Production Of Organoids Research Articles

A Systematically Reduced Mathematical Model for Organoid Expansion.

Organoids are three-dimensional multicellular tissue constructs. When cultured in vitro, they recapitulate the structure, heterogeneity, and function of their in vivo counterparts. As awareness of the multiple uses of organoids has grown, e.g. in drug discovery and personalised medicine, demand has increased for low-cost and efficient methods of producing them in a reproducible manner and at scale. Here we focus on a bioreactor technology for organoid production, which exploits fluid flow to enhance mass transport to and from the organoids. To ensure large numbers of organoids can be grown within the bioreactor in a reproducible manner, nutrient delivery to, and waste product removal from, the organoids must be carefully controlled. We develop a continuum mathematical model to investigate how mass transport within the bioreactor depends on the inlet flow rate and cell seeding density, focusing on the transport of two key metabolites: glucose and lactate. We exploit the thin geometry of the bioreactor to systematically simplify our model. This significantly reduces the computational cost of generating model solutions, and provides insight into the dominant mass transport mechanisms. We test the validity of the reduced models by comparison with simulations of the full model. We then exploit our reduced mathematical model to determine, for a given inlet flow rate and cell seeding density, the evolution of the spatial metabolite distributions throughout the bioreactor. To assess the bioreactor transport characteristics, we introduce metrics quantifying glucose conversion (the ratio between the total amounts of consumed and supplied glucose), the maximum lactate concentration, the proportion of the bioreactor with intolerable lactate concentrations, and the time when intolerable lactate concentrations are first experienced within the bioreactor. We determine the dependence of these metrics on organoid-line characteristics such as proliferation rate and rate of glucose consumption per cell. Finally, for a given organoid line, we determine how the distribution of metabolites and the associated metrics depend on the inlet flow rate. Insights from this study can be used to inform bioreactor operating conditions, ultimately improving the quality and number of bioreactor-expanded organoids.

GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration.

Cerebral organoids generated from human pluripotent stem cells (hiPSCs) are unique in their ability to recapitulate human-specific neurodevelopmental events. They are capable of modeling the human brain and its cell composition, including human-specific progenitor cell types; ordered laminar compartments; and both cell-specific transcriptional signatures and the broader telencephalic transcriptional landscape. The serine/threonine kinase, GSK3β, plays a critical role in neurodevelopment, controlling processes as varied as neurogenesis, morphological changes, polarization, and migration. In the generation of cerebral organoids, inhibition of GSK3β at low doses has been used to increase organoid size and decrease necrotic core. However, little is known of the effects of GSK3β inhibition on organoid development. Here, we demonstrate that while low dose of GSK3β inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth. To examine the mechanisms that may contribute to the phenotypic size differences observed in these treatment groups, we show that low dose of CHIR 99021 increases cell survival, neural progenitor cell proliferation and neuronal migration. A higher dose, however, decreases not only apoptosis but also proliferation, and arrests neural differentiation, enriching the pool of neuroepithelial cells, and decreasing the pools of early neuronal progenitors and neurons. These results reveal new mechanisms of the pleiotropic effects of GSK3β during organoid development, providing essential information for the improvement of organoid production and ultimately shedding light on the mechanisms of embryonic brain development.

Development of 3D Cerebral Aggregates in the Brain Ventricles of Adult Mice

The cerebral organoids are three-dimensional cell cultures formed from brain-specific cell types arising from embryonic or pluripotent stem cells. Organoids provide an opportunity to study the early stages of brain development and diseases of the central nervous system. However, the modeling of organoids is associated with a number of unsolved problems. Organoid production techniques involve a complex cell culture process that requires special media, growth factors, and often the use of a bioreactor. Even under standardized conditions, structures of different morphology are formed: from disorganized cell aggregates to structured minibrains, which are selected for study. For natural reasons, organoids grown in vitro do not have a blood supply, which limits their development. We tried to obtain cerebral aggregates similar to organoids in an in vivo model, where vascular growth and tissue blood supply are provided, for which we transplanted a cell suspension from the mouse embryonic neocortex into the lateral ventricles of the brain of adult mice. Therefore, the medium for cultivation was the cerebrospinal fluid, and the lateral ventricles of the brain, where it circulates, served as a bioreactor. The results showed that the neocortex from E14.5 is a suitable source of stem/progenitor cells that self-assemble into three-dimensional aggregates and vascularized in vivo. The aggregates consisted of a central layer of mature neurons, the marginal zone free of cells and a glia limitans, which resembled cerebral organoids. Thus, the lateral ventricles of the adult mouse brain can be used to obtain vascularized cell aggregates resembling cerebral organoids.

Engineering spatial-organized cardiac organoids for developmental toxicity testing.

SummaryEmerging technologies in stem cell engineering have produced sophisticated organoid platforms by controlling stem cell fate via biomaterial instructive cues. By micropatterning and differentiating human induced pluripotent stem cells (hiPSCs), we have engineered spatially organized cardiac organoids with contracting cardiomyocytes in the center surrounded by stromal cells distributed along the pattern perimeter. We investigated how geometric confinement directed the structural morphology and contractile functions of the cardiac organoids and tailored the pattern geometry to optimize organoid production. Using modern data-mining techniques, we found that pattern sizes significantly affected contraction functions, particularly in the parameters related to contraction duration and diastolic functions. We applied cardiac organoids generated from 600 μm diameter circles as a developmental toxicity screening assay and quantified the embryotoxic potential of nine pharmaceutical compounds. These cardiac organoids have potential use as an in vitro platform for studying organoid structure-function relationships, developmental processes, and drug-induced cardiac developmental toxicity.

Matrigel is required for efficient differentiation of isolated, stem cell-derived otic vesicles into inner ear organoids

Inner ear organoids derived from pluripotent stem cells could be a useful model system to study development, disease, and regeneration. However, there is considerable heterogeneity in the size, morphology, and efficiency of organoid production using standard protocols. Greater control of the culture microenvironment could decrease heterogeneity and increase the yield of organoids. Animal-derived otic vesicles show some autonomy during development and can differentiate into cochlear and vestibular domains in mesenchyme-free ex vivo culture. Therefore, we investigated whether stem cell-derived otic vesicles can autonomously generate inner ear organoids. Isolated, stem cell-derived vesicles grew into cyst-like organoids with high efficiency, over 90%, when embedded in droplets of the basement membrane matrix Matrigel. Though nearly all vesicles within the aggregate were competent to mature into organoids, the efficiency of organoid production depended on the stage of vesicle isolation and required supplementation with Matrigel.

Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here we apply extrusion-based 3D cellular bioprinting to deliver rapid and high throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, 3D bioprinting enabled precise manipulation of biophysical properties including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitated the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production deliver improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Generation of human induced pluripotent stem cell-derived liver buds with chemically defined and animal origin-free media

Advances in organoid technology have broadened the number of target diseases and conditions in which human induced pluripotent stem cell (iPSC)-based regenerative medicine can be applied; however, mass production of organoids and the development of chemically defined, animal origin-free (CD-AOF) media and supplements are unresolved issues that hamper the clinical applicability of these approaches. CD-AOF media and supplements ensure the quality and reproducibility of culture systems by lowering lot-to-lot variations and the risk of contamination with viruses or toxins. We previously generated liver organoids from iPSCs, namely iPSC-liver buds (iPSC-LBs), by mimicking the organogenic interactions among hepatocytes, endothelial cells (ECs), and mesenchymal cells (MCs) and recently reported the mass production of iPSC-LBs derived entirely from iPSCs (all iPSC-LBs), which should facilitate their large-scale production for the treatment of liver failure. However, in previous studies we used media originating from animals for differentiation except for the maintenance of undifferentiated iPSCs. Therefore, we developed a CD-AOF medium to generate all iPSC-LBs. We first developed a CD-AOF medium for hepatocytes, ECs, and stage-matched MCs, i.e., septum transversum mesenchyme (STM), in 2D cultures. We next generated all iPSC-LBs by incubating individual cell types in ultra-low attachment micro-dimple plates. The hepatic functions of all iPSC-LBs generated using the CD-AOF medium were equivalent to those of all iPSC-LBs generated using the conventional medium both in vitro and in vivo. Furthermore, we found that this CD-AOF medium could be used in several cell culture settings. Taken together, these results demonstrate the successful development of a CD-AOF medium suitable for all iPSC-LBs. The protocol developed in this study will facilitate the clinical applicability of all iPSC-LBs in the treatment of liver diseases.

Development of 3D Hepatic Constructs Within Polysaccharide-Based Scaffolds with Tunable Properties.

Organoids production is a key tool for in vitro studies of physiopathological conditions, drug-induced toxicity assays, and for a potential use in regenerative medicine. Hence, it prompted studies on hepatic organoids and liver regeneration. Numerous attempts to produce hepatic constructs had often limited success due to a lack of viability or functionality. Moreover, most products could not be translated for clinical studies. The aim of this study was to develop functional and viable hepatic constructs using a 3D porous scaffold with an adjustable structure, devoid of any animal component, that could also be used as an in vivo implantable system. We used a combination of pharmaceutical grade pullulan and dextran with different porogen formulations to form crosslinked scaffolds with macroporosity ranging from 30 µm to several hundreds of microns. Polysaccharide scaffolds were easy to prepare and to handle, and allowed confocal observations thanks to their transparency. A simple seeding method allowed a rapid impregnation of the scaffolds with HepG2 cells and a homogeneous cell distribution within the scaffolds. Cells were viable over seven days and form spheroids of various geometries and sizes. Cells in 3D express hepatic markers albumin, HNF4α and CYP3A4, start to polarize and were sensitive to acetaminophen in a concentration-dependant manner. Therefore, this study depicts a proof of concept for organoid production in 3D scaffolds that could be prepared under GMP conditions for reliable drug-induced toxicity studies and for liver tissue engineering.

Robust production of uniform human cerebral organoids from pluripotent stem cells.

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

Standardized GMP-compliant scalable production of human pancreas organoids

BackgroundOrganoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach.MethodsStarting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis.ResultsThis work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%).ConclusionThis work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

Pluripotent-Stem-Cell-Derived Hepatic Cells: Hepatocytes and Organoids for Liver Therapy and Regeneration.

The liver is a very complex organ that ensures numerous functions; it is thus susceptible to multiple types of damage and dysfunction. Since 1983, orthotopic liver transplantation (OLT) has been considered the only medical solution available to patients when most of their liver function is lost. Unfortunately, the number of patients waiting for OLT is worryingly increasing, and extracorporeal liver support devices are not yet able to counteract the problem. In this review, the current and expected methodologies in liver regeneration are briefly analyzed. In particular, human pluripotent stem cells (hPSCs) as a source of hepatic cells for liver therapy and regeneration are discussed. Principles of hPSC differentiation into hepatocytes are explored, along with the current limitations that have led to the development of 3D culture systems and organoid production. Expected applications of these organoids are discussed with particular attention paid to bio artificial liver (BAL) devices and liver bio-fabrication.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Biomaterial-Guided Organoid Engineering for Modeling Development and Diseases

Organoids are miniature models of organs to recapitulate spatiotemporal cellular organization and tissue functionality. The production of organoids has revolutionized the field of developmental biology, providing the possibility to study and guide human development and diseases in a dish. More recently, novel bio-material-based culture systems demonstrated the feasibility and versatility to engineer and produce the organoids in a consistent and reproducible manner. By engineering proper tissue micro-environment, functional organoids have been able to exhibit spatial-distinct tissue patterning and morphogenesis. This review will focus on enabling technologies in the field of organoid engineering, including chemical-defined hydrogels, micro-fabrication techniques and organoid-on-chip platforms, which facilitate the assembly of in vitro modeling of development and diseases.

Differentiation of Retinal Organoids from Human Pluripotent Stem Cells.

This unit describes a protocol for generating retinal organoids that contain all major retinal cell types and are responsive to light from human pluripotent stem cells (hPSCs). hPSCs are differentiated in 96-well plates to allow large-scale production of organoids that could be used for multiple applications, including study of human retinal development, disease modeling, and compound screening. The differentiation approach is based on the knowledge that insulin-like growth factor 1 signaling together with retinoic acid and triiodothyronine is important for retinal development. After 22 weeks in culture, the organoids form a thick layer of neuroepithelium containing photoreceptors and bipolar, horizontal, amacrine, Müller, and retinal ganglion cells. Differentiation progress can be tracked by morphological observations and protein localization, as detected with immunocytochemistry. © 2019 by John Wiley & Sons, Inc.

Abstract 469: Patient-derived sarcoma models: First results from the SARQMA study

Abstract Objective: The development of new targeted therapeutics has allowed an essential improvement in carcinoma treatment. For sarcomas, however, the main approach is still the combination of surgery, chemotherapy and radiation, which is due to their high heterogeneity, with more than 70 histopathological subtypes, and the limited knowledge of the molecular drivers of tumor development and progression. Here we show that patient-derived 3D (PD3D) cell culture models allow for an in vitro system to systematically test compounds and combos in a semi-automated way, generating a pre-clinical dataset that in combination with clinical data, genomic and proteomics profiles may help to better understand the biology and ultimately identify more potent treatment regimens. Methods: Fresh surgical specimen underwent several steps of dissociation. Cell aggregates were then seeded into 24w plates in matrix-like scaffolds and grown until >100μm. Organoids were then harvested, transferred to 384w plates and treated with a set of compounds that resemble standard-of-care treatments and novel compounds. Finally, viability was calculated. In parallel, protein extracts were used for DigiWest, a multiplexed protein profiling assay allowing to interrogate up to 800 (phospho)-proteins. Results: Of 49 cases with a biopsy, 23 had too little material available. Of these 23 samples, 5 were reported as sarcoma tissue afterwards, while 5 of the taken 26 samples were not sarcomas according to the final histopathology. Of all sarcomas, 90% (19/21) were taken from tumors localized at the extremities, the rest was located at the trunk. 52% (11/21) were growing in short term cell culture at least to passage 1 (p1), 28% (6/21) were growing for long term analyses. Two of those six were myxoid liposarcomas, two were undifferentiated pleomorphic sarcomas or classified as not otherwise specified (UPS/NOS), one was a myxoid liposarcoma and one a biphasic synovial sarcoma. Here we show that it is feasible to generate organoids from sarcoma tissues for extensive characterization in order to better understand their biology and mechanisms of treatment. High-throughput drug screening allows for an profiling of pharmacokinetic properties of individual sarcomas. Using the same material for additional (phospho)-proteomics provides multiple layers of understanding. Conclusion: At present, the structure of clinical trials is not amenable to N of 1 studies, so applying the information garnered from this platform, particularly combination therapy drug screens, remains a significant hurdle. The major limitation to the establishment of organoid cultures was insufficient amounts of fresh tissue with viable tumor cells. Increasing the tumor tissue available for organoid production would lead to a greater success rate. Nevertheless, by their recapitulation of the donor tissue architecture, they provide an interesting and important tool to study the huge variety of soft-tissue tumors. Citation Format: Manuela Gaebler, Alessandra Silvestri, Peter Reichardt, Eva Wardelmann, Guido Gambara, Johannes Haybaeck, Philipp Stroebel, Maya Niethard, Gerrit Erdmann, Christian R. Regenbrecht. Patient-derived sarcoma models: First results from the SARQMA study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 469.

Morphological and Molecular Analysis of In Vitro Tubular Structures from Bovine Yolk Sac-Derived MSCs.

The yolk sac is an extraembryonic membrane, of saccular form, connected to the ventral region of the embryo. It is the main source of nutrition for the embryo during the period when the placenta is not fully formed. The aim of this study was to generate tubular structures using mesenchymal stem cells from the bovine yolk sac (bYS-MSCs) and determine if these structures can be a model for in vitro vasculogenesis. The evaluation of this tissue by histochemistry revealed a strong marking of collagen fibers and PAS technique negativity. In transmission electron microscopy, cytoplasmic organelles with large nuclei were observed. The vessel formation assay on a Matrigel substrate showed that the mesenchymal cells of the yolk sac without growth factors (VEGF) are capable of forming branches, sprouting cells, and tubular structures similar to capillary blood. These tubular structures were xenotransplanted subcutaneously into the mesentery of BALB/c/nude mice; after 45 days, vascularized tissue and extensions of blood vessels around the tubular structures could be observed. Real-time PCR (qPCR) demonstrated an expression of the VEGF gene in different gestational age groups. No difference in distribution or expression was detected among groups. Our results suggest that the spontaneous formation of tubules from the yolk sac can be an experimental model to elucidate initial organogenesis and the possible formation of blood capillaries from in vitro mesenchymal cells and possible route of organoid production.

A Simple Bioreactor-Based Method to Generate Kidney Organoids fromPluripotent Stem Cells.

SummaryKidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.

Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.

Towards Multi-Organoid Systems for Drug Screening Applications.

A low percentage of novel drug candidates succeed and reach the end of the drug discovery pipeline, mainly due to poor initial screening and assessment of the effects of the drug and its metabolites over various tissues in the human body. For that, emerging technologies involving the production of organoids from human pluripotent stem cells (hPSCs) and the use of organ-on-a-chip devices are showing great promise for developing a more reliable, rapid and cost-effective drug discovery process when compared with the current use of animal models. In particular, the possibility of virtually obtaining any type of cell within the human body, in combination with the ability to create patient-specific tissues using human induced pluripotent stem cells (hiPSCs), broadens the horizons in the fields of drug discovery and personalized medicine. In this review, we address the current progress and challenges related to the process of obtaining organoids from different cell lineages emerging from hPSCs, as well as how to create devices that will allow a precise examination of the in vitro effects generated by potential drugs in different organ systems.

From Otic Induction to Hair Cell Production: Pax2EGFP Cell Line Illuminates Key Stages of Development in Mouse Inner Ear Organoid Model.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.