Enhancing organoid culture: harnessing the potential of decellularized extracellular matrix hydrogels for mimicking microenvironments.

Over the past decade, organoids have emerged as a prevalent and promising research tool, mirroring the physiological architecture of the human body. However, as the field advances, the traditional use of animal or tumor-derived extracellular matrix (ECM) as scaffolds has become increasingly inadequate. This shift has led to a focus on developing synthetic scaffolds, particularly hydrogels, that more accurately mimic three-dimensional (3D) tissue structures and dynamics in vitro. The ECM-cell interaction is crucial for organoid growth, necessitating hydrogels that meet organoid-specific requirements through modifiable physical and compositional properties. Advanced composite hydrogels have been engineered to more effectively replicate in vivo conditions, offering a more accurate representation of human organs compared to traditional matrices. This review explores the evolution and current uses of decellularized ECM scaffolds, emphasizing the application of decellularized ECM hydrogels in organoid culture. It also explores the fabrication of composite hydrogels and the prospects for their future use in organoid systems.

Establishment of the microscope incubation system and its application in evaluating tumor treatment effects through real-time live cellular imaging.

Introduction: Long-term imaging of live cells is commonly used for the study of dynamic cell behaviors. It is crucial to keep the cell viability during the investigation of physiological and biological processes by live cell imaging. Conventional incubators that providing stable temperature, carbon dioxide (CO2) concentration, and humidity are often incompatible with most imaging tools. Available commercial or custom-made stage-top incubators are bulky or unable to provide constant environmental conditions during long time culture. Methods: In this study, we reported the development of the microscope incubation system (MIS) that can be easily adapted to any inverted microscope stage. Incremental PID control algorithm was introduced to keep stable temperature and gas concentration of the system. Moreover, efficient translucent materials were applied for the top and bottom of the incubator which make it possible for images taken during culture. Results: The MIS could support cell viability comparable to standard incubators. When used in real time imaging, the MIS was able to trace single cell migration in scratch assay, T cell mediated tumor cells killing in co-culture assay, inflation-collapse and fusion of organoids in 3D culture. And the viability and drug responses of cells cultured in the MIS were able to be calculated by a label-free methods based on long term imaging. Discussion: We offer new insights into monitoring cell behaviors during long term culture by using the stage adapted MIS. This study illustrates that the newly developed MIS is a viable solution for long-term imaging during in vitro cell culture and demonstrates its potential in cell biology, cancer biology and drug discovery research where long-term real-time recording is required.

MUL1 identified as mitochondria-linked biomarker promoting cisplatin resistance in OC cells.

Ovarian cancer (OC) ranks among the prevalent tumors affecting the female reproductive system. The aim of this study was to evaluate mitochondria-associated platinum resistance genes using organoid models. Univariate Cox regression, LASSO and multivariate Cox regression analyses were performed on The Cancer Genome Atlas (TCGA) database to construct 2-gene prognostic signature (MUL1 and SSBP1), and GSE26712 dataset was used for external validation. In addition, the relationship between MUL1 and platinum resistance was examined by organoid culture, lentiviral transduction, CCK8 assay, and Western blot. The results showed that patients in the high-risk group exhibited significantly worse OS (P = 0.002, P = 0.017). Drug sensitivity analysis revealed that platinum resistance increased with the upregulation of MUL1 expression (Cor = 0.5154, P = 0.02). Our experimental findings demonstrated that knockout of the MUL1 gene significantly increased apoptosis and enhanced the sensitivity of the OC cell line A2780 to cisplatin. Through this study, we have provided strong evidence for further research on prognostic risk factors and individualized treatment in OC patients, and provided new insights into addressing platinum resistance in OC.

Modeling Radiation-Induced Epithelial Cell Injury in Murine Three-Dimensional Esophageal Organoids.

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis.

Lipid Deficiency Contributes to Impaired Alveolar Progenitor Cell Function in Aging and Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated interstitial lung disease resulting from repeated epithelial injury and inadequate epithelial repair. Alveolar type II cells (AEC2s) are progenitor cells that maintain epithelial homeostasis and repair the lung after injury. In the current study, we assessed lipid metabolism in AEC2s from human lungs of patients with IPF and healthy donors, as well as AEC2s from bleomycin-injured young and old mice. Through single-cell RNA sequencing, we observed that lipid metabolism-related genes were downregulated in IPF AEC2s and bleomycin-injured mouse AEC2s. Aging aggravated this decrease and hindered recovery of lipid metabolism gene expression in AEC2s after bleomycin injury. Pathway analyses revealed downregulation of genes related to lipid biosynthesis and fatty acid ß-oxidation in AEC2s from IPF lungs and bleomycin-injured, old mouse lungs compared with the respective controls. We confirmed decreased cellular lipid content in AEC2s from IPF lungs and bleomycin-injured, old mouse lungs using immunofluorescence staining and flow cytometry. Futhermore, we show that lipid metabolism was associated with AEC2 progenitor function. Lipid supplementation and PPARγ (peroxisome proliferator activated receptor γ) activation promoted progenitor renewal capacity of both human and mouse AEC2s in three-dimensional organoid cultures. Lipid supplementation also increased AEC2 proliferation and expression of SFTPC in AEC2s. In summary, we identified a lipid metabolism deficiency in AEC2s from lungs of patients with IPF and bleomycin-injured old mice. Restoration of lipid metabolism homeostasis in AEC2s might promote AEC2 progenitor function and offer new opportunities for therapeutic approaches to IPF.

Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids.

Human papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high-risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR-GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR-GFP vectors were transduced into patient-derived squamocolumnar junction organoids, and the presence of GFP-positive cells was evaluated. Single-cell RNA sequencing of GFP-positive and GFP-negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP-positive and GFP-negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP-positive cells, whereas keratinization and mitophagy/autophagy-related pathways were upregulated in GFP-negative cells. siRNA-mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV-related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18-infected cells downregulated stem cell-related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18-infected cervical lesions.

Live Imaging of 3D Hanging Drop Arrays through Manipulation of Light-Responsive Pyroelectric Slippery Surface and Chip Adhesion.

Three-dimensional (3D) hanging drop cell culture is widely used in organoid culture because of its lack of selection pressure and rapid cell aggregation. However, current hanging drop technology has limitations, such as a dependence on complex microfluidic transport channels or specific capillary force templates for drop formation, which leads to unchangeable drop features. These methods also hinder live imaging because of space and complexity constraints. Here, we have developed a hanging drop construction method and created a flexible 3D hanging drop construction platform composed of a manipulation module and an adhesion module. Their harmonious operation allows for the easy construction of hanging drops of varying sizes, types, and patterns. Our platform produces a cell hanging drop chip with small sizes and clear fields of view, thereby making it compatible with live imaging. This platform has great potential for personalized medicine, cancer and drug discovery, tissue engineering, and stem cell research.

From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid.

Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro. Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination.

Clonal evolution of long-term expanding head and neck cancer organoid: Impact on treatment response for personalized therapeutic screening.

OBJECTIVES: In biobanking based on patient-derived organoids (PDO), the genetic stability of organoid lines is critical for the clinical relevance of PDO with parental tumors. However, data on mutational heterogeneity and clonal evolution of PDO and their effects on treatment response are insufficient. METHODS: To investigate whether head and neck cancer organoids (HNCOs) could maintain the genetic characteristics of their original tumors and elucidate the clonal evolution process during a long-term passage, we performed targeted sequencing, covering 377 cancer-related genes and adopted a sub-clonal fraction model. To explore therapeutic response variability between an early and late passage (>passage 6), we generated dose-response curves for drugs and radiation using two HNCO lines. RESULTS: Using 3D ex vivo organoid culture protocol, we successfully established 27 HNCOs from 39 patients with an overall success rate of 70% (27/39). Their mutational profiles were highly concordant, with three of the HNCOs analyzed showing greater than 70% concordance. Only one HNCO displayed less than 50% concordance. However, many of these organoid lines displayed clonal evolution during serial passaging, although major cancer driver genes and VAF distributions were shared between early and later passages. We also found that all late passages of HNCOs tended to be more sensitive to radiation than early passages, similar to drug response results. CONCLUSIONS: We report the establishment of HNCO lines derived from 27 patients and demonstrate their genetic concordance with corresponding parental tumors. Furthermore, we show serial changes in mutational profiles of HNCO along with long passage culture and the impact of these clonal evolutions on response to radiotherapy.

Advancing Cancer Therapy Predictions with Patient-Derived Organoid Models of Metastatic Breast Cancer.

The poor outcome of metastasized breast cancer (BC) stresses the need for reliable personalized oncology and the significance of models recapitulating the heterogeneous nature of BC. Here, we cultured metastatic tumor cells derived from advanced BC patients with malignant ascites (MA) or malignant pleural effusion (MPE) using organoid technology. We identified the characteristics of tumor organoids by applying immunohistochemistry and mutation analysis. Tumor organoids preserved their expression patterns and hotspot mutations when compared to their original metastatic counterpart and are consequently a well-suited in vitro model for metastasized BC. We treated the tumor organoids to implement a reliable application for drug screenings of metastasized cells. Drug assays revealed that responses are not always in accord with expression patterns, pathway activation, and hotspot mutations. The discrepancy between characterization and functional testing underlines the relevance of linking IHC stainings and mutational analysis of metastasized BC with in vitro drug assays. Our metastatic BC organoids recapitulate the characteristics of their original sample derived from MA and MPE and serve as an invaluable tool that can be utilized in a preclinical setting for guiding therapy decisions.

Effects of Follicular Fluid on Physiological Characteristics and Differentiation of Fallopian Tube Epithelial Cells Implicating for Ovarian Cancer Pathogenesis.

The fallopian tube (FT) is an important reproductive organ in females. Ample evidence suggests that the distal end of FT is the original site of high-grade serous ovarian carcinoma (HGSC). FT may suffer from repeated injury and repair stimulated by follicular fluid (FF); however, this hypothesis has not been examined. In fact, the molecular mechanism of homeostasis, differentiation, and the transformation of fallopian tube epithelial cells (FTECs) resulting from the stimulation of FF are still enigmatic. In this study, we examined the effects of FF along with factors present in the FF on a variety of FTEC models, including primary cell culture, ALI (air-liquid interface) culture, and 3D organ spheroid culture. We found that FF plays a similar role to estrogen in promoting cell differentiation and organoid formation. Moreover, FF significantly promotes cell proliferation and induces cell injury and apoptosis in high concentrations. These observations may help us to investigate the mechanisms of the initiation of HGSC.

Establishing patient-derived organoids from human endometrial cancer and normal endometrium.

Endometrial cancer is the most common gynecologic malignancy in the United States and is one of the few malignancies that had an increasing incidence and mortality rate over the last 10 years. Current research models fail to recapitulate actual characteristics of the tumor that are necessary for the proper understanding and treatment of this heterogenous disease. Patient-derived organoids provide a durable and versatile culture system that can capture patient-specific characteristics such as the mutational profile and response to therapy of the primary tumor. Here we describe the methods for establishing, expansion and banking of endometrial cancer organoids to develop a living biobank. Samples of both endometrial tumor tissue and matched normal endometrium were collected from 10 patients. The tissue was digested into single cells and then cultured in optimized media to establish matched patient endometrial cancer and normal endometrial tissue organoids. Organoids were created from all major endometrial cancer histologic subtypes. These organoids are passaged long term, banked and can be utilized for downstream histological and genomic characterization as well as functional assays such as assessing the response to therapeutic drugs.

Multipotent Embryonic Lung Progenitors: Foundational Units of In Vitro and In Vivo Lung Organogenesis.

Transient, tissue-specific, embryonic progenitors are important cell populations in vertebrate development. In the course of respiratory system development, multipotent mesenchymal and epithelial progenitors drive the diversification of fates that results to the plethora of cell types that compose the airways and alveolar space of the adult lungs. Use of mouse genetic models, including lineage tracing and loss-of-function studies, has elucidated signaling pathways that guide proliferation and differentiation of embryonic lung progenitors as well as transcription factors that underlie lung progenitor identity. Furthermore, pluripotent stem cell-derived and ex vivo expanded respiratory progenitors offer novel, tractable, high-fidelity systems that allow for mechanistic studies of cell fate decisions and developmental processes. As our understanding of embryonic progenitor biology deepens, we move closer to the goal of in vitro lung organogenesis and resulting applications in developmental biology and medicine.

Standardization of organoid culture in cancer research.

Establishing a valid in vitro model to represent tumor heterogeneity and biology is critical but challenging. Tumor organoids are self-assembled three-dimensional cell clusters which are of great significance for recapitulating the histopathological, genetic, and phenotypic characteristics of primary tissues. The organoid has emerged as an attractive in vitro platform for tumor biology research and high-throughput drug screening in cancer medicine. Organoids offer unique advantages over cell lines and patient-derived xenograft models, but there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may affect accurate judgments of tumor biology. This review summarizes the shortcomings of current organoid culture methods, presents the latest research findings on organoid standardization, and proposes an outlook for organoid modeling.

Establishment of experimental salivary gland cancer models using organoid culture and patient-derived xenografting.

PURPOSE: Depending on its histological subtype, salivary gland carcinoma (SGC) may have a poor prognosis. Due to the scarcity of preclinical experimental models, its molecular biology has so far remained largely unknown, hampering the development of new treatment modalities for patients with these malignancies. The aim of this study was to generate experimental human SGC models of multiple histological subtypes using patient-derived xenograft (PDX) and organoid culture techniques. METHODS: Tumor specimens from surgically resected SGCs were processed for the preparation of PDXs and patient-derived organoids (PDOs). Specimens from SGC PDXs were also processed for PDX-derived organoid (PDXO) generation. In vivo tumorigenicity was assessed using orthotopic transplantation of SGC organoids. The pathological characteristics of each model were compared to those of the original tumors using immunohistochemistry. RNA-seq was used to analyze the genetic traits of our models. RESULTS: Three series of PDOs, PDXs and PDXOs of salivary duct carcinomas, one series of PDOs, PDXs and PDXOs of mucoepidermoid carcinomas and PDXs of myoepithelial carcinomas were successfully generated. We found that PDXs and orthotopic transplants from PDOs/PDXOs showed similar histological features as the original tumors. Our models also retained their genetic traits, i.e., transcription profiles, genomic variants and fusion genes of the corresponding histological subtypes. CONCLUSION: We report the generation of SGC PDOs, PDXs and PDXOs of multiple histological subtypes, recapitulating the histological and genetical characteristics of the original tumors. These experimental SGC models may serve as a useful resource for the development of novel therapeutic strategies and for investigating the molecular mechanisms underlying the development of these malignancies.

Precision Immunotherapy Utilizing Adapter CAR-T Cells (AdCAR-T) in Metastatic Breast Cancer Leads to Target Specific Lysis.

A frequent symptom of metastasized breast cancer (BC) includes the development of malignant pleural effusion (MPE), which contains malignant cells derived from the primary tumor site. The poor prognosis of MPE in metastasized BC indicates the necessity for dependable precision oncology and the importance of models representing the heterogenous nature of metastatic BC. In this study, we cultured MPE-derived metastatic tumor cells from four advanced BC patients using organoid technology. We assessed the expression of tumor-associated antigens on MPE-derived organoid lines by flow cytometry (FC). Based on an individual antigen expression pattern, patient-derived organoids were treated with adapter CAR-T cells (AdCAR-T) and biotinylated monoclonal antibodies targeting CD276, HER2, EGFR, TROP2, or EpCAM. Co-culture assays revealed specific organoid lysis by AdCAR-T depending on individual antigen expression patterns. Our results demonstrate that MPE-derived organoids can serve as a reliable tool for assessing the efficacy of AdCAR-T on metastatic BC in a patient-individualized manner. This approach could potentially be applied in a preclinical setting to instruct therapy decisions. Further, our study demonstrates the feasibility of precision immunotherapy utilizing AdCAR-T to target patient-individualized antigen patterns.

Tumor Organoids as a Research Tool: How to Exploit Them.

Organoid models allow for the study of key pathophysiological processes such as cancer biology in vitro. They offer insights into all aspects covering tumor development, progression and response to the treatment of tissue obtained from individual patients. Tumor organoids are therefore not only a better tumor model than classical monolayer cell cultures but can be used as personalized avatars for translational studies. In this review, we discuss recent developments in using organoid models for cancer research and what kinds of advanced models, testing procedures and readouts can be considered.

Organoids From Mucinous Appendiceal Adenocarcinomas as High-Fidelity Models for Individual Therapy.

Background: Mucinous appendiceal adenocarcinoma (MAA) is a rare, heterogeneous disease. Patients with unrespectable mucinous appendiceal adenocarcinoma presenting with peritoneal spread are treated by intraperitoneal chemotherapy, hyperthermic intraperitoneal chemotherapy, systemic chemotherapy, or targeted therapy. However, there are no guidelines for efficacious drugs against mucinous appendiceal adenocarcinoma. Therefore, relevant high-fidelity models should be investigated to identify effective drugs for individual therapy. Methods: Surgical tumor specimens were obtained from a mucinous appendiceal adenocarcinoma patient. The tissue was digested and organoid culture was established. H&E and immunohistochemistry staining as well as DNA sequencing was performed on tissue and organoid. The pathological characteristics and gene mutations of the organoid were compared to those of the original tumor. Drug sensitivity tests were performed on organoid and the patient clinical responds to chemotherapy and targeted therapy was compared. Results: Organoids were successfully established and stably passaged. Pathological characteristics of organoids including H&E staining and expression of protein markers (CK20, CDX-2, STAB2, CD7, PAX8) were consistent to those of the original tumor. Moreover, the organoids carried the same gene mutations as the primary tumor. Sensitivity of the organoids to chemotherapeutic drugs and tyrosine kinase inhibitors included: 5-FU (IC50 43.95 µM), Oxaliplatin (IC50 23.49 µM), SN38 (IC50 1.02 µM), Apatinib (IC50 0.10 µM), Dasatinib (IC50 2.27 µM), Docetaxel (IC50 5.26 µM), Regorafenib (IC50 18.90 µM), and Everolimus (IC50 9.20 µM). The sensitivities of organoid to these drugs were comparable to those of the patient's clinical responses. Conclusion: The mucinous appendiceal adenocarcinoma organoid model which retained the characteristics of the primary tumor was successfully established. Combined organoid-based drug screening and high throughput sequencing provided a promising way for mucinous appendiceal adenocarcinoma treatment.

Establishment of Organoids From Human Epithelioid Sarcoma With the Air-Liquid Interface Organoid Cultures.

Background: Although biological resources are essential for basic and preclinical research in the oncological field, those of sarcoma are not sufficient for rapid development of the treatment. So far, some sarcoma cell lines have been established, however, the success rate was low and the established sarcoma types were frequently biased. Therefore, an efficient culture method is needed to determine the various types of sarcomas. Organoid culture is a 3-dimentional culture method that enables the recapitulation of the tumor microenvironment and the success rate reported is higher than the 2-dimentional culture. The purpose of this study was to report our newly established organoids from human epithelioid sarcoma using the air-liquid interface organoid culture method. Methods: We treated 2 patients with epithelioid sarcoma in our institute. The remaining sarcoma specimens after surgical resection were embedded in collagen type 1 gels according to the air-liquid interface organoid culture method. After serial passages, we xenografted the organoids to NOD-scid IL2Rgnull (NSG) mice. Using the developed tumors, we performed histological and genomic analyses to compare the similarities and differences with the original epithelioid sarcoma from the patient. Results: Organoids from the epithelioid sarcoma could be serially cultured and maintained in collagen type 1 gels for more than 3 passages. Developed orthotopic tumor xenografts were detected in the NSG mice. After the process was repeated severally, the patient derived organoid lines from the epithelioid sarcoma were established. The established organoids showed loss of integrase interactor 1 expression with polymerase chain reaction and immunohistochemical analyses. The xenografted organoids of the epithelioid sarcoma had histologically similar phenotypes with the original tumor and genetically resembled it to some degree. Conclusions: The present study demonstrated 2 novel established organoid models of epithelioid sarcoma, and our organoid models could be used to investigate the molecular pathogenesis and develop a novel treatment.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR T cells.

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αß T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αß-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.