Malignant tumors notably decrease life expectancy. Despite advances in cancer diagnosis and treatment, the mechanisms underlying tumorigenesis, progression and drug resistance have not been fully elucidated. An emerging method to study tumors is tumor organoids, which are a three‑dimensional miniature structure. These retain the patient‑specific tumor heterogeneity while demonstrating the histological, genetic and molecular features of original tumors. Compared with conventional cancer cell lines and animal models, patient‑derived tumor organoids are more advanced at physiological and clinical levels. Their synergistic combination with other technologies, such as organ‑on‑a‑chip, 3D‑bioprinting, tissue‑engineered cell scaffolds and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9, may overcome limitations of the conventional 3D organoid culture and result in the development of more appropriate model systems that preserve the complex tumor stroma, inter‑organ and intra‑organ communications. The present review summarizes the evolution of tumor organoids and their combination with advanced technologies, as well as the application of tumor organoids in basic and clinical research.

Porcine reproductive and respiratory syndrome (PRRS) and pseudorabies (PR) are still the major problems of the worldwide pork industry. Previous studies showed that PR virus (PRV) and PRRS virus (PRRSV) commercial vaccines available in China could not provide complete protection against PRV variants and currently prevalent PRRSV strains. In the present study, a recombinant pseudorabies virus rPRV-GP5/HP-GP5/NA expressing the GP5 of the highly pathogenic PRRSV (HP-PRRSV) and NADC30-like PRRSV was constructed by transfecting the transfer plasmid pG-GP5/HP-GP5/NA-EGFP into ST cells inoculated with gE/gI/TK-gene-deleted rPRV NY-gE-/gI-/TK- using homologous recombination and CRISPR/Cas9 gene editing technique. The recombinant virus rPRV-GP5/HP was also constructed. The expression of the GP5 protein was confirmed by Western blot and indirect immunofluorescence assay. These two viruses were similar to the parental virus rPRV-gE-/gI-/TK- in terms of growth curve, morphogenesis and virus plaque sizes, and proliferated in different cell types. The animal test results showed that ELISA antibodies against PRRSV could be detected in piglets immunized with these two recombinant viruses, and the antibody levels were slightly lower than those of commercial vaccines, but these two recombinant viruses elicited high levels of PRV ELISA antibody and neutralizing antibody, as is the case with commercial vaccine. These two recombinant viruses could provide some protection against virulent PRRSV and PRV, and could effectively inhibit virus proliferation in tissues. These findings provide insights that these two viruses need to be optimally engineered as promising bivalent vaccine candidates against PRV and PRRSV for the control and eradication of the variant PRV and currently prevalent PRRSV. IMPORTANT: Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine pseudorabies virus (PRV) can infect pigs of all ages with high mortality. Due to the appearance of the PRRSV variant (HP-PRRSV and NADC30-Like-PRRSV) and the outbreak of PRV variant in China, the current commercial vaccines available cannot provide complete protection against PRV variants and prevalent PRRSV strains, which causes a major economic loss in pig industry worldwide. Therefore, safe and effective new vaccines are urgently developed to simultaneously control and even eradicate the two viruses. This study intends to use the modified attenuated PRV strain as the carrier and the main antigen gene ORF5 of PRRSV as the exogenous gene to construct the recombinant virus strain, and further validate the protective effect of recombinant strains in vitro and in vivo against the challenge of PRRSV and PRV, which is expected to become a candidate vaccine strain.

The liver fulfils a plethora of vital functions and, due to their importance, liver dysfunction has life-threatening consequences. Liver disorders currently account for more than two million deaths annually worldwide and can be classified broadly into three groups, considering their onset and aetiology, as acute liver diseases, inherited metabolic disorders and chronic liver diseases. In the most advanced and severe forms leading to liver failure, liver transplantation is the only treatment available, which has many associated drawbacks, including a shortage of organ donors. Cell therapy via fully mature cell transplantation is an advantageous alternative that may be able to restore a damaged organ's functionality or serve as a bridge until regeneration can occur. Pioneering work has shown that transplanting adult hepatocytes can support liver recovery. However, primary hepatocytes cannot be grown extensively in vitro as they rapidly lose their metabolic activity. Therefore, different cell sources are currently being tested as alternatives to primary cells. Human pluripotent stem cell-derived cells, chemically induced liver progenitors, or 'liver' organoids, hold great promise for developing new cell therapies for acute and chronic liver diseases. This Review focuses on the advantages and drawbacks of distinct cell sources and the relative strategies to address different therapeutic needs in distinct liver diseases.

Over the last decade, a plethora of organoid models have been generated to recapitulate aspects of human development, disease, tissue homeostasis, and repair. Organoids representing multiple tissues have emerged and are typically categorized based on their origin. Tissue-derived organoids are established directly from tissue-resident stem/progenitor cells of either adult or fetal origin. Starting from pluripotent stem cells (PSCs), PSC-derived organoids instead recapitulate the developmental trajectory of a given organ. Gene editing technologies, particularly the CRISPR-Cas toolbox, have greatly facilitated gene manipulation experiments with considerable ease and scalability, revolutionizing organoid-based human biology research. Here, we review the recent adaptation of CRISPR-based screenings in organoids. We examine the strategies adopted to perform CRISPR screenings in organoids, discuss different screening scopes and readouts, and highlight organoid-specific challenges. We then discuss individual organoid-based genome screening studies that have uncovered novel genes involved in a variety of biological processes. We close by providing an outlook on how widespread adaptation of CRISPR screenings across the organoid field may be achieved, to ultimately leverage our understanding of human biology.

BEST4/CA7+ cells of the human intestine were recently identified by single-cell RNA sequencing. While their gene expression profile predicts a role in electrolyte balance, BEST4/CA7+ cell function has not been explored experimentally owing to the absence of BEST4/CA7+ cells in mice and the paucity of human in vitro models. Here, we establish a protocol that allows the emergence of BEST4/CA7+ cells in human intestinal organoids. Differentiation of BEST4/CA7+ cells requires activation of Notch signaling and the transcription factor SPIB. BEST4/CA7+ cell numbers strongly increase in response to the cytokine interferon-γ, supporting a role in immunity. Indeed, we demonstrate that BEST4/CA7+ cells generate robust CFTR-mediated fluid efflux when stimulated with bacterial diarrhea-causing toxins and find the norepinephrine-ADRA2A axis as a potential mechanism in blocking BEST4/CA7+ cell-mediated fluid secretion. Our observations identify a central role of BEST4/CA7+ cells in fluid homeostasis in response to bacterial infections.

Cardiac organoids offer sophisticated 3D structures that emulate key aspects of human heart development and function. This review traces the evolution of cardiac organoid technology, from early stem cell differentiation protocols to advanced bioengineering approaches. We discuss the methodologies for creating cardiac organoids, including self-organization techniques, biomaterial-based scaffolds, 3D bioprinting, and organ-on-chip platforms, which have significantly enhanced the structural complexity and physiological relevance of in vitro cardiac models. We examine their applications in fundamental research and medical innovations, highlighting their potential to transform our understanding of cardiac biology and pathology. The integration of multiple cell types, vascularization strategies, and maturation protocols has led to more faithful representations of the adult human heart. However, challenges remain in achieving full functional maturity and scalability. We critically assess the current limitations and outline future directions for advancing cardiac organoid technology. By providing a comprehensive analysis of the field, this review aims to catalyze further innovation in cardiac tissue engineering and facilitate its translation to clinical applications.

Site-directed exogenous gene knock-in for stable cell line generation remains a multi-step procedure that heavily relies on expertise. Therefore, there is a need for a competent and easily manageable method, particularly when there is an urgent demand for cell lines, especially for emerging infection research. We present here a universal construct called CutIn that expresses the Cas9 protein and dual sgRNAs targeting a host cell genome locus and the ampicillin resistance (AmpR) gene of a cotransfected donor plasmid commercially available. This construct specifically induces double-strand breaks (DSBs) in cotransfected plasmids and host cell genomes, thereby facilitating whole plasmid integration through nonhomologous end joining (NHEJ) repair mechanisms. As pilot tests, adeno-associated virus integration site 1 (AAVS1) or hypoxanthine phosphoribosyl transferase (HPRT) locus was selected as host genome target, commonly used human cell lines 293T, HeLa and HCT116 were employed. CutIn was subjected for reporter plasmid knock-in in all three cell lines, either AAVS1 and AmpR or HPRT and AmpR loci were efficiently targeted. Fluorescent protein, human angiotensin-converting enzyme 2 (ACE2) and dengue virus (DENV) infection reporter transgenic cells were rapidly obtained via CutIn-mediated whole expression vector integration. This method is designed to be user-friendly and shows potential for supporting the investigation of emerging/re-emerging infectious diseases. Further validation in diverse research contexts will be necessary to fully assess its applicability and effectiveness.

The importance of the gut in regulating the brain-body immune axis is becoming increasingly evident. Interestingly, the brain and gut share many common signaling molecules, with serotonin being one of the most notable. In fact, the gut is the primary source of serotonin in the body. However, studying serotonin dynamics in a human-specific context remains a challenge. Human stem cell-derived models provide a promising avenue for studying signal transmission in well-controlled, in vitro environments. In this study, we report the first fast-scan cyclic voltammetry (FSCV) measurements of serotonin signaling in a newly developed enterochromaffin cell (ECC)-enriched gut organoid model. First, we characterize the stem cell-derived gut organoids and confirm they are enriched with ECCs, the key cell type responsible for producing and releasing serotonin in the gut. We then optimize an in vitro buffer that maintains cell viability while supporting FSCV measurements. Using this system, we detect spontaneous release events, which increase in frequency and amplitude following stimulation with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX). Finally, we confirm the identity of the signal as serotonin using a selective serotonin reuptake inhibitor (SSRI), which significantly delayed the reuptake profile. Our study introduces the first real-time measurement of serotonin signaling in a human-derived gut model. We believe this system will be essential for future research on serotonin's role in the gut and for potential novel drug target identification.

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

Genome-wide functional genetic screening has been widely used in the biomedicine field, which makes it possible to find a needle in a haystack at the genetic level. In cancer research, gene mutations are closely related to tumor development, metastasis, and recurrence, and the use of state-of-the-art powerful screening technologies, such as clustered regularly interspaced short palindromic repeat (CRISPR), to search for the most critical genes or coding products provides us with a new possibility to further refine the cancer mapping and provide new possibilities for the treatment of cancer patients. The use of CRISPR screening for the most critical genes or coding products has further refined the cancer atlas and provided new possibilities for the treatment of cancer patients. Immunotherapy, as a highly promising cancer treatment method, has been widely validated in the clinic, but it could only meet the needs of a small proportion of cancer patients. Finding new immunotherapy targets is the key to the future of tumor immunotherapy. Here, we revisit the application of functional screening in cancer immunology from different perspectives, from the selection of diverse in vitro and in vivo screening models to the screening of potential immune checkpoints and potentiating genes for CAR-T cells. The data will offer fresh therapeutic clues for cancer patients.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer-related mortality worldwide1,2. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulate progressive driver mutations3, with hepatocytes the most likely cell of origin2. However, the landscape of driver mutations in HCC is broadly independent of the underlying aetiologies4. Despite an increasing range of systemic treatment options for advanced HCC, outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations5,6. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC7. Here we generated a suite of genetically driven immunocompetent in vivo and matched in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance and metastasis. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with the human histopathology. In a proof-of-principle analysis, we verified response to standard-of-care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not previously been linked to HCC treatment. Cladribine acts in a highly effective subtype-specific manner in combination with standard-of-care therapy.

Taxanes and platinum molecules, specifically paclitaxel and carboplatin, are widely used anticancer drugs that induce cell death and serve as first-line chemotherapy for various cancer types. Despite the efficient effect of both drugs on cancer cell proliferation, many tumours have innate resistance against paclitaxel and carboplatin, which leads to inefficient treatment and poor survival rates. Haploid human embryonic stem cells (hESCs) are a novel and robust platform for genetic screening. To gain a comprehensive view of genes that affect or regulate paclitaxel and carboplatin resistance, genome-wide loss-of-function screens in haploid hESCs were performed. Both paclitaxel and carboplatin screens have yielded selected plausible gene lists and pathways relevant to resistance prediction. The effects of mutations in selected genes on the resistance to the drugs were demonstrated. Based on the results, an algorithm that can predict resistance to paclitaxel or carboplatin was developed. Applying the algorithm to the DNA mutation profile of patients' tumours enabled the separation of sensitive versus resistant patients, thus, providing a prediction tool. As the anticancer drugs arsenal can offer alternatives in case of resistance to either paclitaxel or carboplatin, an early prediction can provide a significant advantage and should improve treatment. The algorithm assists this unmet need and helps predict whether a patient will respond to the treatment and may have an immediate clinically actionable application.

Organoids have revolutionized in vitro research by offering three-dimensional, multicellular systems that recapitulate the structure, function, and genetics of human tissues. Initially developed from both pluripotent stem cells (PSCs) and adult stem cells (AdSCs), organoids have expanded to model nearly every major human organ, significantly advancing developmental biology, disease modeling, and therapeutic screening. This review highlights the progression of organoid technologies, emphasizing the integration of genetic tools, including CRISPR-Cas9, prime editing, and lineage tracing. These advancements have facilitated precise modeling of human-specific pathologies and drug responses, often surpassing traditional 2D cultures and animal models in accuracy. Emerging technologies, such as organoid fusion, xenografting, and optogenetics, are expected to further enhance our understanding of cellular interactions and microenvironmental dynamics. As organoid complexity and genetic engineering methods continue to evolve, they will become increasingly indispensable for personalized medicine and translational research, bridging gaps between in vitro and in vivo systems.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

Organoid culture has emerged as a forefront technology in the life sciences field. As "in vitro micro-organs", organoids can faithfully recapitulate the organogenesis process, and conserve the key structure, physiological function and pathological state of the original tissue or organ. Consequently, it is widely used in basic and clinical studies, becoming important preclinical models for studying diseases and developing therapies. Here, we introduced the definition and advantages of organoids and described the development and advances in hepatobiliary organoids research. We focus on applying hepatobiliary organoids in benign and malignant diseases of the liver and biliary tract, drug research, and regenerative medicine to provide valuable reference information for the application of hepatobiliary organoids. Despite advances in research and treatment, hepatobiliary diseases including carcinoma, viral hepatitis, fatty liver and bile duct defects have still been conundrums of the hepatobiliary field. It is necessary and crucial to study disease mechanisms, establish efficient and accurate research models and find effective treatment strategies. The organoid culture technology shed new light on solving these issues. However, the technology is not yet mature, and many hurdles still exist that need to be overcome. The combination with new technologies such as CRISPR-HOT, organ-on-a-chip may inject new vitality into future development.

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.

The liver is a vital organ responsible for numerous metabolic processes in the human body, including the metabolism of drugs and nutrients. After liver damage, the organ can rapidly return to its original size if the causative factor is promptly eliminated. However, when the harmful stimulus persists, the liver's regenerative capacity becomes compromised. Substantial theoretical feasibility has been demonstrated at the levels of gene expression, molecular interactions, and intercellular dynamics, complemented by numerous successful animal studies. However, a robust model and carrier that closely resemble human physiology are still lacking for translating these theories into practice. The potential for liver regeneration has been a central focus of ongoing research. Over the past decade, the advent of organoid technology has provided improved models and materials for advancing research efforts. Liver organoid technology represents a novel in vitro culture system. After several years of refinement, human liver organoids can now accurately replicate the liver's morphological structure, nutrient and drug metabolism, gene expression, and secretory functions, providing a robust model for liver disease research. Regenerative medicine aims to replicate human organ or tissue functions to repair or replace damaged tissues, restore their structure or function, or stimulate the regeneration of tissues or organs within the body. Liver organoids possess the same structure and function as liver tissue, offering the potential to serve as a viable replacement for the liver, aligning with the goals of regenerative medicine. This review examines the role of liver organoids in regenerative medicine.

Stem cells (SCs) can self-replicate and differentiate into multiple lineages. Organoids, 3D cultures derived from SCs, can replicate the spatial structure and physiological characteristics of organs in vitro. Skin organoids can effectively simulate the physiological structure and function of skin tissue, reliably restoring the natural skin ecology in various in vitro environments. Skin organoids have been employed extensively in skin development and pathology research, offering valuable insights for drug screening. Moreover, they play crucial roles in skin regeneration and tissue repair. This in-depth review explores the construction and applications of skin organoids in wound healing, with a focus on their construction process, including skin appendage integration, and significant advancements in wound-healing research.

People with cystic fibrosis carrying two nonsense alleles lack CFTR-specific treatment. Growing evidence supports the hypothesis that nonsense mutation identity affects therapeutic response, calling for mutation-specific CF models. We describe a novel W1282X mouse model and compare it to an existing G542X mouse.

The W1282X mouse was created using CRISPR/Cas9 to edit mouse Cftr. In this model, Cftr transcription was assessed using qRT-PCR and CFTR function was measured in the airway by nasal potential difference and in the intestine by short circuit current. Growth, survival, and intestinal motility were examined as well. Correction of W1282X CFTR was assessed pharmacologically and by gene-editing using a forskolin-induced swelling (FIS) assay in small intestine-derived organoids.

Homozygous W1282X mice demonstrate decreased Cftr mRNA, little to no CFTR function, and reduced survival, growth, and intestinal motility. W1282X organoids treated with various combinations of pharmacologic correctors display a significantly different amount of CFTR function than that of organoids from G542X mice. Successful gene editing of W1282X to wildtype sequence in intestinal organoids was achieved leading to restoration of CFTR function.

The W1282X mouse model recapitulates common human manifestations of CF similar to other CFTR null mice. Despite the similarities between the congenic W1282X and G542X models, they differ meaningfully in their response to identical pharmacological treatments. This heterogeneity highlights the importance of studying therapeutics across genotypes.

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8-17 (8-17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency in vitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion in vitro and of generating all three pancreatic cell lineages.

Organoids are three-dimensional models of microscopic organisms created through the self-organization of various types of stem cells. They are widely unitized in personalized medicine due to their capacity to replicate the structure and functionality of native organs. Meanwhile, nanotechnology has been integrated into diagnostic and therapeutic tools to manage an array of medical conditions, given its unique characteristics of nanoscale. Nanomaterials have demonstrated potential in developing innovative and effective organoids. With a focus on studying the interaction of nanomaterials and organoid technology in personalized medicine, this Review examines the role of nanomaterials in regulating the fate of stem cells to construct different types of organoids. It also explores the potential of nanotechnology to create 3D microenvironments for organoids. Finally, perspectives and challenges of applying nanotechnology for organoids development toward the translation of personalized medicine are discussed.

Colorectal cancer (CRC) ranks among the most prevalent gastrointestinal tumors globally and poses a significant threat to human health. In recent years, tumor organoids have emerged as ideal models for clinical disease research owing to their ability to closely mimic the original tumor tissue and maintain a stable phenotypic structure. Organoid technology has found widespread application in basic tumor research, precision therapy, and new drug development, establishing itself as a reliable preclinical model in CRC research. This has significantly advanced individualized and precise tumor therapies. Additionally, the integration of single-cell technology has enhanced the precision of organoid studies, offering deeper insights into tumor heterogeneity and treatment response, thereby contributing to the development of personalized treatment approaches. This review outlines the evolution of colorectal cancer organoid technology and highlights its strengths in modeling colorectal malignancies. This review also summarizes the progress made in precision tumor medicine and addresses the challenges in organoid research, particularly when organoid research is combined with single-cell technology. Furthermore, this review explores the future potential of organoid technology in the standardization of culture techniques, high-throughput screening applications, and single-cell multi-omics integration, offering novel directions for future colorectal cancer research.

All the information essential for life is encoded within our genome and epigenome, which orchestrates diverse cellular states spatially and temporally. In particular, the epigenome interacts with internal and external stimuli, encoding and preserving cellular experiences, and it serves as the regulatory base of the transcriptome across diverse cell types. The emergence of single-cell transcriptomic and epigenomic data collection has revealed unique omics signatures in diverse tissues, highlighting cellular heterogeneity. Recent research has documented age-related epigenetic changes at the single-cell level, alongside the validation of cellular rejuvenation through partial reprogramming, which involves simultaneous epigenetic modifications. These dynamic shifts, primarily fueled by stem cell plasticity, have catalyzed significant interest and cross-disciplinary research endeavors. This review explores the genomic and epigenomic alterations with aging, elucidating their reciprocal interactions. Additionally, it seeks to discuss the evolving landscape of rejuvenation research, with a particular emphasis on dissecting stem cell behavior through the lens of single-cell analysis. Moreover, it proposes potential research methodologies for future studies.

One of the most famous and heterogeneous cancers worldwide is breast cancer (BC). Owing to differences in the gene expression profiles and clinical features of distinct BC subtypes, different treatments are prescribed for patients. However, even with more thorough pathological evaluations of tumors than in the past, available treatments do not perform equally well for all individuals. Precision medicine is a new approach that considers the effects of patients' genes, lifestyle, and environment to choose the right treatment for an individual patient. As a powerful tool, the organoid culture system can maintain the morphological and genetic characteristics of patients' tumors. Evidence also shows that organoids have high predictive value for patient treatment. In this review, a variety of BC studies performed on organoid culture systems are evaluated. Additionally, the potential of using organoid models in BC translational research, especially in immunotherapy, drug screening, and precision medicine, has been reported.

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.

Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remains unexplored in plants. Here, we introduce the in locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, a result attributable to low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence up to 6.9 fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in situ visualization using a cross-breeding strategy, and effective immunoprecipitation for analysis of protein interactions. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

Caco-2 cells, a human colorectal adenocarcinoma cell line, are widely used to model small intestinal epithelial cells in the drug development process because they can predict drug absorption with high accuracy. However, Caco-2 cells have several issues. First, Caco-2 cells have little expression of cytochrome P450 3A4 (CYP3A4), which is a major drug-metabolizing enzyme in the human intestine. We previously developed Caco-2 cells whose expression of CYP3A4 can be controlled using doxycycline (Dox) (CYP3A4-Caco-2 cells) (Ichikawa et al., Sci. Rep, 2021). However, since the Tet-On system was used to regulate CYP3A4 expression in these cells, there was concern about drug-drug interactions. The second issue is that in the human small intestine, carboxylesterase 2 (CES2) is more highly expressed than carboxylesterase 1 (CES1), while in Caco-2 cells CES1 is more highly expressed. The third issue is the low level expression of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a phase II drug-metabolizing enzyme. In this study, we used genome-editing technology to establish CES1-knockout Caco-2 cells whose CYP3A4 and UGT1A1 expression can be regulated by the Tet-Off system. These cell lines would be useful in pharmaceutical researches, including intestinal toxicological studies, as an in vitro model for orally administered drugs.

There are lots of gene editing tools for targeting genome sequences. Some are almost known, and most are a complete mystery and undiscovered. CRISPR/Cas editing tools have brought about a major revolution in medicine. Researchers have shown that CRISPR can modify DNA much more accurately, economically and easily than previous methods. CRISPR has proven itself effective for the deletion, replacement and insertion of DNA fragments into cell types, tissues and organisms. Recently, combining CRISPR/Cas with factors (transcription factors/repressors, exonucleases, endonucleases, transposons, caspase, fluorescent proteins, oxidoreductive enzymes, DNA/RNA polymerases), and elements (aptamers, barcodes, fluorescent probes, Trigger) have provided genome, transcriptome, proteome and epigenome modification. These modules are being investigated for cancer prevention and therapy and this review focuses on such innovative combinations that hopefully will become a clinical reality in the near future.

Enteroendocrine cells (EECs) are gut epithelial cells that respond to intestinal contents by secreting hormones, including the incretins glucagon-like peptide 1 (GLP-1) and gastric inhibitory protein (GIP), which regulate multiple physiological processes. Hormone release is controlled through metabolite-sensing proteins. Low expression, interspecies differences, and the existence of multiple EEC subtypes have posed challenges to the study of these sensors. We describe differentiation of stomach EECs to complement existing intestinal organoid protocols. CD200 emerged as a pan-EEC surface marker, allowing deep transcriptomic profiling from primary human tissue along the stomach-intestinal tract. We generated loss-of-function mutations in 22 receptors and subjected organoids to ligand-induced secretion experiments. We delineate the role of individual human EEC sensors in the secretion of hormones, including GLP-1. These represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity, and mucosal immunity.

In mice, intestinal tuft cells have been described as a long-lived, postmitotic cell type. Two distinct subsets have been identified: tuft-1 and tuft-2 (ref. 1). By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase on interleukin-4 (IL-4) and IL-13 exposure, as reported previously in mice2-4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

Diseases of the female reproductive system, especially malignant tumors, pose a serious threat to women's health worldwide. One of the key factors limiting research progress in this area is the lack of representative models. Organoid technology, especially tumor organoids, has been increasingly applied in the study of female reproductive system tumors due to their high heterogeneity, close resemblance to the physiological state, easy acquisition and cultivation advantages. They play a significant role in understanding the origin and causes of tumors, drug screening, and personalized treatment and more. This article reviews the organoid models for the female reproductive system, focusing on the cancer research advancements. It discusses the methods for constructing tumor organoids of the female reproductive tract and summarizes the limitations of current research. The aim is to offer a reference for future development and application of these organoid models, contributing to the advancement of anti-tumor drugs and treatment strategies for female reproductive tract cancer patients.

The intertwined interactions various immune cells have with epithelial cells in our body require sophisticated experimental approaches to be studied. Due to the limitations of immortalized cell lines and animal models, there is an increasing demand for human in vitro model systems to investigate the microenvironment of immune cells in normal and in pathological conditions. Organoids, which are self-renewing, 3D cellular structures that are derived from stem cells, have started to provide gap-filling tissue modelling solutions. In this review, we first demonstrate with some of the available examples how organoid-based immune cell co-culture experiments can advance disease modelling of cancer, inflammatory bowel disease, and tissue regeneration. Then, we argue that to achieve both complexity and scale, organ-on-chip models combined with cutting-edge microfluidics-based technologies can provide more precise manipulation and readouts. Finally, we discuss how genome editing techniques and the use of patient-derived organoids and immune cells can improve disease modelling and facilitate precision medicine. To achieve maximum impact and efficiency, these efforts should be supported by novel infrastructures such as organoid biobanks, organoid facilities, as well as drug screening and host-microbe interaction testing platforms. All these together or in combination can allow researchers to shed more detailed, and often patient-specific, light on the crosstalk between immune cells and epithelial cells in health and disease.

Organoids refer to 3D cultures established to recapitulate histology, pathology, architecture, and genetic traits of various organs and tissues in the body, thereby replacing 2D cell cultures, xenograft, and animal models. Organoids form a 3D in vitro mimic of original tissues like the liver and are derived from embryonic or adult tissue stem cells. Liver and bile duct tumor organoids, also called, tumoroids capture genetic diversity, cellular, and pathophysiological properties of original tumors. Moreover, co-culture techniques along with genetic modulation of organoids allow for using tumoroids in liver and bile duct cancer research and drug screening/testing. Therefore, tumoroids are promising platforms for studying liver and bile duct cancer, which paves the way for the new era of personalized therapies. In the current review, we aimed to discuss liver and bile duct organoids with special emphasis on tumoroids and their applications, advantages, and shortcomings.

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

Organoid technology has been developed rapidly in the past decade, which involves the exploration of the mechanism of development, regeneration and various diseases, and intersects among multiple disciplines. Thousands of literature on 3D-culture or organoids have been published in the research areas of cell biology tissue engineering, nanoscience, oncology and so on, resulting in it being challenging for researchers to timely summarize these studies. Bibliometric statistics is a helpful way to help researchers clarify the above issues efficiently and manage the whole landscape systematically. In our study, all original articles on organoids were included in the Web of Science database from January 2009 to May 2024, and related information was collected and analyzed using Excel software, "bibliometrix" packages of the R software, VOSviewer and CiteSpace. As results, a total of 6222 papers were included to classify the status quo of the organoids and predict future research areas. Our findings highlight a growing trend in publications related to organoids, with the United States and Netherlands leading in this field. The University of California System, Harvard University, Utrecht University and Utrecht University Medical Center have emerged as pivotal contributors and the key authors in the field include Clevers, H, Beekman, JM and Spence JR. Our results also revealed that the research hotspots and trends of organoids mainly focused on clinical treatment, drug screening, and the application of materials and technologies such as "hydrogel" and "microfluidic technology" in organoids. Next, we had an in-depth interpretation of the development process of organoid research area, including the emergence of technology, the translation from bench to bedsides, the profiles of the most widely studied types of organoids, the application of materials and technologies, and the emerging organoid-immune co-cultures trends. Furthermore, we also discussed the pitfalls, challenges and prospects of organoid technology. In conclusion, this study provides readers straightforward and convenient access to the organoid research field.