Pluripotent Stem Cell-derived Organoids Research Articles

Organalysis: Multifunctional Image Preprocessing and Analysis Software for Cardiac Organoid Studies.

Due to a growing need in visualizing human pluripotent stem cell-derived organoids from recent advancements in the field, an efficient bulk-processing application is necessary to provide preprocessing and image analysis services. In this study, we developed Organalysis, a high-accuracy, multifunctional, and accessible application that meets these needs by providing the functionality of image manipulation and enhancement, organoid area and intensity calculation, fractal analysis, noise removal, and feature importance computation. The image manipulation feature includes brightness and contrast adjustment. The area and intensity calculation computes six values for each image: organoid area, total image area, percentage of the image covered by organoid, the total intensity of organoid, the total intensity of organoid-by-organoid area, and total intensity of organoid by total image area. The fractal analysis function computes the fractal dimension value for each image. The noise removal function removes superfluous marks from the input images, such as bubbles and other unwanted noise. The feature importance function trains a lasso-regularized linear regression machine learning algorithm to identify cardiac growth factors that are the strongest determinants for cell differentiation. The batch processing of this application further builds on existing services like ImageJ to provide a more convenient way to process multiple images. Collectively, the versatility and preciseness of Organalysis demonstrate novelty, since no other current imaging software combines the capability of batch processing and the breadth of feature analysis. Therefore, Organalysis provides unique functions in cardiac organoid research and proves to be invaluable in regenerative medicine.

Use of induced pluripotent stem cell-derived brain cells, organoids, assembloids, and blood-brain barrier models in understanding alcohol and anesthetic-induced brain injuries: an emerging perspective.

Use of induced pluripotent stem cell-derived brain cells, organoids, assembloids, and blood-brain barrier models in understanding alcohol and anesthetic-induced brain injuries: an emerging perspective.

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System.

Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 FAN1+/-), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease.

Free Bilirubin Induces Neuro-Inflammation in an Induced Pluripotent Stem Cell-Derived Cortical Organoid Model of Crigler-Najjar Syndrome.

Bilirubin-induced neurological damage (BIND), which might progress to kernicterus, occurs as a consequence of defects in the bilirubin conjugation machinery, thus enabling albumin-unbound free bilirubin (BF) to cross the blood-brain barrier and accumulate within. A defect in the UGT1A1 enzyme-encoding gene, which is directly responsible for bilirubin conjugation, can cause Crigler-Najjar syndrome (CNS) and Gilbert's syndrome. We used human-induced pluripotent stem cell (hiPSC)-derived 3D brain organoids to model BIND in vitro and unveil the molecular basis of the detrimental effects of BF in the developing human brain. Healthy and patient-derived iPSCs were differentiated into day-20 brain organoids, and then stimulated with 200 nM BF. Analyses at 24 and 72 h post-treatment point to BF-induced neuro-inflammation in both cell lines. Transcriptome, associated KEGG, and Gene Ontology analyses unveiled the activation of distinct inflammatory pathways, such as cytokine-cytokine receptor interaction, MAPK signaling, and NFκB activation. Furthermore, the mRNA expression and secretome analysis confirmed an upregulation of pro-inflammatory cytokines such as IL-6 and IL-8 upon BF stimulation. This novel study has provided insights into how a human iPSC-derived 3D brain organoid model can serve as a prospective platform for studying the etiology of BIND kernicterus.

Single-cell brain organoid screening identifies developmental defects in autism

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1–4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR–human organoids–single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR–Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

Engineering Highly Vascularized Bone Tissues by 3D Bioprinting of Granular Prevascularized Spheroids.

The convergence of 3D bioprinting with powerful manufacturing capability and cellular self-organization that can reproduce intricate tissue microarchitecture and function is a promising direction toward building functional tissues and has yet to be demonstrated. Here, we develop a granular aggregate-prevascularized (GAP) bioink for engineering highly vascularized bone tissues by capitalizing on the condensate-mimicking, self-organization, and angiogenic properties of prevascularized mesenchymal spheroids. The GAP bioink utilizes prevascularized aggregates as building blocks, which are embedded densely in extracellular matrices conducive to spontaneous self-organization. We printed various complex structures with high cell density (∼1.5 × 108 cells/cm3), viability (∼80%), and shape fidelity using GAP bioink. After printing, the prevascularized mesenchymal spheroids developed an interconnected vascular network through angiogenic sprouting. We printed highly vascularized bone tissues using GAP bioink and found that prevascularized spheroids were more conducive to osteogenesis and angiogenesis. We envision that the design of the GAP bioink could be further integrated with human-induced pluripotent stem cell-derived organoids, which opens new avenues to create patient-specific vascularized tissues for therapeutic applications..

Emerging Landscape of Preclinical Models for Studying COVID-19 Neurologic Diseases.

COVID-19 (Coronavirus Disease 2019) is an infectious disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and has globally infected 768 million people and caused over 6 million deaths. COVID-19 primarily affects the respiratory system but increasing reports of neurologic symptoms associated with COVID-19 have been reported in the literature. The exact mechanism behind COVID-19 neurologic pathophysiology remains poorly understood due to difficulty quantifying clinical neurologic symptoms in humans and correlating them to findings in human post-mortem samples and animal models. Thus, robust preclinical experimental models for COVID-19 neurologic manifestations are urgently needed. Here, we review recent advances in in vitro, in vivo, and other models and technologies for studying COVID-19 including primary cell cultures, pluripotent stem cell-derived neurons and organoids, rodents, nonhuman primates, 3D bioprinting, artificial intelligence, and multiomics. We specifically focus our discussion on the contribution, recent advancements, and limitations these preclinical models have on furthering our understanding of COVID-19's neuropathic physiology. We also discuss these models' roles in the screening and development of therapeutics, vaccines, antiviral drugs, and herbal medicine, and on future opportunities for COVID-19 neurologic research and clinical management.

Integrative analysis of single-cell RNA-seq and ATAC-seq reveals heterogeneity of induced pluripotent stem cell-derived hepatic organoids

To gain deeper insights into transcriptomes and epigenomes of organoids, liver organoids from two states (expandable and more differentiated) were subjected to single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) analyses. Mitochondrial gene expression was higher in differentiated than in non-differentiated hepatocytes, with ATAC-seq peaks increasing near the mitochondrial control region. Differentiation of liver organoids resulted in the expression of transcription factors that act as enhancers and repressors. In addition, epigenetic mechanisms regulating the expression of alpha-fetoprotein (AFP) and albumin (ALB) differed in liver organoids and adult liver. Knockdown of PDX1, an essential transcription factor for pancreas development, led to the hepatic maturation of liver organoids through regulation of AFP and ALB expression. This integrative analysis of the transcriptomes and epigenomes of liver organoids at the single-cell level may contribute to a better understanding of the regulatory networks during liver development and the further development of mature invitro human liver models.

The Activation of the LIMK/Cofilin Signaling Pathway via Extracellular Matrix-Integrin Interactions Is Critical for the Generation of Mature and Vascularized Cardiac Organoids.

The generation of mature and vascularized human pluripotent stem cell-derived cardiac organoids (hPSC-COs) is necessary to ensure the validity of drug screening and disease modeling. This study investigates the effects of cellular aggregate (CA) stemness and self-organization on the generation of mature and vascularized hPSC-COs and elucidates the mechanisms underlying cardiac organoid (CO) maturation and vascularization. COs derived from 2-day-old CAs with high stemness (H-COs) and COs derived from 5-day-old CAs with low stemness (L-COs) were generated in a self-organized microenvironment via Wnt signaling induction. This study finds that H-COs exhibit ventricular, structural, metabolic, and functional cardiomyocyte maturation and vessel networks consisting of endothelial cells, smooth muscle cells, pericytes, and basement membranes compared to L-COs. Transcriptional profiling shows the upregulation of genes associated with cardiac maturation and vessel formation in H-COs compared with the genes in L-COs. Through experiments with LIMK inhibitors, the activation of ROCK-LIMK-pCofilin via ECM-integrin interactions leads to cardiomyocyte maturation and vessel formation in H-COs. Furthermore, the LIMK/Cofilin signaling pathway induces TGFβ/NODAL and PDGF pathway activation for the maturation and vascularization of H-COs. The study demonstrates for the first time that LIMK/Cofilin axis activation plays an important role in the generation of mature and vascularized COs.

Modifications of lipid pathways restrict SARS-CoV-2 propagation in human induced pluripotent stem cell-derived 3D airway organoids

BackgroundModifications of lipid metabolism were closely associated with the manifestations and prognosis of coronavirus disease of 2019 (COVID-19). Pre-existing metabolic conditions exacerbated the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection while modulations of aberrant lipid metabolisms alleviated the manifestations. To elucidate the underlying mechanisms, an experimental platform that reproduces human respiratory physiology is required. MethodsHere we generated induced pluripotent stem cell-derived airway organoids (iPSC-AOs) that resemble the human native airway. Single-cell sequencing (ScRNAseq) and microscopic examination verified the cellular heterogeneity and microstructures of iPSC-AOs, respectively. We subjected iPSC-AOs to SARS-CoV-2 infection and investigated the treatment effect of lipid modifiers statin drugs on viral pathogenesis, gene expression, and the intracellular trafficking of the SARS-CoV-2 entry receptor angiotensin-converting enzyme-2 (ACE-2). ResultsIn SARS-CoV-2-infected iPSC-AOs, immunofluorescence staining detected the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins and bioinformatics analysis further showed the aberrant enrichment of lipid-associated pathways. In addition, SARS-CoV-2 hijacked the host RNA replication machinery and generated the new isoforms of a high-density lipoprotein constituent apolipoprotein A1 (APOA1) and the virus-scavenging protein deleted in malignant brain tumors 1 (DMBT1). Manipulating lipid homeostasis using cholesterol-lowering drugs (e.g. Statins) relocated the viral entry receptor angiotensin-converting enzyme-2 (ACE-2) and decreased N protein expression, leading to the reduction of SARS-CoV-2 entry and replication. The same lipid modifications suppressed the entry of luciferase-expressing SARS-CoV-2 pseudoviruses containing the S proteins derived from different SARS-CoV-2 variants, i.e. wild-type, alpha, delta, and omicron. ConclusionsTogether, our data demonstrated that modifications of lipid pathways restrict SARS-CoV-2 propagation in the iPSC-AOs, which the inhibition is speculated through the translocation of ACE2 from the cell membrane to the cytosol. Considering the highly frequent mutation and generation of SARS-CoV-2 variants, targeting host metabolisms of cholesterol or other lipids may represent an alternative approach against SARS-CoV-2 infection.

Novel human pluripotent stem cell-derived hypothalamus organoids demonstrate cellular diversity

The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our invitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus.

Modelling host- Trypanosoma brucei gambiense interactions in vitro using human induced pluripotent stem cell-derived cortical brain organoids.

Background: Sleeping sickness is caused by the extracellular parasite Trypanosoma brucei and is associated with neuroinflammation and neuropsychiatric disorders, including disruption of sleep/wake patterns, and is now recognised as a circadian disorder. Sleeping sickness is traditionally studied using murine models of infection due to the lack of alternative in vitro systems that fully recapitulate the cellular diversity and functionality of the human brain. The aim of this study is to develop a much-needed in vitro system that reduces and replaces live animals for the study of infections in the central nervous system, using sleeping sickness as a model infection. Methods: We developed a co-culture system using induced pluripotent stem cell (iPSC)-derived cortical human brain organoids and the human pathogen T. b. gambiense to model host-pathogen interactions in vitro. Upon co-culture, we analysed the transcriptional responses of the brain organoids to T. b. gambiense over two time points. Results: We detected broad transcriptional changes in brain organoids exposed to T. b. gambiense, mainly associated with innate immune responses, chemotaxis, and blood vessel differentiation compared to untreated organoids. Conclusions: Our co-culture system provides novel, more ethical avenues to study host-pathogen interactions in the brain as alternative models to experimental infections in mice. Although our data support the use of brain organoids to model host-pathogen interactions during T. brucei infection as an alternative to in vivo models, future work is required to increase the complexity of the organoids ( e.g., addition of microglia and vasculature). We envision that the adoption of organoid systems is beneficial to researchers studying mechanisms of brain infection by protozoan parasites. Furthermore, organoid systems have the potential to be used to study other parasites that affect the brain significantly reducing the number of animals undergoing moderate and/or severe protocols associated with the study of neuroinflammation and brain infections.

BACE2: A Promising Neuroprotective Candidate for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia that affects millions of predominantly elderly individuals worldwide. Despite intensive research over several decades, controversies still surround the etiology of AD and the disease remains incurable. Meanwhile, new molecular players of the central amyloid cascade hypothesis have emerged and among these is a protease known as β-site APP cleavage enzyme 2 (BACE2). Unlike BACE1, BACE2 cleaves the amyloid-β protein precursor within the Aβ domain that accordingly prevents the generation of Aβ42 peptides, the aggregation of which is commonly regarded as the toxic entity that drives neurodegeneration in AD. Given this non-amyloidogenic role of BACE2, it is attractive to position BACE2 as a therapeutic target for AD. Indeed, several groups including ours have demonstrated a neuroprotective role for BACE2 in AD. In this review, we discuss emerging evidence supporting the ability of BACE2 in mitigating AD-associated pathology in various experimental systems including human pluripotent stem cell-derived cerebral organoid disease models. Alongside this, we also provide an update on the identification of single nucleotide polymorphisms occurring in the BACE2 gene that are linked to increased risk and earlier disease onset in the general population. In particular, we highlight a recently identified point mutation on BACE2 that apparently leads to sporadic early-onset AD. We believe that a better understanding of the role of BACE2 in AD would provide new insights for the development of viable therapeutic strategies for individuals with dementia.

Spatially controlled construction of assembloids using bioprinting

The biofabrication of three-dimensional (3D) tissues that recapitulate organ-specific architecture and function would benefit from temporal and spatial control of cell-cell interactions. Bioprinting, while potentially capable of achieving such control, is poorly suited to organoids with conserved cytoarchitectures that are susceptible to plastic deformation. Here, we develop a platform, termed Spatially Patterned Organoid Transfer (SPOT), consisting of an iron-oxide nanoparticle laden hydrogel and magnetized 3D printer to enable the controlled lifting, transport, and deposition of organoids. We identify cellulose nanofibers as both an ideal biomaterial for encasing organoids with magnetic nanoparticles and a shear-thinning, self-healing support hydrogel for maintaining the spatial positioning of organoids to facilitate the generation of assembloids. We leverage SPOT to create precisely arranged assembloids composed of human pluripotent stem cell-derived neural organoids and patient-derived glioma organoids. In doing so, we demonstrate the potential for the SPOT platform to construct assembloids which recapitulate key developmental processes and disease etiologies.

Schizophrenia-associated NRXN1 deletions induce developmental-timing- and cell-type-specific vulnerabilities in human brain organoids

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

Engineering Extracellular Matrix-Bound Nanovesicles Secreted by Three-Dimensional Human Mesenchymal Stem Cells.

Extracellular matrix (ECM) in the human tissue contains vesicles, which are defined as matrix-bound nanovesicles (MBVs). MBVs serve as one of the functional components in ECM, recapitulating part of the regulatory roles and in vivo microenvironment. In this study, extracellular vesicles from culture supernatants (SuEVs) and MBVs are isolated from the conditioned medium or ECM, respectively, of 3D human mesenchymal stem cells. Nanoparticle tracking analysis shows that MBVs are smaller than SuEVs (100-150nm). Transmission electron microscopy captures the typical cup shape morphology for both SuEVs and MBVs. Western blot reveals that MBVs have low detection of some SuEV markers such as syntenin-1. miRNA analysis of MBVs shows that 3D microenvironment enhances the expression of miRNAs such as miR-19a and miR-21. In vitro functional analysis shows that MBVs can facilitate human pluripotent stem cell-derived forebrain organoid recovery after starvation and promote high passage fibroblast proliferation. In macrophage polarization, 2D MBVs tend to suppress the pro-inflammatory cytokine IL-12β, while 3D MBVs tend to enhance the anti-inflammatory cytokine IL-10. This study has the significance in advancing the understanding of the bio-interface of nanovesicles with human tissue and the design of cell-free therapy for treating neurological disorders such as ischemic stroke.

A single-cell level comparison of human inner ear organoids with the human cochlea and vestibular organs

Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demonstrate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of human fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its potential applications in studying inner ear development and disorders.

TOP-056 - Modeling Alagille syndrome in vitro using complex induced pluripotent stem cell-derived human liver organoids

TOP-056 - Modeling Alagille syndrome in vitro using complex induced pluripotent stem cell-derived human liver organoids

Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids.

Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5 - /KRT17 + aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.

Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia.

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0-5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5-10). Then, upon transfer to spinner flasks containing OVB medium (days 10-30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.