulated genetically. Given the differences between species regarding developmental kinetics, the maturation of the lung at birth, the distribution of the different cell populations along the respiratory tract and species barriers for infectious diseases, there is a need for species-specific lung models capable of mimicking mammal lungs as they are of great interest for animal health and production, following the One Health approach. This paper reviews the latest developments in the growing field of lung organoids.

Cerebral organoids and their potential for studies of brain diseases in domestic animals.

The brain is a complex organ and any model for studying it in its normal and pathological aspects becomes a tool of choice for neuroscientists. The mastering and dissemination of protocols allowing brain organoids development have paved the way for a whole range of new studies in the field of brain development, modeling of neurodegenerative or neurodevelopmental diseases, understanding tumors as well as infectious diseases that affect the brain. While studies are so far limited to the use of human cerebral organoids, there is a growing interest in having similar models in other species. This review presents what is currently developed in this field, with a particular focus on the potential of cerebral organoids for studying neuro-infectious diseases in human and domestic animals.

In Vitro Human Joint Models Combining Advanced 3D Cell Culture and Cutting-Edge 3D Bioprinting Technologies.

Joint-on-a-chip is a new technology able to replicate the joint functions into microscale systems close to pathophysiological conditions. Recent advances in 3D printing techniques allow the precise control of the architecture of the cellular compartments (including chondrocytes, stromal cells, osteocytes and synoviocytes). These tools integrate fluid circulation, the delivery of growth factors, physical stimulation including oxygen level, external pressure, and mobility. All of these structures must be able to mimic the specific functions of the diarthrodial joint: mobility, biomechanical aspects and cellular interactions. All the elements must be grouped together in space and reorganized in a manner close to the joint organ. This will allow the study of rheumatic disease physiopathology, the development of biomarkers and the screening of new drugs.

Patient-derived organoids and high grade serous ovarian cancer: from disease modeling to personalized medicine.

BACKGROUND: High grade serous ovarian cancer (HGSOC) is among the deadliest human cancers and its prognosis remains extremely poor. Tumor heterogeneity and rapid acquisition of resistance to conventional chemotherapeutic approaches strongly contribute to poor outcome of patients. The clinical landscape of HGSOC has been radically transformed since the advent of targeted therapies in the last decade. Nevertheless, the lack of predictive biomarkers informing on the differential clinical benefit in select subgroups, and allowing patient-centric approaches, currently limits the efficacy of these novel therapies. Thus, rational selection of the best possible treatment for each patient represents a clinical priority in order to improve outcome, while limiting undesirable effects. MAIN BODY: In this review, we describe the state of the art and the unmet needs in HGSOC management, illustrate the treatment options that are available and the biomarkers that are currently employed to orient clinical decisions. We also describe the ongoing clinical trials that are testing new therapeutic approaches for HGSOC. Next, we introduce the organoid technology as a promising, expanding strategy to study cancer and to develop personalized therapeutic approaches. In particular, we discuss recent studies that have characterized the translational potential of Patient's Derived Organoids (PDOs) to inform on drug sensitivity of HGSOC patients. CONCLUSIONS: PDOs can predict the response of patients to treatments and may therefore guide therapeutic decisions. Although preliminary results appear encouraging, organoids still need to be generated and expanded efficiently to enable drug screening in a clinically meaningful time window. A new generation of clinical trials based on the organoid technology should guarantee tailored approaches to ovarian cancer management, as it is now clear that the one-size-fits-all approach cannot lead to efficient and meaningful therapeutic advancements.

Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids. We showed successful separation of organoids exhibiting a crypt-like proliferative phenotype, and organoids exhibiting a villus-like phenotype, enriched for enterocytes and goblet cells. Functional enrichment analysis of significantly changing proteins during HR revealed that processes related to mitochondrial metabolism and organization, other metabolic processes, and the immune response were altered in both organoid phenotypes. Changes in protein metabolism, as well as mitophagy pathway and protection against oxidative stress were more pronounced in crypt-like organoids, whereas cellular stress and cell death associated protein changes were more pronounced in villus-like organoids. Profile analysis highlighted several interesting proteins showing a consistent temporal profile during HR in organoids from different origin, such as NDRG1, SDF4 or DMBT1. This study demonstrates that the HR response in human intestinal organoids recapitulates properties of the in vivo IR response. Our findings provide a framework for further investigations to elucidate underlying mechanisms of IR injury in crypt and/or villus separately, and a model to test therapeutics to prevent IR injury.

Controllable Fabrication of Composite Core-Shell Capsules at a Macroscale as Organoid Biocarriers.

The cell encapsulation technology is promising for generation of functional carriers with well-tailored structures for efficient transplantation and immunoprotection of cells/tissues. Stem cell organoids are highly potential for recapitulating the intricate architectures and functionalities of native organs and also providing an unlimited cell source for cellular replacement therapy. However, it remains challenging for loading the organoids with hundreds of micrometers size by current existing cell carriers. Herein, a simple and facile coextrusion strategy is developed for controllable fabrication of Ca-alginate/poly(ethylene imine) (Alg/PEI) macrocapsules for efficient encapsulation and cultivation of organoids. Human-induced pluripotent stem cell (hiPSC)-derived islet organoids are encapsulated in the aqueous compartments of the capsules and immunoisolated by a semipermeable Alg/PEI shell. Via electrostatic interactions, a PEI polyelectrolyte can be incorporated in the shell for restricting its swelling, thus effectively improving the stability of the capsules. The Alg/PEI macrocapsules are featured with desirable selective permeability for immunoisolation of antibodies from reaching the loaded organoids. Meanwhile, they also exhibit excellent permeability for mass transfer due to their well-defined core-shell structure. As such, the encapsulated islet organoids contain islet-specific multicellular components, with high viability and sensitive glucose-stimulated insulin secretion function. The proposed approach provides a versatile encapsulation system for tissue engineering and regenerative medicine applications.

An organoid-based drug screening identified a menin-MLL inhibitor for endometrial cancer through regulating the HIF pathway.

Tumor organoids recapitulate pathological properties and would serve as an excellent ex vivo model for drug discovery. Here, we performed an unbiased drug screening on drivers-defined tumor organoids from mouse endometrial cancer, the most prevalent gynecological malignancy in human, with a small molecule library targeting epigenetic factors. Among them, menin-MLL inhibitors MI-136 and MI-463 scored. The therapeutic capacity of MI-136 was further validated in tumor organoids in vitro and an orthotopic model in vivo. CRISPR/cas9-mediated mutations of major components of the menin-MLL complex, Men1, Kmt2a and Ash2l, inhibited the growth of tumor organoids, suggesting that the complex was the target of MI-136. Transcriptome analysis showed that the hypoxia-inducible factor (HIF) pathway was the most significantly downregulated pathway by MI-136 treatment. Consistently, Men1, Kmt2a, and Ash2l knockout also repressed the expressions of the HIF target genes. Loss of Hif1a or Hif1b partially phenocopied the inhibition of the menin-MLL complex by MI-136 or mutations in term of tumor organoid growth. Further, we found that MEN1 was upregulated in human endometrial cancers, which were tightly correlated with the expression levels of HIF1A, and associated with poor prognosis. Importantly, MI-136 also significantly inhibited the growth of endometrial cancer organoids derived from patients. Thus, our study identified MI-136 as a potential inhibitor for endometrial cancer through regulating the HIF pathway, a novel molecular mechanism distinguished from those in AML and prostate cancer.

Colonoids From Patients With Pediatric Inflammatory Bowel Disease Exhibit Decreased Growth Associated With Inflammation Severity and Durable Upregulation of Antigen Presentation Genes.

BACKGROUND: Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids. METHODS: We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture. RESULTS: Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD. CONCLUSIONS: We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.

Creation of bladder assembloids mimicking tissue regeneration and cancer.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

The Microfluidic Environment Reveals a Hidden Role of Self-Organizing Extracellular Matrix in Hepatic Commitment and Organoid Formation of hiPSCs.

The specification of the hepatic identity during human liver development is strictly controlled by extrinsic signals, yet it is still not clear how cells respond to these exogenous signals by activating secretory cascades, which are extremely relevant, especially in 3D self-organizing systems. Here, we investigate how the proteins secreted by human pluripotent stem cells (hPSCs) in response to developmental exogenous signals affect the progression from endoderm to the hepatic lineage, including their competence to generate nascent hepatic organoids. By using microfluidic confined environment and stable isotope labeling with amino acids in cell culture-coupled mass spectrometry (SILAC-MS) quantitative proteomic analysis, we find high abundancy of extracellular matrix (ECM)-associated proteins. Hepatic progenitor cells either derived in microfluidics or exposed to exogenous ECM stimuli show a significantly higher potential of forming hepatic organoids that can be rapidly expanded for several passages and further differentiated into functional hepatocytes. These results prove an additional control over the efficiency of hepatic organoid formation and differentiation for downstream applications.

SMARCB1 loss interacts with neuronal differentiation state to block maturation and impact cell stability.

Atypical teratoid rhabdoid tumors (ATRTs) are challenging pediatric brain cancers that are predominantly associated with inactivation of the gene SMARCB1, a conserved subunit of the chromatin remodeling BAF complex, which has known contributions to developmental processes. To identify potential interactions between SMARCB1 loss and the process of neural development, we introduced an inducible SMARCB1 loss-of-function system into human induced pluripotent stem cells (iPSCs) that were subjected to either directed neuronal differentiation or differentiation into cerebral organoids. Using this system, we identified substantial differences in the downstream effects of SMARCB1 loss depending on differentiation state and identified an interaction between SMARCB1 loss and neural differentiation pressure that causes a resistance to terminal differentiation and a defect in maintenance of a normal cell state. Our results provide insight into how SMARCB1 loss might interact with neural development in the process of ATRT tumorigenesis.

Rapid and Efficient Invasion Assay of Glioblastoma in Human Brain Organoids.

Glioblastoma (GBM) possesses glioma stem cells (GSCs) that exhibit aggressive invasion behavior in the brain. Current preclinical GBM invasion assays using mouse brain xenografts are time consuming and less efficient. Here, we demonstrate an array of methods that allow rapid and efficient assaying of GSCs invasion in human brain organoids. The assays are versatile to characterize various aspects of GSCs, such as invasion, integration, and interaction with mature neurons of brain organoids. Tissue clearing and quantitative 3D imaging of GSCs in host organoids reveal that invasiveness is inversely correlated with the organoids' age. Importantly, the described invasion assays can distinguish the invasive behaviors of primary and recurrent GSCs. The assays are also amenable to test pharmacological agents. As an example, we show that GI254023X, an inhibitor of ADAM10, could prevent the integration of GSCs into the organoids.

Epithelial-mesenchymal crosstalk in COPD: An update from in vitro model studies.

Chronic Obstructive Pulmonary disease (COPD) involves airway inflammation and remodeling leading to small airways disease and emphysema, which results in irreversible airflow obstruction. During lung development, reciprocal interactions between the endoderm and mesoderm (epithelial-mesenchymal trophic unit (EMTU)) are essential for morphogenetic cues that direct cell proliferation, differentiation, and extracellular (ECM) production. In COPD, a significant number of the inflammation and remodeling mediators resemble those released during lung development, which has led to the hypothesis that aberrant activation of the EMTU may occur in the disease. Studies assessing lung epithelial and fibroblast function in COPD, have been primarily focused on monoculture studies. To capture the in vivo environment of the human lung and aid in the understanding of mechanisms and mediators involved in abnormal epithelial-fibroblast communication in COPD, complex co-culture models are required. In this review, we describe the studies that have used co-culture models to assess epithelial-fibroblast interactions and their role in the pathogenesis of COPD.

Tissue engineering to better understand senescence: Organotypics come of age.

The recent advent of 'organs in a dish' has revolutionised the research landscape. These 3D culture systems have paved the way for translational, post genomics research by enabling scientists to model diseases in the laboratory, grow patient-derived organoids, and unite this technology with other cutting-edge methodologies such as drug discovery. Fields such as dermatology and neuroscience have revolutionised the development of robust 3D models, which faithfully recapitulate native physiology in vivo to provide important functional and mechanistic insights. These models have underpinned a rapid growth in the number of organs and myriad of human diseases that can be modelled in 3D, which currently includes breast, cerebral cortex, heart, intestine, kidney, liver, lung, neural tube, pancreas, prostate, skin and stomach, as well as patient derived tumours. However, so far, they have not yet been employed extensively in the study of fundamental cellular programmes such as senescence. Thus, tissue engineering and 3D culture offer an exciting opportunity to further understand the bright and dark sides of senescence in a more complex and physiologically relevant environment. Below, we will discuss previous approaches to investigating senescence and ageing using organotypic models, and some potential opportunities for future research.

Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids.

High-grade serous ovarian cancer (HG-SOC)-often referred to as a "silent killer"-is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients.