Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids

Neural tube defects(NTDs)are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure.Although folate supplementation has been shown to mitigate the incidence of NTDs,some cases,often attributable to genetic factors,remain unpreventable.The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation;at present,however,the underlying mechanism remains unclear.Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate.To determine the role of SHROOM3 in early developmental morphogenesis,we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase.Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei.These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins,namely fibrous actin(F-actin),myosin II,and phospho-myosin light chain(PMLC),to the apical side of the neuroepithelial cells.Notably,these defects were not rescued by folate supplementation.RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis.In summary,we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Human pluripotent stem cell-derived kidney organoids:Current progress and challenges

Human pluripotent stem cell(hPSC)-derived kidney organoids share similarities with the fetal kidney.However,the current hPSC-derived kidney organoids have some limitations,including the inability to perform nephrogenesis and lack of a corticomedullary definition,uniform vascular system,and coordinated exit path-way for urinary filtrate.Therefore,further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development,regeneration,disease modeling,and drug screening.In this review,we discussed recent advances in the generation of hPSC-derived kidney organoids,how these organoids contribute to the understanding of human kidney development and research in disease modeling.Additionally,the limitations,future research focus,and applications of hPSC-derived kidney organoids were highlighted.

Bioengineered skin organoids:from development to applications

Signifcant advancements have been made in recent years in the development of highly sophisticated skin organoids.Serving as three-dimensional(3D)models that mimic human skin,these organoids have evolved into complex structures and are increasingly recognized as efective alternatives to traditional culture models and human skin due to their ability to overcome the limitations of two-dimensional(2D)systems and ethical concerns.The inherent plasticity of skin organoids allows for their construction into physiological and pathological models,enabling the study of skin development and dynamic changes.This review provides an overview of the pivotal work in the progression from 3D layered epidermis to cyst-like skin organoids with appendages.Furthermore,it highlights the latest advancements in organoid construction facilitated by state-of-the-art engineering techniques,such as 3D printing and microfuidic devices.The review also summarizes and discusses the diverse applications of skin organoids in developmental biology,disease modelling,regenerative medicine,and personalized medicine,while considering their prospects and limitations.

Success rate of current human-derived gastric cancer organoids establishment and influencing factors:A systematic review and meta-analysis

BACKGROUND Human-derived gastric cancer organoids(GCOs)are widely used in gastric cancer research;however,the culture success rate is generally low.AIM To explore the potential influencing factors,and the literature on successful culture rates of GCOs was reviewed using meta-analysis.METHODS PubMed,Web of Science,and EMBASE were searched for studies.Two trained researchers selected the studies and extracted data.STATA 17.0 software was used for meta-analysis of the incidence of each outcome event.The adjusted Methodological Index for Non-Randomized Studies scale was used to assess the quality of the included studies.Funnel plots and Egger’s test were used to detect publication bias.Subgroup analyses were conducted for sex,tissue source,histo-logical classification,and the pathological tumor-node-metastasis(pTNM)cancer staging system.RESULTS Eight studies with a pooled success rate of 66.6%were included.GCOs derived from women and men had success rates of 67%and 46.7%,respectively.GCOs from surgery or biopsy/endoscopic submucosal dissection showed success rates of 70.9%and 53.7%,respectively.GCOs of poorly-differentiated,moderately-differentiated and signet-ring cell cancer showed success rates of 64.6%,31%,and 32.7%,respectively.GCOs with pTNM stages I-II and III-IV showed success rates of 38.3%and 65.2%,respectively.Y-27632 and non-Y-27632 use showed success rates of 58.2%and 70%,respectively.GCOs generated with collagenase were more successful than those constructed with Liberase TH and TrypLE(72.1%vs 71%,respectively).EDTA digestion showed a 50%lower success rate than other methods(P=0.04).CONCLUSION GCO establishment rate is low and varies by sex,tissue source,histological type,and pTNM stage.Omitting Y-27632,and using Liberase TH,TrypLE,or collagenase yields greater success than EDTA.

Non-small cell lung cancer organoids:Advances and challenges in current applications

Lung cancer is emerging as a common malignancy worldwide,with non-small cell lung cancer(NSCLC)accounting for approximately 85%of all cases.Two-dimensional(2D)in vitro cell line cultures and animal models are currently used to study NSCLC.However,2D cell cultures fail to replicate the medication response and neoplastic heterogeneity of parental tumors.Animal models are expensive and require lengthy modeling cycles.The generation of in vitro three-dimensional(3D)tissue cultures called organoids,which exhibit multicellular,anatomical,and functional properties of real organs,is now achievable owing to advancements in stem cell culturing.The genetic,proteomic,morphological,and pharmacological characteristics of tumors are largely preserved in tumor organoids grown in vitro.The design and physiology of human organs can be precisely reconstructed in tumor organoids,opening new possibilities for complementing the use of animal models and studying human diseases.This review summarizes the development of NSCLC organoids and their applications in basic research,drug testing,immunotherapy,and individualized treatments.

Advances in the study of gastric organoids as disease models

Gastric organoids are models created in the laboratory using stem cells and sophisticated three-dimensional cell culture techniques.These models have shown great promise in providing valuable insights into gastric physiology and advanced disease research.This review comprehensively summarizes and analyzes the research advances in culture methods and techniques for adult stem cells and induced pluripotent stem cell-derived organoids,and patient-derived organoids.The potential value of gastric organoids in studying the pathogenesis of stomach-related diseases and facilitating drug screening is initially discussed.The construction of gastric organoids involves several key steps,including cell extraction and culture,three-dimensional structure formation,and functional expression.Simulating the structure and function of the human stomach by disease modeling with gastric organoids provides a platform to study the mechanism of gastric cancer induction by Helicobacter pylori.In addition,in drug screening and development,gastric organoids can be used as a key tool to evaluate drug efficacy and toxicity in preclinical trials.They can also be used for precision medicine according to the specific conditions of patients with gastric cancer,to assess drug resistance,and to predict the possibility of adverse reactions.However,despite the impressive progress in the field of gastric organoids,there are still many unknowns that need to be addressed,especially in the field of regenerative medicine.Meanwhile,the reproducibility and consistency of organoid cultures are major challenges that must be overcome.These challenges have had a significant impact on the development of gastric organoids.Nonetheless,as technology continues to advance,we can foresee more comprehensive research in the construction of gastric organoids.Such research will provide better solutions for the treatment of stomach-related diseases and personalized medicine.

Progress in the generation of spinal cord organoids over the past decade and future perspectives

Spinal cord organoids are three-dimensional tissues derived from stem cells that recapitulate the primary morphological and functional characteristics of the spinal cord in vivo.As emerging bioengineering methods have led to the optimization of cell culture protocols,spinal cord organoids technology has made remarkable advancements in the past decade.Our literature search found that current spinal cord organoids do not only dynamically simulate neural tube formation but also exhibit diverse cytoarchitecture along the dorsal-ventral and rostral-caudal axes.Moreover,fused organoids that integrate motor neurons and other regionally specific organoids exhibit intricate neural circuits that allows for functional assessment.These qualities make spinal cord organoids valuable tools for disease modeling,drug screening,and tissue regeneration.By utilizing this emergent technology,researchers have made significant progress in investigating the pathogenesis and potential therapeutic targets of spinal cord diseases.However,at present,spinal cord organoid technology remains in its infancy and has not been widely applied in translational medicine.Establishment of the next generation of spinal cord organoids will depend on good manufacturing practice standards and needs to focus on diverse cell phenotypes and electrophysiological functionality evaluation.

Visualization analysis of research hotspots and trends on gastrointestinal tumor organoids

BACKGROUND Gastrointestinal tumor organoids serve as an effective model for simulating cancer in vitro and have been applied in basic biology and preclinical research.Despite over a decade of development and increasing research achievements in this field,a systematic and comprehensive analysis of the research hotspots and future trends is lacking.AIM To address this problem by employing bibliometric tools to explore the publication years,countries/regions,institutions,journals,authors,keywords,and references in this field.METHODS The literature was collected from Web of Science databases.CiteSpace-6.2R4,a widely used bibliometric analysis software package,was used for institutional analysis and reference burst analysis.VOSviewer 1.6.19 was used for journal cocitation analysis,author co-authorship and co-citation analysis.The‘online platform for bibliometric analysis(https://bibliometric.com/app)’was used to assess the total number of publications and the cooperation relationships between countries.Finally,we employed the bibliometric R software package(version R.4.3.1)in R-studio,for a comprehensive scientific analysis of the literature.RESULTS Our analysis included a total of 1466 publications,revealing a significant yearly increase in articles on the study of gastrointestinal tumor organoids.The United States(n=393)and Helmholtz Association(n=93)have emerged as the leading countries and institutions,respectively,in this field,with Hans Clevers and Toshiro Sato being the most contributing authors.The most influential journal in this field is Gastroenterology.The most impactful reference is"Long term expansion of epithelial organs from human colon,adenoma,adenocarcinoma,and Barrett's epithelium".Keywords analysis and citation burst analysis indicate that precision medicine,disease modeling,drug development and screening,and regenerative medicine are the most cutting-edge directions.These focal points were further detailed based on the literature.CONCLUSION This bibliometric study offers an objective and quantitative analysis of the research in this field,which can be considered as an important guide for next scientific research.

Conversion therapy in advanced perihilar cholangiocarcinoma based on patient-derived organoids:A case report

BACKGROUND Patient-derived organoids(PDOs)have been demonstrated to predict the response to drugs in multiple cancer types.However,it remains unclear about its application in cholangiocarcinoma.CASE SUMMARY A 59-year-old woman was admitted to the hospital due to upper abdominal pain for over 8 months.According to relevant examinations,she was diagnosed as perihilar cholangiocarcinoma(pCCA)with intrahepatic metastasis and perihilar lymphatic metastasis.After multidisciplinary team discussion,percutaneous transhepatic cholangiodrainage was performed to relieve biliary obstruction,and puncture biopsy was conducted to confirm the pathological diagnosis.Transarterial chemoembolization with nab-paclitaxel was used in combination with toripalimab and lenvatinib,but the levels of tumor markers including alpha fetal protein,carcinoembryonic antigen,carbohydrate antigen 15-3 and cancer antigen 125 were still raised.The PDO for drug screening showed sensitive to gemcitabine and cisplatin.Accordingly,the chemotherapy regimen was adjusted to gemcitabine and cisplatin in combination with toripalimab and lenvatinib.After 4 cycles of treatment,the tumor was assessed resectable,and radical surgical resection was performed successfully.One year after surgery,the patient was still alive,and no recurrence or occurred.CONCLUSION PDOs for drug sensitivity contribute to screening effective chemotherapy drugs for advanced pCCA,promoting conversion therapy and improving the prognosis.

Pathologic complete response to conversion therapy in hepatocellular carcinoma using patient-derived organoids:A case report

BACKGROUND For primary liver cancer,the key to conversion therapy depends on the effectiveness of drug treatment.Patient-derived tumor organoids have been demonstrated to improve the efficacy of conversion therapy by identifying individualtargeted effective drugs,but their clinical effects in liver cancer remain unknown.CASE SUMMARY We described a patient with hepatocellular carcinoma(HCC)who achieved pathologic complete response(pCR)to conversion therapy guided by the patientderived organoid(PDO)drug sensitivity testing.Despite insufficiency of the remaining liver volume after hepatectomy,the patient obtained tumor reduction after treatment with the PDO-sensitive drugs and successfully underwent radical surgical resection.Postoperatively,pCR was observed.CONCLUSION PDOs contributes to screening sensitive drugs for HCC patients to realize the personalized treatment and improve the conversion therapy efficacy.

Strategic insights into the cultivation of pancreatic cancer organoids from endoscopic ultrasonography-guided biopsy tissue

BACKGROUND The frequent suboptimal efficacy of endoscopic ultrasound-guided fine-needle biopsy(EUS-FNB)to culture pancreatic cancer(PC)organoids(PCOs)poses a major challenge in the advancement of personalized medicine for advanced PC.AIM To explore how to obtain appropriate puncture tissues from EUS-FNB and optimize the strategy for efficiently constructing PCOs,providing an efficient tool for the advancement of personalized medicine.METHODS Patients who underwent EUS-FNB for the diagnosis of PC tissue were prospectively enrolled.We refined the endoscopic biopsy procedures and organoid cultivation techniques.All tissue specimens verified by on-site pathological assessment were cultured in a semi-suspended medium in a microfluidic environment.We assessed differences in PCOs cultured beyond and below five generations examining patient demographics,specimen and organoid attributes,and the sensitivity of organoids to a panel of clinical drugs through cell viability assays.RESULTS In this study,16 patients with PC were recruited,one sample was excluded because onsite cytopathology showed no tumor cells.Successful organoid generation occurred in 93.3%(14 of 15)of the EUS-FNB specimens,with 60%(9 of 15)sustaining over five generations.Among these patients,those with a history of diabetes,familial cancer,or larger tumors exhibited enhanced PCO expandability.The key factors influencing longterm PCOs expansion included initial needle sample quality(P=0.005),rapid initiation of organoid culture postisolation(P≤0.001),and high organoid activity(P=0.031).Drug sensitivity analysis revealed a partial response in two patients following therapeutic intervention and surgery and stable disease in four patients,indicating a moderate correlation between organoid response and clinical outcomes.CONCLUSION Optimal initial needle sampling,rapid and precise biopsy sample processing,process isolated samples as soon as possible,and sufficient cellular material are crucial for successful cultivating PCOs.High organoid activity is an important factor in maintaining their long-term expansion,which is essential for shortening the time of drug sensitivity analysis and is the basis of PC research.

The generation and properties of human cortical organoids as a disease model for malformations of cortical development

As three-dimensional“organ-like”aggregates,human cortical organoids have emerged as powerful models for studying human brain evolution and brain disorders with unique advantages of humanspecificity,fidelity and manipulation.Human cortical organoids derived from human pluripotent stem cells can elaborately replicate many of the key properties of human cortical development at the molecular,cellular,structural,and functional levels,including the anatomy,functional neural network,and interaction among different brain regions,thus facilitating the discovery of brain development and evolution.In addition to studying the neuro-electrophysiological features of brain cortex development,human cortical organoids have been widely used to mimic the pathophysiological features of cortical-related disease,especially in mimicking malformations of cortical development,thus revealing pathological mechanism and identifying effective drugs.In this review,we provide an overview of the generation of human cortical organoids and the properties of recapitulated cortical development and further outline their applications in modeling malformations of cortical development including pathological phenotype,underlying mechanisms and rescue strategies.

A quick and reliable image-based AI algorithm for evaluating cellular senescence of gastric organoids

Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passages for which organoids maintain cellular vitality ex vivo remains unclear.Methods:Herein,we constructed 55 gastric organoids from 35 individuals,serially passaged the organoids,and captured microscopic images for phenotypic evaluation.Senescence-associatedβ-galactosidase(SA-β-Gal),cell diameter in suspension,and gene expression reflecting cell cycle regulation were examined.The YOLOv3 object detection algorithm integrated with a convolutional block attention module(CBAM)was used to evaluate organoid vitality.Results:SA-β-Gal staining intensity;single-cell diameter;and expression of p15,p16,p21,CCNA2,CCNE2,and LMNB1 reflected the progression of aging in organoids during passaging.The CBAM-YOLOv3 algorithm precisely evaluated aging organoids on the basis of organoid average diameter,organoid number,and number×diameter,and the findings positively correlated with SA-β-Gal staining and single-cell diameter.Organoids derived from normal gastric mucosa had limited passaging ability(passages 1–5),before aging,whereas tumor organoids showed unlimited passaging potential for more than 45 passages(511 days)without showing clear senescence.Conclusions:Given the lack of indicators for evaluating organoid growth status,we established a reliable approach for integrated analysis of phenotypic parameters that uses an artificial intelligence algorithm to indicate organoid vitality.This method enables precise evaluation of organoid status in biomedical studies and monitoring of living biobanks.

Organoids revealed:morphological analysis of the profound next generation in-vitro model with artificial intelligence

In modern terminology,“organoids”refer to cells that grow in a specific three-dimensional(3D)environment in vitro,sharing similar structures with their source organs or tissues.Observing themorphology or growth characteristics of organoids through a microscope is a commonly used method of organoid analysis.However,it is difficult,time-consuming,and inaccurate to screen and analyze organoids only manually,a problem which cannot be easily solved with traditional technology.Artificial intelligence(AI)technology has proven to be effective in many biological and medical research fields,especially in the analysis of single-cell or hematoxylin/eosin stained tissue slices.When used to analyze organoids,AI should also provide more efficient,quantitative,accurate,and fast solutions.In this review,we will first briefly outline the application areas of organoids and then discuss the shortcomings of traditional organoid measurement and analysis methods.Secondly,we will summarize the development from machine learning to deep learning and the advantages of the latter,and then describe how to utilize a convolutional neural network to solve the challenges in organoid observation and analysis.Finally,we will discuss the limitations of current AI used in organoid research,as well as opportunities and future research directions.

Development of glioblastoma organoids and their applications in personalized therapy

Glioblastomas(GBMs)are the brain tumors with the highest malignancy and poorest prognoses.GBM is characterized by high heterogeneity and resistance to drug treatment.Organoids are 3-dimensional cultures that are constructed in vitro and comprise cell types highly similar to those in organs or tissues in vivo,thus simulating specific structures and physiological functions of organs.Organoids have been technically developed into an advanced ex vivo disease model used in basic and preclinical research on tumors.Brain organoids,which simulate the brain microenvironment while preserving tumor heterogeneity,have been used to predict patients’therapeutic responses to antitumor drugs,thus enabling a breakthrough in glioma research.GBM organoids provide an effective supplementary model that reflects human tumors’biological characteristics and functions in vitro more directly and accurately than traditional experimental models.Therefore,GBM organoids are widely applicable in disease mechanism research,drug development and screening,and glioma precision treatments.This review focuses on the development of various GBM organoid models and their applications in identifying new individualized therapies against drug-resistant GBM.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Mini-organs with big impact:Organoids in liver cancer studies

Hepatocellular carcinoma,the most common primary liver cancer and a leading cause of death,is a difficult disease to treat due to its heterogeneous nature.Traditional models,such as 2D culture and patient-derived xenografts,have not proven effective.However,the development of 3D culture techniques,such as organoids,which can mimic the tumor microenvironment(TME)and preserve heterogeneity and pathophysiological properties of tumor cells,offers new opportunities for treatment and research.Organoids also have the potential for biomarker detection and personalized medication,as well as genome editing using CRISPR/Cas9 to study the behavior of certain genes and therapeutic interventions.This review explores to-the-date development of organoids with a focus on TME modeling in 3D organoid cultures.Further,it discusses gene editing using CRISPR/Cas9 in organoids,the challenges faced,and the prospects in the field of organoids.

Benchmark for establishment of organoids from gastrointestinal epithelium and cancer based on available consumables and reagents

Gastrointestinal cancers are a public health problem that threatens the lives of human being. A good experimental model is a powerful tool to promote the uncovering pathogenesis and establish novel treatment methods. High-quality biomedical research requires experimental models to recapitulate the physiological and pathological states of their parental tissues as much as possible. Organoids are such experimental models. Organoids refer to small organlike cellular clusters formed by the expansion and passaging of living tissues in 3D culture medium in vitro.Organoids are highly similar to the original tissues in terms of cellular composition, cell functions, and genomic profiling. Organoids have many advantages, such as short preparation cycles, long-term storage based on cryopreservation, and reusability. In recent years, researchers carried out the establishment of organoids from gastrointestinal mucosa and cancer tissues, and accumulated valuable experiences. In order to promote effective usage and further development of organoid-related technologies in the research of gastrointestinal diseases, this study proposes a benchmark based on utilization of available experimental consumables and reagents, which are involved in the key steps such as collection and pretreatment of biospecimen, organoid construction, organoid cryopreservation and recovery, growth status evaluation, and organoid quality control. We believe that the standard for the construction and preservation of organoids derived from human gastrointestinal epithelium and cancer tissues can provide an important reference for the majority of scientific researchers.

Optimization of Culture Medium and Transfection Method for Head and Neck Squamous Cell Carcinoma Organoids

[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.

Influence of hypoxia on retinal progenitor and ganglion cells in human induced pluripotent stem cell-derived retinal organoids

AIM:To observe the effect of low oxygen concentration on the neural retina in human induced pluripotent stem cell(hiPSC)-derived retinal organoids(ROs).METHODS:The hiPSC and a three-dimensional culture method were used for the experiments.Generated embryoid bodies(EBs)were randomly and equally divided into hypoxic and normoxic groups.Photographs of the EBs were taken on days 38,45,and 52,and the corresponding volume of EBs was calculated.Simultaneously,samples were collected at these three timepoints,followed by fixation,sectioning,and immunofluorescence.RESULTS:The proportion of Ki67-positive proliferating cells increased steadily on day 38;this proliferationpromoting effect tended to increase tissue density rather than tissue volume.On days 45 and 52,the two groups had relatively similar ratios of Ki67-positive cells.Further immunofluorescence analysis showed that the ratio of SOX2-positive cells significantly increased within the neural retina on day 52(P<0.05).In contrast,the percentage of PAX6-and CHX10-positive cells significantly decreased following hypoxia treatment at all three timepoints(P<0.01),except for CHX10 at day 45(P>0.05).Moreover,the proportion of PAX6-/TUJ1+cells within the neural retinas increased considerably(P<0.01,<0.05,<0.05 respectively).CONCLUSION:Low oxygen promotes stemness and proliferation of neural retinas,suggesting that hypoxic conditions can enlarge the retinal progenitor cell pool in hiPSC-derived ROs.