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Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies.

Cardiovascular organ-on-a-chip (OoC) devices are composed of engineered or native functional tissues that are cultured under controlled microenvironments inside microchips. These systems employ microfabrication and tissue engineering techniques to recapitulate human physiology. This review focuses on human OoC systems to model cardiovascular diseases, to perform drug screening, and to advance personalized medicine. We also address the challenges in the generation of organ chips that can revolutionize the large-scale application of these systems for drug development and personalized therapy.

In vivo tendon and ligament research can be limited by the difficultly of obtaining tissue samples that can be biochemically analyzed. In this study, we characterize the most widely used in vitro engineered ligament model. Despite previous works suggesting multiple passages change gene expression in 2D primary tenocytes, we found no relationship between passage number and expression of classical tendon fibroblast markers across different biological donors. When engineered into 3D ligaments, there was an increase in maximal tensile load between 7 and 14 days in culture, that corresponded with an increase in collagen content. By contrast, percent collagen increased logarithmically from Day 7 to Day 14, and this was similar to the increase in the modulus of the tissue. Importantly, there was no relationship between passage number and mechanical function or collagen content in the two independent donors tested. These results suggest that the model develops quickly and is reliable across differing passage numbers. This provides the field with the ability to 1) consistently determine functional changes of interventions out to passage number 10; and 2) to time interventions to the appropriate developmental stage: developing/regenerating (Day 7) or mature (Day 14) tissue.

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.

Alzheimer's disease (AD) is marked by the presence of intraneuronal neurofibrillary tangles (NFTs), which are primarily composed of hyperphosphorylated tau protein. The locus coeruleus (LC), the brain's main source of norepinephrine (NE), is one of the earliest regions to develop NFTs and experience neurodegeneration in AD. While LC‐derived NE plays beneficial roles in cognition, emotion, locomotion, and the sleep–wake cycle, its impact on tau pathology is unclear. To explore this relationship, we administered intraperitoneal injections of either N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP4), a selective neurotoxin for noradrenergic neurons, or reboxetine (RBX), a norepinephrine reuptake inhibitor, to decrease or increase NE levels, respectively, in early tau transgenic mice expressing mutant human P301L tau (ADLPTau) for two months. Only the RBX‐treated mice exhibited cognitive deficits, as evidenced by their performance in the Y‐maze, novel object recognition, and contextual fear conditioning tests. Immunohistochemical analysis revealed increased hyperphosphorylated tau aggregates in the LC and hippocampus of the RBX‐treated mice. Furthermore, neuronal apoptosis was observed in the hippocampal CA1 region of these mice. Western blotting showed that RBX injections led to the overactivation of tau kinases PKA and GSK3β, resulting in hyperphosphorylated tau, neuronal loss, and cognitive impairments. Consistent with these findings, human brain organoids exposed to higher NE concentrations also displayed elevated hyperphosphorylated tau and increased activity of the same tau kinases. These findings suggest that excessive NE exposure accelerates tau pathology by overactivating the tau kinases. Thus, modulating NE levels in the brain via the LC‐NE system could be a potential therapeutic strategy for tau‐related AD. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is one of the deadliest malignant tumors. Despite significant advances in RCC treatment over the past decade, complete remission is rarely achieved. Consequently, there is an urgent need to explore and develop new therapies to improve the survival rates and quality of life for patients. In recent years, the development of tumor organoid technology has attracted widespread attention as it can more accurately simulate the spatial structure and physiological characteristics of tumors within the human body. In this review, we summarize the main methods currently used to construct kidney cancer organoids, as well as their various biological and clinical applications. Furthermore, combining organoids with other technologies, such as co-culture techniques and microfluidic technologies, can further develop organoids and address their limitations, creating more practical models. This approach summarizes the interactions between different tissues or organs during tumor progression. Finally, we also provide an outlook on the construction and application of kidney cancer organoids. These rapidly evolving kidney cancer organoids may soon become a focal point in the development of in vitro clinical models and therapeutic research for kidney cancer. [ABSTRACT FROM AUTHOR]

Impaired interaction of fibroblasts with pneumocytes contributes to the progression of chronic lung disease such as idiopathic pulmonary fibrosis (IPF). Mucin 5B (MUC5B) is associated with IPF. Here we analyzed the interaction of primary fibroblasts and alveolar type 2 (AT2) pneumocytes in the organoid model. Single-cell analysis, histology, and qRT-PCR revealed that fibroblasts expressing high levels of fibrosis markers regulate STAT3 signaling in AT2 cells, which is accompanied by cystic organoid growth and MUC5B expression. Cystic growth and MUC5B expression were also caused by the cytokine IL-6. The PI3K-Akt signaling pathway was activated in fibroblasts. The drug dasatinib prevented the formation of MUC5B-expressing cystic organoids. MUC5B associated with AT2 cells in samples obtained from IPF patients. Our model shows that fibrotic primary fibroblasts induce impaired differentiation of AT2 cells via STAT3 signaling pathways, as observed in IPF patients. It can be used for mechanistic studies and drug development. [ABSTRACT FROM AUTHOR]

The primary role of the parathyroid glands is to maintain calcium homeostasis through the secretion of parathyroid hormone (PTH). The limited proliferative capacity and differentiation of parathyroid cells hinder the generation of cell therapy options. In this study, parathyroid organoids are successfully generated from human‐induced pluripotent stem cells (hiPSCs). At the end of the 20 days of differentiation, the parathyroid organoids exhibited distinct parathyroid morphology. Stereomicroscope, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analysis demonstrated the 3D arrangement of the cell layers in which intracellular structures of parathyroid cells resemble human parathyroid cellular morphology. Comprehensive molecular analyses, including RNA sequencing (RNA‐Seq) and liquid chromatography/mass spectrometry (LC‐MS/MS), confirmed the expression of key parathyroid‐related markers. Protein expression of CasR, CxCr4, Gcm2, and PTH are observed in parathyroid organoids. Parathyroid organoids secrete PTH, demonstrate active intercellular calcium signaling, and induce osteogenic differentiation via their secretome. The tissue integration potential of parathyroid organoids is determined by transplantation into parathyroidectomized rats. The organoid transplanted animals showed significant elevations in PTH‐related markers (CasR, CxCr4, Foxn1, Gcm2, and PTH). PTH secretion is detected in organoid‐transplanted animals. The findings represent a significant advancement in parathyroid organoid culture and may offer a cellular therapy for treating PTH‐related diseases, including hypoparathyroidism. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction-associated steatohepatitis (MASH) is a form of chronic liver inflammation associated with metabolic syndrome, such as obesity and a major cause of hepatocellular carcinoma (HCC). Multi-biotics, a soymilk fermented with lactic acid bacteria, are known to alleviate obesity by lowering lipid profile. This study aimed to establish and characterize mouse organoids derived from MASH-related HCC models to evaluate drug responses, particularly focusing on Lenvatinib resistance. Organoids were developed using mouse liver tissues subjected to a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) to mimic MASH-related HCC. The study evaluated the effect of multi-biotics, a fermented product, on tumor regression and drug sensitivity. While multi-biotics did not reduce tumor burden, they enhanced the response to Lenvatinib. Additionally, repeated treatment with Lenvatinib led to the development of drug-resistant organoids. Transcriptomic analysis of these resistant organoids identified key pathways related to KRAS signaling, inflammation, and epithelial-mesenchymal transition (EMT), revealing potential targets for overcoming Lenvatinib resistance. This study provides valuable insights into MASH-related HCC progression and drug resistance, offering a model for further therapeutic research. [ABSTRACT FROM AUTHOR]

Spatial-transcriptomics technologies have demonstrated exceptional performance in characterizing brain and visceral organ tissues, as well as brain and retinal organoids. However, it has not yet been proven whether spatial transcriptomics can effectively characterize primary tissue-derived organoids, as the standardized tissue sectioning or slicing methods are not applicable for such organoids. Herein, we present a technique, lamination-based organoid spatially resolved transcriptomics (LOSRT), for organoid-spatially resolved transcriptomics based on organoid lamination. Primary mouse lung and liver-derived organoids were used in this study. The organoids were formulated using the droplet-engineering method and laminated using a homemade device with weight compression. This technique preserved most cells in individual organoids while maintaining delicate epithelium structures in laminated domains that can be recognized through visual segmentation. The mouse lung and liver organoids were resolved comprising various cell types, including alveolar cells, damage-associated transient progenitor cells, basal cells, macrophages, endothelial cells, fibroblasts, hepatocytes, and hepatic stellate cells. The distribution and count of cells were confirmed using immunohistology and identified with spatial transcriptomic features. This study reports an automated and integrated spatial transcriptomics method for primary organoids. It has the potential to standardize and rapidly characterize primary tissue-derived organoids. [ABSTRACT FROM AUTHOR]

Mutations of KRAS, CDKN2A, TP53, and SMAD4 are the four major driver genes for pancreatic ductal adenocarcinoma (PDAC), of which mutations of KRAS and TP53 are the most frequently recognized. However, molecular‐targeted therapies for mutations of KRAS and TP53 have not yet been developed. To identify novel molecular targets, we newly established organoids with the Kras mutation (KrasmuOR) and Trp53 loss of function using Cre transduction and CRISPR/Cas9 (Krasmu/p53muOR) from murine epithelia of the pancreatic duct in KrasLSL‐G12D mice, and then analyzed the proteomic and metabolomic profiles in both organoids by mass spectrometry. Hyperfunction of the glycolysis pathway was recognized in Krasmu/p53muOR compared with KrasmuOR. Loss of function of triosephosphate isomerase (TPI1), which is involved in glycolysis, induced a reduction of cell proliferation in human PDAC cell lines with the TP53 mutation, but not in PDAC or in human fibroblasts without TP53 mutation. The TP53 mutation is clinically recognized in 70% of patients with PDAC. In the present study, protein expression of TPI1 and nuclear accumulation of p53 were recognized in the same patients with PDAC. TPI1 is a potential candidate therapeutic target for PDAC with the TP53 mutation. [ABSTRACT FROM AUTHOR]

Immunotherapies are a revolutionary approach to treating cancer by utilizing the body's immune system to target and combat cancer cells. This approach offers promising alternatives to traditional chemotherapies. Its potential to induce long-lasting remissions and specificity for cancer cells, which minimizes side effects, makes it a cutting-edge treatment with tremendous potential. With the increase of the clinical usage of immunotherapy, evidence emerges of the microbiome's impact on both tumor growth and response to immunotherapy. The proposed involvement of the microbiome can change treatment efficacy by altering drug metabolism and reshaping the immune system response. Understanding the specific interactions between tumor cells, immune cells, and the microbiome is a critical step in the advancement of immunotherapy. To study the complex interaction between cancer immunity and the microbiome, various preclinical in vivo and in vitro models have been developed. We have recently described the use of an ex vivo preclinical model for anti-cancer treatment outcome prediction -tumor tissue equivalents (organoids). Specifically, immune-reactive tumor organoids are proposed as a novel tool for understanding how the microbiome influences cancer immunity and immunotherapy. More importantly, this platform can utilize patient samples to dissect patient-specific elements regulating cancer immune response and microbiome influence. This review presents the rationale for using the immunereactive tumor organoids model to study the interactions between the microbiome and cancer immunotherapy. It will discuss available components of the model and analyze their interplay, summarize relevant experimental data, and assess their validity. Additionally, it explores the potential of immune-reactive organoids for personalized treatment approaches. Understanding the microbiome's role in immunotherapy outcomes will lead to transformative cancer treatment via a simple change of diet or other microbiome manipulations. Ongoing research on microbiome-cancer interactions utilizing the described model systems will lead to innovative treatment strategies and improved patient outcomes. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) has a fatal prognosis because of its aggressive and invasive characteristics. Understanding the mechanism of invasion necessitates an elucidation of the relationship between tumor cells and the tumor microenvironment. However, there has been a scarcity of suitable models to investigate this. In this study, we established a glioblastoma‐cerebral organoid assembloid (GCOA) model by co‐culturing patient‐derived GBM tumoroids and human cerebral organoids. Tumor cells from the tumoroids infiltrated the cerebral organoids, mimicking the invasive nature of the parental tumors. Using time‐lapse imaging, various invasion patterns of cancer cells within cerebral organoids resembling a normal tissue milieu were monitored. Both single‐ and collective‐cell invasion was captured in real‐time. We also confirmed the formation of an intercellular tumor network and tumor–normal‐cell interactions. Furthermore, the transcriptomic characterization of GCOAs revealed distinct features of invasive tumor cells. Overall, this study established the GCOA as a three‐dimensional (3D) in vitro assembloid model to investigate invasion mechanisms and interactions between tumor cells and their microenvironment. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) obtained from patient-derived human induced pluripotent stem cells (iPSCs) are a promising tool for curing various hematological disorders. We previously demonstrated that enforced expression of the LIM-homeobox transcription factor Lhx2, which is essential for mouse embryonic hematopoiesis, leads to generation of engraftable and expandable hematopoietic stem cells (HSCs) from mouse iPSCs. However, it remained unknown whether Lhx2 can induce HSCs from human iPSCs. Here, we investigated the effect of Lhx2 overexpression on hematopoietic differentiation of human iPSCs. Unexpectedly, Lhx2 severely inhibited proliferation of human iPSC-derived hematopoietic cells. Thus, Lhx2 exhibited differential effects on mouse and human hematopoietic cells. Further studies implied that the inhibitory effect of Lhx2 on human iPSC-derived hematopoietic cells was due to insufficient transcriptional activation ability. Therefore, we modified Lhx2 to strengthen its activity as a transcriptional activator. This modified Lhx2 could induce ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells (HSPCs). We believe that these findings will facilitate the development of a method to efficiently produce HSCs from human iPSCs. [ABSTRACT FROM AUTHOR]

The increasing prevalence of neurodevelopmental disorders has highlighted the need for improved testing methods to determine developmental neurotoxicity (DNT) hazard for thousands of chemicals. This paper proposes the integration of organoid intelligence (OI); leveraging brain organoids to study neuroplasticity in vitro, into the DNT testing paradigm. OI brings a new approach to measure the impacts of xenobiotics on plasticity mechanisms - a critical biological process that is not adequately covered in current DNT in vitro assays. Finally, the integration of artificial intelligence (AI) techniques will further facilitate the analysis of complex brain organoid data to study these plasticity mechanisms. [ABSTRACT FROM AUTHOR]

Soluble biochemical agents are being employed to generate kidney organoids from human‐induced pluripotent stem cells. However, this soluble factor approach does not consider the effect of the mechanical environment on lineage commitment. Here, a mechanoresponsive nano‐environment with cell‐adhesive properties composed of supramolecular hydrogelators that co‐assemble into fibrous superstructures to form a transient network is presented, which is used to encapsulate kidney organoids. The delayed sol‐gel transition of the transient network enables fibrous superstructures to diffuse into the densely packed extracellular organoid space during the encapsulation procedure. This allows the mechanoresponsive matrix to induce a biological response beyond the organoid‐hydrogel border, and tune glomerulogenesis inside kidney organoids. In this manner, biomaterials are used as a complementary tool to soluble biochemical agents in tuning lineage commitment and refining organoid maturation. [ABSTRACT FROM AUTHOR]

Background: Cervical squamous cell carcinoma (CSCC) is a prevalent gynecological malignancy worldwide. Current treatments for CSCC can impact fertility and cause long-term complications, underscoring the need for new therapeutic strategies. Oncolytic virotherapy has emerged as a promising option for cancer treatment. Previous research has demonstrated the oncolytic activity of the coxsackievirus B3 strain 2035 A (CVB3/2035A) against various tumor types. This study aims to evaluate the clinical viability of CVB3/2035A for CSCC treatment, focusing on its oncolytic effect in patient-derived CSCC organoids. Methods: The oncolytic effects of CVB3/2035A were investigated using human CSCC cell lines in vitro and mouse xenograft models in vivo. Preliminary tests for tumor-selectivity were conducted on patient-derived CSCC tissue samples and compared to normal cervical tissues ex vivo. Three patient-derived CSCC organoid lines were developed and treated with CVB3/2035A alone and in combination with paclitaxel. Both cytotoxicity and virus replication were evaluated in vitro. Results: CVB3/2035A exhibited significant cytotoxic effects in human CSCC cell lines and xenograft mouse models. The virus selectively induced oncolysis in patient-derived CSCC tissue samples while sparing normal cervical tissues ex vivo. In patient-derived CSCC organoids, which retained the immunohistological characteristics of the original tumors, CVB3/2035A also demonstrated significant cytotoxic effects and efficient replication, as evidenced by increased viral titers and presence of viral nucleic acids and proteins. Notably, the combination of CVB3/2035A and paclitaxel resulted in enhanced cytotoxicity and viral replication. Conclusions: CVB3/2035A showed oncolytic activity in CSCC cell lines, xenografts, and patient-derived tissue cultures and organoids. Furthermore, the virus exhibited synergistic anti-tumor effects with paclitaxel against CSCC. These results suggest CVB3/2035A could serve as an alternative or adjunct to current CSCC chemotherapy regimens. [ABSTRACT FROM AUTHOR]

Research on embryonic development is entering into a new era. As a traditionally descriptive discipline within anatomy, embryologists have formed international consortia and digitized important histological collections for preservation and open access. Embryonic development has recently received a wider attention in context with temporo-spatial transcriptomics at single cell level. These can be expected to fuel the realization of the transdisciplinary significance of efforts to decipher embryonic development. Addressing its complexities encompasses a wealth of challenges that intersect across the domains of science, society, and politics underlining its outstanding importance as well as its inherently interdisciplinary nature. The challenges of this field are by no means confined to understanding the intricate biological mechanisms but also have humanitarian implications. To fully appreciate the mechanisms underlying human development, principles of embryogenesis have predominantly been analyzed employing animal models which allow us to broaden our view on developmental processes. As a result of recent pioneering work and technical progress centered around stem cell-based 3D approaches, we are entering into a historical new phase of learning about mammalian embryonic development. In vertebrates, a growing concern now focuses the reduction of animal experimentation. This perspective article outlines the major challenges in this amazing field that offer an enormous potential for basic biomedical sciences as well as related translational approaches if they are tackled in a multidisciplinary discourse. [ABSTRACT FROM AUTHOR]

Background and purpose: Amplification of the urokinase plasminogen activator receptor (uPAR) gene is closely associated with poor prognosis in pancreatic cancer patients. uPAR regulates epithelial-mesenchymal transition (EMT) and chemoresistance in pancreatic cancer cells through the mitogen-activated protein kinases (MAPK) signaling pathway, though the specific mechanisms remain unclear. This study aimed to investigate the mechanism by which uPAR promotes proliferation, invasion, and chemoresistance of pancreatic cancer cells by inhibiting autophagy. Methods: Pancreatic cancer tissue samples were collected from patients who underwent surgical resection and biopsy at the Changsha Central Hospital, Affiliated to University of South China (Changsha Central Hospital), between December 2021 and Jun 2022. The study was approved by the Ethics Committee of Changsha Central Hospital (Approval No.: 2021-S0182, 2022-S0084). Patient-derived organoids (PDOs) from pancreatic cancer samples were cultured in vitro. Six pancreatic cancer cell lines (AsPC-1, PANC-1, CAPAN-1, CAPAN-2, MIA PaCa-2 and PaTu8988T) were used in this study. uPAR-deficient models were constructed using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 technology. Cell proliferation and invasion abilities were measured using confocal microscopy, Western blot, enzyme-linked immunosorbent assay (ELISA), and MTS assays. Changes in MAPK and autophagy signaling pathways and gemcitabine-induced cell death were analyzed. The synergistic effects of combined treatments were evaluated using gene silencing (siRNA) or autophagy inhibitors. Results: In AsPC-1 cells, uPAR knockout significantly reduced the proliferation and migration abilities of clone cells compared to wild-type cells, as shown by MTS assays and wound healing experiments, and decreased sensitivity to gemcitabine (P＜0.05). Re-expression of uPAR restored the proliferation and invasion abilities of clone cells and partially restored sensitivity to gemcitabine (P＜0.05). Confocal microscopy revealed reduced F-actin and a rounded morphology in clone cells. Western blot analysis showed increased expressions of E-cadherin and Slug, decreased expression of vimentin, and increased expressions of phospho-focal adhesion kinase (p-FAK), p-p38MAPK, and the microtubule-associated protein light chain 3B (LC3B) in clone cells compared to wild-type cells. siRNA results indicated that silencing FAK or p38MAPK or combining autophagy inhibition could resensitize clone cells to gemcitabine (P＜0.05), with p38MAPK silencing reducing LC3B expression. Organoid studies showed varying responses to gemcitabine among 8 organoids, all expressing uPAR. uPAR expression levels were negatively correlated with gemcitabine IC50 (r2=0.66, P＜0.05). Three organoids responded well to the combination of gemcitabine and autophagy inhibitors (P＜0.05). Conclusion: uPAR promotes pancreatic cancer cell activity through the p38MAPK signaling pathway, preventing FAKmediated resistance and cell dormancy. The study suggests that pancreatic cancer patients with high uPAR expression respond better to gemcitabine, while tumors with low uPAR and high p38MAPK expressions may benefit from combined treatment with autophagy inhibitors and cytotoxic chemotherapy. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases are a major cause of death worldwide. Advanced in vitro models can be the key stone for a better understanding of the mechanisms at the basis of the different pathologies, supporting the development of novel therapeutic protocols. In particular, the implementation of induced pluripotent stem cell (iPSC) technology allows for the generation of a patient-specific pluripotent cell line that is able to differentiate in several organ-specific cell subsets while retaining the patient genetic background, thus putting the basis for personalized in vitro modeling toward personalized medicine. The design of iPSC-based models able to recapitulate the complexity of the cardiac environment is a critical goal. Here, we review some of the published efforts to exploit three dimensional (3D) iPSC-based methods to recapitulate the relevant cardiomyopathies, including genetically and non-genetically determined cardiomyopathies and cardiotoxicity studies. Finally, we discuss the actual method limitations and the future perspectives in the field. [ABSTRACT FROM AUTHOR]

Around 13% of women will likely develop breast cancer during their lifetime. Advances in cancer metabolism research have identified a range of metabolic reprogramming events, such as altered glucose and amino acid uptake, increased reliance on glycolysis, and interactions with the tumor microenvironment (TME), all of which present new opportunities for targeted therapies. However, studying these metabolic networks is challenging in traditional 2D cell cultures, which often fail to replicate the three-dimensional architecture and dynamic interactions of real tumors. To address this, organoid models have emerged as powerful tools. Tumor organoids are 3D cultures, often derived from patient tissue, that more accurately mimic the structural and functional properties of actual tumor tissues in vivo, offering a more realistic model for investigating cancer metabolism. This review explores the unique metabolic adaptations of breast cancer and discusses how organoid models can provide deeper insights into these processes. We evaluate the most advanced tools for studying cancer metabolism in three-dimensional culture models, including optical metabolic imaging (OMI), matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and recent advances in conventional techniques applied to 3D cultures. Finally, we explore the progress made in identifying and targeting potential therapeutic targets in breast cancer metabolism. [ABSTRACT FROM AUTHOR]

Background: Although several cell culture systems have been developed to investigate the function of the mammary gland in dairy livestock, they have potential limitations, such as the loss of alveolar structure or genetic and phenotypic differences from their native counterparts. Overcoming these challenges is crucial for lactation research. Development of protocols to establish lactating organoid of livestock represents a promising goal for the future. In this study, we developed a protocol to establish a culture system for mammary organoids in dairy goats to model the mammary gland development and lactation process. Results: The organoids cultured within an extracellular matrix gel maintained a bilayer structure that closely resembled the native architecture of mammary tissue. The expansion of mammary organoids was significantly promoted by growth factors containing epidermal growth factor and fibroblast growth factor 2 whereas the proliferative index of the organoids was significantly inhibited by the treatment with WNT inhibitors. Upon stimulation with a lactogenic medium containing prolactin, the mammary organoids exhibited efficient lactation, characterized by the accumulation of lipid droplets in the lumen space. The lactation could be sustained for more than 3 weeks. Importantly, the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in mammary tissues. These observations were confirmed by data from proteomic analysis that the bulk of milk proteins was produced in the lactating organoids. Conclusion: This study is the first to establish a mammary organoid culture system modeling the mammary gland development and lactation process in ruminants. The efficient induction of lactation in ruminant mammary organoids holds promises for advancing the field of cell-based milk bio-manufacture in the food industry. [ABSTRACT FROM AUTHOR]

Purpose: The overall survival rate for metastatic osteosarcoma hovers around 20%. Responses to second-line chemotherapy, targeted therapies, and immunotherapies have demonstrated limited efficacy in metastatic osteosarcoma. Our objective is to validate differentially expressed genes and signaling pathways between non-metastatic and metastatic osteosarcoma, employing single-cell RNA sequencing (scRNA-seq) and additional functional investigations. We aim to enhance comprehension of metastatic mechanisms and potentially unveil a therapeutic target. Methods: scRNA-seq was performed on two primary osteosarcoma lesions (1 non-metastatic and 1 metastatic). Seurat package facilitated dimensionality reduction and cluster identification. Copy number variation (CNV) was predicted using InferCNV. CellChat characterized ligand-receptor-based intercellular communication networks. Differentially expressed genes underwent GO function enrichment analysis and GSEA. Validation was achieved through the GSE152048 dataset, which identified PDGFD-PDGFRB as a common ligand-receptor pair with significant contribution. Immunohistochemistry assessed PDGFD and PDGFRB expression, while multicolor immunofluorescence and flow cytometry provided insight into spatial relationships and the tumor immune microenvironment. Kaplan-Meier survival analysis compared metastasis-free survival and overall survival between high and low levels of PDGFD and PDGFRB. Manipulation of PDGFD expression in primary osteosarcoma cells examined invasion abilities and related markers. Results: Ten clusters encompassing osteoblasts, osteoclasts, osteocytes, fibroblasts, pericytes, endothelial cells, myeloid cells, T cells, B cells, and proliferating cells were identified. Osteoblasts, osteoclasts, and osteocytes exhibited heightened CNV levels. Ligand-receptor-based communication networks exposed significant fibroblast crosstalk with other cell types, and the PDGF signaling pathway was activated in non-metastatic osteosarcoma primary lesion. These results were corroborated by the GSE152048 dataset, confirming the prominence of PDGFD-PDGFRB as a common ligand-receptor pair. Immunohistochemistry demonstrated considerably greater PDGFD expression in non-metastatic osteosarcoma tissues and organoids, correlating with extended metastasis-free and overall survival. PDGFRB expression showed no significant variation between non-metastatic and metastatic osteosarcoma, nor strong correlations with survival times. Multicolor immunofluorescence suggested co-localization of PDGFD with PDGFRB. Flow cytometry unveiled a highly immunosuppressive microenvironment in metastatic osteosarcoma. Manipulating PDGFD expression demonstrated altered invasive abilities and marker expressions in primary osteosarcoma cells from both non-metastatic and metastatic lesions. Conclusions: scRNA-seq illuminated the activation of the PDGF signaling pathway in primary lesion of non-metastatic osteosarcoma. PDGFD displayed an inhibitory effect on osteosarcoma metastasis, likely through the suppression of the EMT signaling pathway. [ABSTRACT FROM AUTHOR]

Purpose: Early dissemination of primary pancreatic ductal adenocarcinoma (PDAC) is the main cause of dismal prognosis as it highly limits possible treatment options. A number of PDAC patients experience distant metastasis even after treatment due to the metastatic clones. We aimed to demonstrate the molecular architecture of borderline resectable PDAC manifests cancer dissemination of PDAC. Methods: Here, 36 organoids isolated from primary tumor masses of PDAC patients with diverse metastatic statues are presented. Whole-exome sequencing and RNA sequencing were performed and drug responses to clinically relevant 18 compounds were assessed. Results: Our results revealed that borderline resectable PDAC organoids exhibited distinct patterns according to their metastatic potency highlighted by multiple genetic and transcriptional factors and strong variances in drug responses. Conclusions: These data suggest that the presence of metastatic PDAC can be identified by integrating molecular compositions and drug responses of borderline resectable PDAC. [ABSTRACT FROM AUTHOR]

Primary sclerosing cholangitis (PSC) is a challenging cholestatic liver disease marked by progressive bile duct inflammation and fibrosis that has no FDA-approved therapy. Although obeticholic acid (OCA) has been sanctioned for PSC, its clinical utility in PSC is constrained by its potential hepatotoxicity. Here, we introduce a novel therapeutic construct consisting of OCA encapsulated within a reactive oxygen species (ROS)-responsive, biodegradable polymer, further cloaked with human placenta-derived mesenchymal stem cell (hP-MSC) membrane (MPPFTU@OCA). Using PSC patient-derived organoid models, we assessed its cellular uptake and cytotoxicity. Moreover, using a PSC mouse model induced by 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC), we demonstrated that intravenous administration of MPPFTU@OCA not only improved cholestasis via the FXR-SHP pathway but also reduced macrophage infiltration and the accumulation of intracellular ROS, and alleviated mitochondria-induced apoptosis. Finally, we verified the ability of MPPFTU@OCA to inhibit mitochondrial ROS thereby alleviating apoptosis by measuring the mitochondrial adenosine triphosphate (ATP) concentration, ROS levels, and membrane potential (ΔΨm) using H 2 O 2 -stimulated PSC-derived organoids. These results illuminate the synergistic benefits of integrating an ROS-responsive biomimetic platform with OCA, offering a promising therapeutic avenue for PSC. ROS-responsive and self-positioning biomimetic nanoparticle delivery of obeticholic acid mitigates primary sclerosing cholangitis via mitochondria-induced apoptosis. [Display omitted] • ROS-responsive PPFTU material loaded with OCA alleviated hepatobiliary damage. • MSC membrane encapsulation enables powerful targeting of the liver and inflammation. • Organoids and animal models reveal that MPPFTU@OCA mitigates disease progression via mitochondrial-induced apoptosis. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare the formation of organoid structures by co-culturing of human endometrial mesenchymal stem cells (hEnMSCs) and mouse germinal vesicle (GV) oocytes in hanging drop and sodium alginate hydrogel co-culture methods. Following the preparation of hEnMSCs and partially denuded mouse germinal vesicle oocytes, they were co-cultured in hanging drop and sodium alginate hydrogel systems as two experimental groups. In respected control groups the hEnMSCs were cultured without oocytes. The organoid formation was evaluated under the inverted microscope in all studied groups during the culture period. The hematoxylin and eosin, alcian blue, periodic acid Schiff, and Masson's trichrome methods, were applied for morphological evaluation and extracellular matrix components staining such as glycosaminoglycan, carbohydrate, and collagen fibers. In addition, the germ cell-like characteristics within the organoid structures were investigated via alkaline phosphatase activity immunocytochemistry for DEAD-box polypeptide 4 (DDX4), and the expression of octamer-binding transcription factor 4 (OCT4), DDX4, and synaptonemal complex protein 3 (SYCP3) genes by real-time RT-PCR. The culturing of hEnMSCs in the hanging drop method led to the formation of organoid structures while this structure was not seen in sodium alginate hydrogel culture. The mean diameter of organoid structures was increased during 4 days of culture in both the experimental and control groups in the hanging drop method, reaching 675.50 ± 18.55 µm and 670.25 ± 21.40 µm, respectively (P < 0.05). Morphological staining indicated some large ovoid cells with euchromatin nuclei in the experimental group, whereas, in the control group cells showed dark and dense nuclei. The extracellular matrix components were deposited in organoid structures in both control and experimental groups. The positive alkaline phosphatase activity and immunocytochemistry for DDX4 confirmed the presence of germ cell-like in the experimental group. Real-time RT-PCR showed a significant increase in the expression of DDX4 and SYCP3 genes and a decrease in the level of OCT4 expression in the experimental group compared with its controls. This study successfully generated organoid structures by co-culture of hEnMSCs and oocytes in the hanging drop method and the hEnMSCs could be differentiated into germ cell-like. This organoid structure has potential applications in regenerative medicine and reproductive biology. [ABSTRACT FROM AUTHOR]

Prostate cancer is the most common cancer in over 50% of countries and the third most common malignancy worldwide. The tumor microenvironment includes the non‐cancerous cells and components present in the tumor, including the molecules they produce and release. Prostate cancer proliferation, angiogenesis, metastasis, and drug resistance are closely associated with the tumor microenvironment. With the continuous development of in vitro tumor models, they have gradually become important tools for recapitulating in vivo parental tumors and studying the reciprocal interactions between tumors and their microenvironment. In this perspective, we describe the significant in vitro models of prostate cancer, analyze the research results on incorporating the tumor microenvironment into these models, and compare their advantages and disadvantages. Furthermore, we highlight the future developmental direction of in vitro prostate cancer model research according to the hotspots of in vitro model research in other cancer types to facilitate precision medicine in prostate cancer. [ABSTRACT FROM AUTHOR]

Owing to patient-derived tumor tissues and cells, significant advances have been made in personalized cancer treatment and precision medicine, with cancer stem cell-derived three-dimensional tumor organoids serving as crucial in vitro models that accurately replicate the structural, phenotypic, and genetic characteristics of tumors. However, despite their extensive use in drug testing, genome editing, and transplantation for facilitating personalized treatment approaches in clinical practice, the inadequate capacity of these organoids to effectively model immune cells and stromal components within the tumor microenvironment limits their potential. Additionally, effective clinical immunotherapy has led the tumor immune microenvironment to garner considerable attention, increasing the demand for simulating patient-specific tumor–immune interactions. Consequently, co-culture techniques integrating tumor organoids with immune cells and tumor microenvironment constituents have been developed to expand the possibilities for personalized drug response investigations, with recent advancements enhancing the understanding of the strengths, limitations, and applicability of the co-culture approach. Herein, the recent advancements in the field of tumor organoids have been comprehensively reviewed, specifically highlighting the tumor organoid co-culture-related developments with various immune cell models and their implications for clinical research. Furthermore, this review delineates the current state of research and application of organoid models regarding the therapeutic approaches and related challenges for gynecological tumors. This study may provide a theoretical basis for further research on the use of patient-derived organoids in tumor immunity, drug development, and precision medicine. [ABSTRACT FROM AUTHOR]

Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability. Yet, an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis, due to limited in vitro translational lacrimal gland (LG) models. Consequently, this remains a major roadblock to discover effective therapies for the restoration of tear film secretion. Herein, the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states. Using a proof-of-concept approach, porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform. This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity). DNA damage-induced senescence and cell death was induced with etoposide, and LG organoid hypofunction and senescence-associated pathogenesis were observed. To confer DNA protection against aging, a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group, was applied here to prevent LG cellular senescence for the first time. HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic, metabolomic and proteomic levels. Thus, M3DB platforms could generate functional and DNA damage-induced senescence LG organoids, and this latter damage could be prevented with HMGB1-Box A gene therapy. [ABSTRACT FROM AUTHOR]

Glioblastoma organoids (GBOs) serve as a powerful and reliable tool to study glioblastoma stem cells (GSCs) and glioblastoma (GBM). GBOs can be derived from different materials using different methods. To identify the predominant generation methods and the most applications of GBOs, we searched four databases (PubMed, Embase, Web of Science, and Wiley Online Laboratory) from August 2021 to August 2023. After screening, 42 out of 295 articles were included and analyzed. GBOs in these articles were generated using only one material, such as tumor tissues, tumor cells, and gene-edited multifunctional stem cells, or simultaneously using two materials, such as tumor cells and normal organoids. Methodologically, direct cultivation of GBM cells or tissues was the most commonly used method to generate GBOs. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were the frequently used multifunctional stem cells to generate GBOs by simultaneously silencing P53, NF1, and PTEN using CRISPR/Cas9. In terms of applications, GBOs generated by direct cultivation of GBM tissue had the most applications, including molecular mechanisms, therapy, and culture technique. This review provides a theoretical reference for selecting an appropriate method to generate GBOs when studying GSCs and GBM. [ABSTRACT FROM AUTHOR]

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

Non-healing skin wounds pose significant clinical challenges, with biologic products like exosomes showing promise for wound healing. Saliva and saliva-derived exosomes, known to accelerate wound repair, yet their extraction is difficult due to the complex environment of oral cavity. In this study, as a viable alternative, we established human minor salivary gland organoids (hMSG-ORG) to produce exosomes (MsOrg-Exo). In vitro, MsOrg-Exo significantly enhanced cell proliferation, migration, and angiogenesis. When incorporated into a GelMA-based controlled-release system, MsOrg-Exo demonstrated controlled release, effectively improving wound closure, collagen synthesis, angiogenesis, and cellular proliferation in a murine skin wound model. Further molecular analyses revealed that MsOrg-Exo promotes proliferation, angiogenesis and the secretion of growth factors in wound sites. Proteomic profiling showed that MsOrg-Exo's protein composition is similar to human saliva and enriched in proteins essential for wound repair, immune modulation, and coagulation. Additionally, MsOrg-Exo was found to modulate macrophage polarization, inducing a shift towards M1 and M2 phenotypes in vitro within 48 h and predominantly towards the M2 phenotype in vivo after 15 days. In conclusion, our study successfully extracted MsOrg-Exo from hMSG-ORGs, confirmed the effectiveness of the controlled-release system combining MsOrg-Exo with GelMA in promoting skin wound healing, and explored the potential role of macrophages in this action. [ABSTRACT FROM AUTHOR]

Higher levels of aneuploidy, characterized by imbalanced chromosome numbers, are associated with lethal progression in prostate cancer. However, how aneuploidy contributes to prostate cancer aggressiveness remains poorly understood. In this study, we assessed in patients which genes on chromosome 8q, one of the most frequently gained chromosome arms in prostate tumors, were most strongly associated with long-term risk of cancer progression to metastases and death from prostate cancer (lethal disease) in 403 patients and found the strongest candidate was cohesin subunit gene, RAD21, with an odds ratio of 3.7 (95% CI 1.8, 7.6) comparing the highest vs. lowest tertiles of mRNA expression and adjusting for overall aneuploidy burden and Gleason score, both strong prognostic factors in primary prostate cancer. Studying prostate cancer driven by the TMPRSS2-ERG oncogenic fusion, found in about half of all prostate tumors, we found that increased RAD21 alleviated toxic oncogenic stress and DNA damage caused by oncogene expression. Data from both organoids and patients indicate that increased RAD21 thereby enables aggressive tumors to sustain tumor proliferation, and more broadly suggests one path through which tumors benefit from aneuploidy. [ABSTRACT FROM AUTHOR]

Background: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule. Methods: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses. Results: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations. Conclusions: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis. [ABSTRACT FROM AUTHOR]

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

Objective To construct and identify an organoid model of human placental site trophoblastic tumor (PSTT). Methods The tumor cells were obtained by digesting and separating the PSTT tissues and then embedded in Matrigel. The organoids were cultured in the specific organoid medium. The histological morphology of the organoid model was observed by HE staining and the expression levels of the PSTT specific markers ［human placental prolactin (HPL), human leukocyte antigen-G (HLA-G) and placental alkaline phosphatase (PLAP)] were detected by immunohistochemistry and immunofluorescence, so as to evaluate the consistency between the organoid model and the PSTT tissue. Meanwhile, the morphology and forming efficiency of the constructed model were observed under a microscope after primary culture, passage generation and cryopreservation to evaluate its potential application as an organoid model in basic and clinical translational research of PSTT. Results The constructed organoid model could proliferate stably, growing from small microspheres into compact solid spheres or spheres with follicle-like structures, and could passage after fully grown in 7-10 days. The cell state remained stable after passage, frozen storage and recovery. HE staining showed that the morphology of the cells in the organoids was similar to that of the primary PSTT tumor cells, and immunofluorescence staining showed that the organoids highly expressed HLA-G and lowly expressed β-HCG, indicating that the constructed organoid model mainly contained intermediate trophoblast. Conclusion The adult-derived PSTT organoid (ADPO) models were successfully established. [ABSTRACT FROM AUTHOR]

Gastric cancer ranks fifth in global cancer mortality, which is highly aggressive and heterogeneous. However, the research and treatment of gastric cancer is hindered by short of research models that may characterize the developmental properties of gastric cancer. Gastric cancer organoid is a multicellular three-dimensional structure developed in vitro, which can mimic the structure and function of native gastric cancer. Gastric cancer organoids have great application potential and development prospects in establishing gastric cancer research models, mimicking the tumor microenvironment, high-throughput screening of drugs, discovering new therapeutic targets, predicting clinical therapeutic responses, and guiding individualized treatment. In this paper, the progress of gastric cancer organoids in basic research and clinical application is reviewed aiming for promoting the progress of preclinical research and supporting the clinical treatment of gastric cancer. [ABSTRACT FROM AUTHOR]

Traditional in vitro models have inevitable limitations and there are significant differences in assessing drug efficacy and side effects as compared to human trials. Organ-on-a-chip technology simulates human organs in a physiological environment and functional chip with a high fidelity physiological or pathophysiological level, offering great innovative prospects for drug development. This paper mainly introduces the research progress and application of organ chip from the perspective of various systems in vivo. At the same time, the limitations of the current development process of organ chip and the future development direction are proposed. [ABSTRACT FROM AUTHOR]

Objective To establish breast cancer organoids for a long time and to characterize their molecular expression and test their sensitivity to chemotherapy drugs. Methods Breast cancer specimens were digested with collagenase to release cancer cells for organoid culture. The organoids were characterized by morphology and ER, PR, HER-2, CK expression by technology of histoimmunofluorescence. Among them, three were tested for paclitaxel, doxorubicin and cisplatin sensitivity. Results A total of 44 cases of breast cancer organoids were established, all of which showed dense spherical-like growth and ER, PR, HER-2, CK, matching their clinical counterpart. Breast cancer organoids were sensitive to chemotherapy drugs paclitaxel, doxorubicin and cisplatin. Conclusions Organoid model, as a new in vitro may reproduce the pathophysiology features with heterogeneity. [ABSTRACT FROM AUTHOR]

Experimental models play a pivotal role in biomedical research, facilitating the understanding of disease mechanisms and the development of novel therapeutics. This is particularly true for neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and motor neuron disease, which present complex challenges for research and therapy development. In this work, we review the recent literature about experimental models and motor neuron disease. We identified three main categories of models that are highly studied by scientists. In fact, experimental models for investigating these diseases encompass a variety of approaches, including modeling the patient's cell culture, patient-derived induced pluripotent stem cells, and organoids. Each model offers unique advantages and limitations, providing researchers with a range of tools to address complex biological questions. Here, we discuss the characteristics, applications, and recent advancements in terms of each model system, highlighting their contributions to advancing biomedical knowledge and translational research. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD), a type of liver disease for which no treatment is currently approved, remains a major concern worldwide. It is manifested as simple hepatocyte steatosis and can develop into inflammation, fibrosis, cirrhosis and liver cancer in severe cases. However, due to the lack of appropriate in vitro drug testing platforms, an in-depth understanding of the therapeutic activity of ginsenoside Rb1 in NAFLD remains challenging. Here, we proposed a NAFLD model on a liver organoids (LOs)-on-a-chip platform to evaluate the therapeutic effect of ginsenoside Rb1 in a dynamic, multi-condition and high-throughput manner. This platform allowed us to reshape certain features such as multicellular types and liver-specific functions of the physiology of the human-relative liver. Free fatty acids (FFAs)-induced LOs displayed typical pathological characteristics of NAFLD progression, including steatosis, oxidative stress, lipid peroxidation, inflammation and fibrosis. With ginsenoside Rb1 intervention, these pathological features can be significantly improved, which may provide new insights into the potential mechanisms of NAFLD progression and treatment and suggest the clinical implications for humans. The proposed system enables the formation, differentiation, and function of LOs to serve as a scalable, high-throughput and sensitive drug testing model, to potentially expedite the NAFLD drug discovery. [ABSTRACT FROM AUTHOR]

Introduction: Rhodiola species have been utilized as functional foods in Asia and Europe for promoting health. Research has demonstrated that Rhodiola has the potential to alleviate inflammatory bowel disease (IBD) in animal models. However, the specific active components and the underlying mechanism for ameliorating intestinal damage remain unclear. This study aims to explore the relieving effect of Rosavin (Rov), a known active constituent of Rhodiola, in IBD and the regulatory mechanisms. Methods: The therapeutic effect of Rov was evaluated using a murine model of acute colitis induced by dextran sulfate sodium salt (DSS). Inflammatory cytokines and neutrophil activation markers were measured by corresponding kits. Immunohistochemistry, immunofluorescence, TUNEL, and EdU assays were applied to investigate the tight conjunction proteins expression, epithelial marker expression, number of apoptotic cells, and epithelial proliferation, respectively. The protection effect of Rov on gut epithelial injury was assessed using TNF-α-induced intestinal organoids. Additinally, RNA sequencing was applied to observe the genetic alteration profile in these intestinal organoids. Results: Oral administration of Rov significantly attenuated weight loss and restored colon length in mice. Notably, Rov treatment led to decreased levels of pro-inflammatory cytokines and neutrophil activation markers while increasing anti-inflammatory factors. Importantly, Rov restored intestinal despair by increasing the number of Lgr5+ stem cells, Lyz1+ Paneth cells and Muc2+ goblet cells in intestines of colitis mice, displaying reduced epithelial apoptosis and recovered barrier function. In TNF-α-induced intestinal organoids, Rov facilitated epithelial cell differentiation and protected against TNF-α-induced damage. RNA sequencing revealed upregulation in the gene expression associated with epithelial cells (including Lgr5+, Lyz1+ and Muc2+ cells) proliferation and defensin secretion, unveiling the protective mechanisms of Rov on the intestinal epithelial barrier. Discussion: Rov holds potential as a natural prophylactic agent against IBD, with its protective action on the intestinal epithelium being crucial for its therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Ovarian cancer is high recurrence and mortality malignant tumor. The most common ovarian cancer was High‐Grade Serous Ovarian Cancer. However, High‐Grade Serous Ovarian Cancer organoid is rare, which organoid with patient immune microenvironment and blood vessels even absence. Here, we report a novel High‐Grade Serous Ovarian Cancer organoid system derived from patient ovarian cancer samples. These organoids recapitulate High‐Grade Serous Ovarian Cancer organoids' histological and molecular heterogeneity while preserving the critical immune microenvironment and blood vessels, as evidenced by the presence of CD34+ endothelial cells. Whole exome sequencing identifies key mutations (CSMD3, TP53, GABRA6). Organoids show promise in testing cisplatin sensitivity for patients resistant to carboplatin and paclitaxel, with notable responses in cancer proteoglycans and p53 (TP53) signaling, like ACTG/ACTB1/AKT2 genes and BBC3/MDM2/PERP. Integration of immune microenvironment and blood vessels enhances potential for novel therapies like immunotherapies and angiogenesis inhibitors. Our work may provide a new detection system and theoretical basis for ovarian cancer research and individual therapy. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell (hPSC) models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms. In particular, hPSC-based human–animal brain chimeras enable the study of human cell pathophysiology in vivo. In chimeric brains, human neural and immune cells can maintain human-specific features, undergo maturation, and functionally integrate into host brains, allowing scientists to study how human cells impact neural circuits and animal behaviors. The emerging human–animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease, elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels, and testing the efficacy of cell therapy interventions. Here, we discuss recent advances in the generation and applications of using human–animal chimeric brain models for the study of neurological disorders, including disease modeling and cell therapy. [ABSTRACT FROM AUTHOR]

The choroid plexus (CP), a highly vascularized endothelial–epithelial convolute, is placed in the ventricular system of the brain and produces a large part of the cerebrospinal fluid (CSF). Additionally, the CP is the location of a blood–CSF barrier (BCSFB) that separates the CSF from the blood stream in the CP endothelium. In vitro models of the CP and the BCSFB are of high importance to investigate the biological functions of the CP and the BCSFB. Since the CP is involved in several serious diseases, these in vitro models promise help in researching the processes contributing to the diseases and during the development of treatment options. In this review, we provide an overview on the available models and the advances that have been made toward more sophisticated and "in vivo near" systems as organoids and microfluidic lab-on-a-chip approaches. We go into the applications and research objectives for which the various modeling systems can be used and discuss the possible future prospects and perspectives. [ABSTRACT FROM AUTHOR]

Small intestinal neuroendocrine tumors, siNETs, are a group of rare cancers that arise from neuroendocrine cells in the lining of the jejunum and ileum, which are either classified as tumors, siNETs, or small intestinal neuroendocrine carcinomas, siNECs. Current treatment strategies for low-grade tumors include surgical resection, peptide radionucleotide receptor therapy, and somatostatin analogues, while high-grade and recurrent tumors may receive cytotoxic chemotherapy. These limited treatment options are linked to the lack of representative models that can both reflect the biology of the tumor and are amenable to mid-to-high throughput experimentation. Cell line generation is challenging considering the indolent nature of primary lesions, although some attempts have been successful using a variety of methods and include the primary P-STS line and those derived from metastatic lesions, including GOT1, CNDT2.5, and HC45. Patient-derived modeling, including organoids and xenografting, have allowed for multicellular and 3D representations of the original tumor. These specific models allow for multicellular populations derived from the tumor, providing better tumor representation for use in drug screening and in vitro assays. Currently, there are limited, although increasing, published models of siNETs implanted as xenografts in mice and zebrafish. As these cellular and animal models provide insights into siNET biology, theragnostic modeling has provided key information on the clinical progression and treatment of this disease. Significant strides toward more representative models have been made throughout the last decade. In this review, details of these attempts as well as future directions and strategies for more robust models will be addressed.

Abstract Impaired interaction of fibroblasts with pneumocytes contributes to the progression of chronic lung disease such as idiopathic pulmonary fibrosis (IPF). Mucin 5B (MUC5B) is associated with IPF. Here we analyzed the interaction of primary fibroblasts and alveolar type 2 (AT2) pneumocytes in the organoid model. Single-cell analysis, histology, and qRT-PCR revealed that fibroblasts expressing high levels of fibrosis markers regulate STAT3 signaling in AT2 cells, which is accompanied by cystic organoid growth and MUC5B expression. Cystic growth and MUC5B expression were also caused by the cytokine IL-6. The PI3K-Akt signaling pathway was activated in fibroblasts. The drug dasatinib prevented the formation of MUC5B-expressing cystic organoids. MUC5B associated with AT2 cells in samples obtained from IPF patients. Our model shows that fibrotic primary fibroblasts induce impaired differentiation of AT2 cells via STAT3 signaling pathways, as observed in IPF patients. It can be used for mechanistic studies and drug development.

Abstract Metabolic dysfunction-associated steatohepatitis (MASH) is a form of chronic liver inflammation associated with metabolic syndrome, such as obesity and a major cause of hepatocellular carcinoma (HCC). Multi-biotics, a soymilk fermented with lactic acid bacteria, are known to alleviate obesity by lowering lipid profile. This study aimed to establish and characterize mouse organoids derived from MASH-related HCC models to evaluate drug responses, particularly focusing on Lenvatinib resistance. Organoids were developed using mouse liver tissues subjected to a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) to mimic MASH-related HCC. The study evaluated the effect of multi-biotics, a fermented product, on tumor regression and drug sensitivity. While multi-biotics did not reduce tumor burden, they enhanced the response to Lenvatinib. Additionally, repeated treatment with Lenvatinib led to the development of drug-resistant organoids. Transcriptomic analysis of these resistant organoids identified key pathways related to KRAS signaling, inflammation, and epithelial-mesenchymal transition (EMT), revealing potential targets for overcoming Lenvatinib resistance. This study provides valuable insights into MASH-related HCC progression and drug resistance, offering a model for further therapeutic research.

Background and purpose: Amplification of the urokinase plasminogen activator receptor (uPAR) gene is closely associated with poor prognosis in pancreatic cancer patients. uPAR regulates epithelial-mesenchymal transition (EMT) and chemoresistance in pancreatic cancer cells through the mitogen-activated protein kinases (MAPK) signaling pathway, though the specific mechanisms remain unclear. This study aimed to investigate the mechanism by which uPAR promotes proliferation, invasion, and chemoresistance of pancreatic cancer cells by inhibiting autophagy. Methods: Pancreatic cancer tissue samples were collected from patients who underwent surgical resection and biopsy at the Changsha Central Hospital, Affiliated to University of South China (Changsha Central Hospital), between December 2021 and Jun 2022. The study was approved by the Ethics Committee of Changsha Central Hospital (Approval No.: 2021-S0182, 2022-S0084). Patient-derived organoids (PDOs) from pancreatic cancer samples were cultured in vitro. Six pancreatic cancer cell lines (AsPC-1, PANC-1, CAPAN-1, CAPAN-2, MIA PaCa-2 and PaTu8988T) were used in this study. uPAR-deficient models were constructed using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 technology. Cell proliferation and invasion abilities were measured using confocal microscopy, Western blot, enzyme-linked immunosorbent assay (ELISA), and MTS assays. Changes in MAPK and autophagy signaling pathways and gemcitabine-induced cell death were analyzed. The synergistic effects of combined treatments were evaluated using gene silencing (siRNA) or autophagy inhibitors. Results: In AsPC-1 cells, uPAR knockout significantly reduced the proliferation and migration abilities of clone cells compared to wild-type cells, as shown by MTS assays and wound healing experiments, and decreased sensitivity to gemcitabine (P