Generation Of Organoids Research Articles

Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling

Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling

UNITEPANC: Using organoids to predict efficacy of adjuvant treatment to improve outcome in resectable pancreatic cancer—Prospective, multicenter, proof-of-concept trial of the AIO Pancreatic Cancer Group.

TPS791 Background: Pancreatic cancer (PDAC) is a highly heterogeneous disease and there is a lack of predictive biomarker to guide treatment. Patient tumor derived organoids (PDO) have been demonstrated to faithfully recapitulate morphological and genetic features of the parental tumor and also retain patient-specific heterogeneity. Retrospective studies show that PDO can be used to predict treatment sensitivity and can support the selection of an optimal treatment in terms of efficacy and also lower toxicity, e.g. by selecting a double rather than a triple combination. Methods: UNITEPANC is a prospective, proof of concept, multicenter IIT of the German AIO PDAC Group and funded by the German Cancer Aid. UNITEPANC examines the feasibility of an organoid-educated adjuvant treatment approach with respect to organoid establishment, expansion and characterization and its potential role for selecting an optimal adjuvant treatment in PDAC in a multicenter setting. Preparatory activities like harmonizing SOPs and round-robin-tests for generation of organoids and organoid-based pharmacotyping in the trial centers and the different Organoid Facilities has been completed and evaluated. UNITEPANC examines selection of adjuvant chemotherapy by pharmacotyping of tumor organoids. Options for adjuvant treatment are gemcitabine, gemcitabine/capecitabine, gemcitabine/nab-paclitaxel or mFOLFIRINOX. Major inclusion criteria are R0 or R1 resected, histologically confirmed PDAC and patients in principle, suitability for mFOLFIRINOX treatment in the adjuvant setting, postoperative Ca19-9<180 U/ml, and a timely PDO-based chemotherapy recommendation by the UNITEPANC Organoid Board within 11 weeks to guarantee a start of the adjuvant treatment within 12 weeks after resection. Design: UNITEPANC is an interventional, prospective, multicenter, single-arm trial. 95 patients shall be allocated to the trial for the generation of organoids, 38 patients shall be enrolled for PDO-based adjuvant treatment and 34 patients need to be analysed as ITT population in in the follow-up period. Efficacy: The efficacy endpoint of the trial is purely descriptive and intended to obtain a signal for further development of the strategy. This requires the proof of an efficient 1) generation and 2) expansion of PDO and 3) a clear sign that an organoid-educated treatment selection is superior to standard treatment with mFOLFIRINOX. For 1) and 2) we have chosen a rate of 75% and 60%, respectively, according to the literature. For 3): The approach would be considered promising, if the true 18-months-DFS rate is 77% (corresponding to a hazard ratio of 0.5 compared to Prodige-24, 80% power, one-sided type I error 0.1. HR-QoL data will be analyzed (QLQ-C30, Pan26). Additionally, an extensive translational program is enclosed.

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Advances in lacrimal gland organoid development: Techniques and therapeutic applications.

The human lacrimal gland (LG), located above the outer orbital region within the frontal bone socket, is essential in maintaining eye surface health and lubrication. It is firmly anchored to the orbital periosteum by the connective tissue, and it is vital for protecting and lubricating the eye by secreting lacrimal fluid. Disruption in the production, composition, or secretion of lacrimal fluid can lead to dry eye syndrome, a condition characterized by ocular discomfort and potential eye surface damage. This review explores the recent advancements in LG organoid generation using tissues and stem cells, highlighting cutting-edge techniques in biomaterial-based and scaffold-free technologies. Additionally, we shed light on the complex pathophysiology of LG dysfunction, providing insights into the LG physiological roles while identifying strategies for generating LG organoids and exploring their potential clinical applications. Alterations in LG morphology or secretory function can affect the tear film stability and quality, leading to various ocular pathological conditions. This comprehensive review underlines the critical crosslink of LG organoid development with disease modeling and drug screening, underscoring their potential for advancing therapeutic applications.

Engineering Pancreatic Organoids.

Experimental cell culture models that mimic the intricate features of organs offer significant potential for fundamental research and clinical applications. In order to enhance the growth of organoids, various matrices have been developed to replicate the essential character-istics of the tissue microenvironment through physical, chemical, and mechanical cues. Recent advancements in biomaterial technology have further refined the cultivation of organoids. This review provides an overview of the effect of different matrices, whether natural or synthetic, on the fabrication of pancreatic and islet clusters. Through a comprehensive examination of ongoing research, this paper aims to increase an understanding of pancreatic organoid genera-tion, with implications for modeling pancreatic disorders and advancing regenerative medicine.

Establishment and Characterization of Patient-Derived Oral Cancer Organoids.

Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer. The current standard for treating primary OSCC is surgical resection combined with radiotherapy and chemotherapy. Despite improved therapeutic strategies, OSCC has high rates of metastasis and mortality, with one in two patients dying of the disease. Patient-derived organoids have become promising cell culture systems for disease modeling and precision medicine. Here we describe the high-efficiency generation of organoids from OSCC patients, which can be maintained in the culture for the long term. We further provide protocols for characterizing OSCC organoids using histology and immunofluorescence staining.

A novel human specific lncRNA MEK6-AS1 regulates adipogenesis and fatty acid biosynthesis by stabilizing MEK6 mRNA

BackgroundObesity is becoming one of the major non-communicable diseases with increasing incidence and risks that cannot be ignored. However effective and safe clinical treatment strategies still need to be deeply explored. Increased number and volume of adipocytes lead to overweight and obesity. The aim of our work is to identify lncRNAs that have important regulatory in differentiation of human mesenchymal stem cells (MSCs) into adipocytes, and to provide effective targets for clinical prevention and treatment of obesity and related metabolic disorders.MethodsWe extracted primary MSCs from human adipose tissue, and conducted expression profile analysis of lncRNAs during adipogenic differentiation of MSCs to screen changed lncRNAs. Characteristics of lncRNA were revealed mainly by RACE and RNA FISH. Loss- and gain-of function experiments in vivo and in vitro were used to analyze effects of lncRNA. Targeted metabolomics was utilized to detect levels of free fatty acids. RNA pull-down, mRNA stability tests, etc. were employed to explore mechanisms of lncRNA.ResultsHuman-specific lncRNA, we named it MEK6-AS1, was the most up-regulated transcript during adipogenic differentiation of MSCs. MEK6-AS1 was highly expressed in adipose tissue samples from individuals with BMI ≥ 25 and positively correlated with adipogenic marker genes in these samples. Knocking down lncRNA inhibited expression of adipogenic differentiation markers and ectopic adipogenesis, reducing contents of various free fatty acids, as well as promoting osteogenic differentiation. Overexpression of lncRNA had the opposite effects to the above processes. We also found that MEK6-AS1 was elevated during hepatic steatosis organoid generation. Mechanistically, MEK6-AS1 worked partially through stabilization of MEK6 mRNA by NAT10.ConclusionsWe have identified a human-specific lncRNA (MEK6-AS1) with position information in the genomic database but has not been extensively reported. We demonstrated that MEK6-AS1 as a novel lncRNA involved in adipogenic differentiation and adipogenesis, fatty acid metabolism, and osteogenic differentiation. We found that MEK6-AS1 may exert its effect by enhancing MEK6 mRNA stability through NAT10. Our study may provide insights into implication of lncRNAs in stem cell biology and offer a new potential therapeutic target for the prevention and treatment of obesity and other related disease.

Protocol for Immune Cell Isolation, Organoid Generation, and Co-culture Establishment from Cryopreserved Whole Human Intestine.

The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures. Key features • Dissection and tissue analysis of whole human intestines. • Cryopreservation for biobank generation. • Optimized protocols for the isolation of epithelial and immune cells. • Autologous co-culture of organoids and lamina propria-derived immune cells.

Catalpol promotes the generation of cerebral organoids with oRGs through activation of STAT3 signaling

AbstractThe generation of human cortical organoids containing outer radial glia (oRG) cells is crucial for modeling neocortical development. Here we show that Catalpol, an iridoid glucoside derived from Rehmannia glutinosa, significantly enhances the generation of cerebral organoids with expanded oRG populations and increased neurogenic potential. Catalpol‐treated organoids exhibited thicker ventricular zone/subventricular zone (VZ/SVZ) and outer subventricular zone (oSVZ) regions, with increased numbers of SOX2 + HOPX+ and SOX2 + TNC+ oRG cells and elevated expression of oRG markers HOPX and FAM107A. We found that Catalpol promoted oRG generation through non‐vertical divisions of ventricular radial glia (vRG) cells, indicating enhanced oRG generation via asymmetrical divisions. Furthermore, we demonstrated that Catalpol augmented oRG cell numbers through activation of the STAT3 signaling pathway. These findings highlight Catalpol's potential in promoting the generation of cerebral organoids with expanded oRG populations and increased neurogenic potential through STAT3 activation, offering new insights into neocortical development modeling.

The molecular mechanisms of cardiac development and related diseases.

Cardiac development is a complex and intricate process involving numerous molecular signals and pathways. Researchers have explored cardiac development through a long journey, starting with early studies observing morphological changes and progressing to the exploration of molecular mechanisms using various molecular biology methods. Currently, advancements in stem cell technology and sequencing technology, such as the generation of human pluripotent stem cells and cardiac organoids, multi-omics sequencing, and artificial intelligence (AI) technology, have enabled researchers to understand the molecular mechanisms of cardiac development better. Many molecular signals regulate cardiac development, including various growth and transcription factors and signaling pathways, such as WNT signaling, retinoic acid signaling, and Notch signaling pathways. In addition, cilia, the extracellular matrix, epigenetic modifications, and hypoxia conditions also play important roles in cardiac development. These factors play crucial roles at one or even multiple stages of cardiac development. Recent studies have also identified roles for autophagy, metabolic transition, and macrophages in cardiac development. Deficiencies or abnormal expression of these factors can lead to various types of cardiac development abnormalities. Nowadays, congenital heart disease (CHD) management requires lifelong care, primarily involving surgical and pharmacological treatments. Advances in surgical techniques and the development of clinical genetic testing have enabled earlier diagnosis and treatment of CHD. However, these technologies still have significant limitations. The development of new technologies, such as sequencing and AI technologies, will help us better understand the molecular mechanisms of cardiac development and promote earlier prevention and treatment of CHD in the future.

Highly Stretchable 3D Microelectrode Array for Noninvasive Functional Evaluation of Cardiac Spheroids and Midbrain Organoids.

Organoids are 3D biological models that recapitulate the complex structures and functions of human organs. Despite the rapid growth in the generation of organoids, in vitro assay tools are still limited to 2D forms. Thus, a comprehensive and continuous functional evaluation of the electrogenic organoids remains a challenge. Here, a highly stretchable 3D multielectrode array (sMEA) with protruding microelectrodes is presented for functional evaluation of electrogenic organoids. The optimized serpentine structures with bridge structures cover the surface of the organoids conformally even in immersion. The protruding microelectrodes form a stable contact with the organoids and allow electrophysiological recordings with high signal-to-noise ratio (SNR). sMEAs are fabricated in wafer-scale for repeatable, scalable, and mass production and packed into an easy-to-use, user-friendly, and robust microwell for fast dissemination of technology. The versatility of sMEA is validated by measuring electrophysiological signals from cardiac spheroids and midbrain organoids with a wide range of sizes from 500 to 1500µm. Also, electrophysiological signals recorded with high SNR enable functional evaluation of the effects of drugs. The proposed sMEA with high SNR and user-friendly interface could be the key player in high-throughput drug screening, 3D spatiotemporal mapping of electrogenic organoids, and standardization of protocols for quality assessment.

105 Generation of bovine oviductal organoids with apical-out polarity

105 Generation of bovine oviductal organoids with apical-out polarity

Multiplexing cortical brain organoids for the longitudinal dissection of developmental traits at single-cell resolution.

Dissecting human neurobiology at high resolution and with mechanistic precision requires a major leap in scalability, given the need for experimental designs that include multiple individuals and, prospectively, population cohorts. To lay the foundation for this, we have developed and benchmarked complementary strategies to multiplex brain organoids by pooling cells from different pluripotent stem cell (PSC) lines either during organoid generation (mosaic models) or before single-cell RNA sequencing (scRNA-seq) library preparation (downstream multiplexing). We have also developed a new computational method, SCanSNP, and a consensus call to deconvolve cell identities, overcoming current criticalities in doublets and low-quality cell identification. We validated both multiplexing methods for charting neurodevelopmental trajectories at high resolution, thus linking specific individuals' trajectories to genetic variation. Finally, we modeled their scalability across different multiplexing combinations and showed that mosaic organoids represent an enabling method for high-throughput settings. Together, this multiplexing suite of experimental and computational methods provides a highly scalable resource for brain disease and neurodiversity modeling.

Liver organoids: From 3D printing to biomedical applications

AbstractThe liver is an immune organ, especially an immune tolerance organ. The critical shortage of donor organs and disease models for the treatment of end‐stage liver failure underscores the urgent need for the generation of liver organoids from human induced pluripotent stem cells (iPSCs). Notably, significant advancements have been made in the study of liver organoids over the past decade. The construction of liver organoids has transitioned from single cell type to multicellular models, and from two‐dimensional to three‐dimensional cultures. Here we provide the progress surrounding the different liver organoids culture techniques from 3D printing to organ‐on‐chip, as well as focuses on the present and future applications of liver organoids, and then to present challenges and perspectives ahead for further advancement.

Generation, interrogation, and future applications of microglia-containing brain organoids.

Brain organoids encompass a large collection of in vitro stem cell-derived 3D culture systems that aim to recapitulate multiple aspects of in vivo brain development and function. First, this review provides a brief introduction to the current state-of-the-art for neuro-ectoderm brain organoid development, emphasizing their biggest advantages in comparison with classical two-dimensional cell cultures and animal models. However, despite their usefulness for developmental studies, a major limitation for most brain organoid models is the absence of contributing cell types from endodermal and mesodermal origin. As such, current research is highly investing towards the incorporation of a functional vasculature and the microglial immune component. In this review, we will specifically focus on the development of immune-competent brain organoids. By summarizing the different approaches applied to incorporate microglia, it is highlighted that immune-competent brain organoids are not only important for studying neuronal network formation, but also offer a clear future as a new tool to study inflammatory responses in vitro in 3D in a brain-like environment. Therefore, our main focus here is to provide a comprehensive overview of assays to measure microglial phenotype and function within brain organoids, with an outlook on how these findings could better understand neuronal network development or restoration, as well as the influence of physical stress on microglia-containing brain organoids. Finally, we would like to stress that even though the development of immune-competent brain organoids has largely evolved over the past decade, their full potential as a pre-clinical tool to study novel therapeutic approaches to halt or reduce inflammation-mediated neurodegeneration still needs to be explored and validated.

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Generation and long-term culture of human cerebellar organoids from pluripotent stem cells.

The advancement of research on human cerebellar development and diseases has been hindered by the lack of a cell-based system that mirrors the cellular diversity and functional characteristics of the human cerebellum. Here, we describe our protocol for a human pluripotent stem cell-derived human cerebellar organoid (hCerO) model, which successfully replicates the cellular diversity of the fetal cerebellum along with some of its distinct cytoarchitectural features. Our approach involves the patterning of human pluripotent stem cells, resulting in the generation of both cerebellar excitatory and inhibitory progenitor populations-specifically, the rhombic lip and ventricular zone progenitors, respectively. This patterning strategy leads to the reproducible differentiation of the major neurons of the cerebellum such as granule cells and Purkinje cells within just one month of culture. hCerOs serve as platforms for molecular, cellular and functional assays, including single-cell transcriptomics, immunohistochemistry and investigations into calcium dynamics and electrophysiological properties. Remarkably, the cultivation of hCerOs for up to 8 months enables the healthy survival and maturation of Purkinje cells, which exhibit molecular and electrophysiological features akin to their in vivo counterparts. Overall, our protocol generates and allows for the long-term culture of all major cell types within the cerebellum. Consequently, this significant advancement provides the developmental neurobiology field with a robust platform for exploring both cerebellar development and diseases within an all-human system. This protocol can be easily implemented by a technician with cell culture experience and takes 1-2 months to complete with an option for extended maturation over the course of several months.

Organoid generation from trophoblast stem cells highlights distinct roles for cytotrophoblasts and stem cells in organoid formation and expansion.

BackgroundOrganoids are stem-cell derived, self-organised, three-dimensional cultures that improve in vitro recapitulation of tissue structure. The generation of trophoblast organoids using primary placental villous digests (containing cytotrophoblasts and trophoblast stem cells (TSC)) improved high-throughput assessment of early trophoblast differentiation. However, the relative contributions of cytotrophoblasts and TSCs to trophoblast organoid growth and differentiation remain unclear, with implications for model interpretation. Here we sought to generate organoids from side-population trophoblasts (SpTSCs) to better understand the contribution of TSC to trophoblast organoid formation. MethodsMethods were adapted from Haider et al., 2018 to generate organoids from Okae TSCs (OkTSCs) or SpTSCs. Organoid growth was compared with primary villous trophoblast organoids and cellular composition interrogated by immunohistochemistry. ResultsOrganoids can be derived from first-trimester SpTSCs that exhibit similar architecture to those from primary villous trophoblast. However, organoids established from pure TSC populations (OkTSC or SpTSC) have different growth dynamics to primary placental villous digest-derived organoids – with OkTSCs developing faster and spontaneously generating migratory cells, whilst SpTSC organoids grow more slowly. Importantly, depletion of SpTSC from first-trimester villous digests ablates organoid formation. Finally, the capacity of the side-population technique to isolate late-gestation TSC enabled the generation of trophoblast organoids from term placentae, although these were significantly smaller than their first-trimester SpTSC counterparts. DiscussionTogether, this work highlights the requirement of TSC for organoid formation, and the functional distinction between TSC and cytotrophoblasts. Proof-of-principle data demonstrating organoid generation from late gestation TSC isolated directly from the placenta lays the groundwork for future disease models.

Generation of self-organized neuromusculoskeletal tri-tissue organoids from human pluripotent stem cells.

The human body function requires crosstalk between different tissues. An essential crosstalk is in the neuromusculoskeletal (NMS) axis involving neural, muscular, and skeletal tissues, which is challenging to model using human cells. Here, we describe the generation of three-dimensional, NMS tri-tissue organoids (hNMSOs) from human pluripotent stem cells through a co-development strategy. Staining, single-nucleus RNA sequencing, and spatial transcriptome profiling revealed the co-emergence and self-organization of neural, muscular, and skeletal lineages within individual organoids, and the neural domains of hNMSOs obtained a ventral-specific identity and produced motor neurons innervating skeletal muscles. The neural, muscular, and skeletal regions of hNMSOs exhibited maturation and established functional connections during development. Notably, structural, functional, and transcriptomic analyses revealed that skeletal support in hNMSOs benefited human muscular development. Modeling with hNMSOs also unveiled the neuromuscular alterations following pathological skeletal degeneration. Together, our study provides an accessible experimental model for future studies of human NMS crosstalk and abnormality.