Human Pluripotent Stem Cell-derived Organoids Research Articles

Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

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.

CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids

Mutations in CHD7 cause CHARGE syndrome, affecting multiple organs including the inner ear in humans. We investigate how CHD7 mutations affect inner ear development using human pluripotent stem cell-derived organoids as a model system. We find that loss of CHD7 or its chromatin remodeling activity leads to complete absence of hair cells and supporting cells, which can be explained by dysregulation of key otic development-associated genes in mutant otic progenitors. Further analysis of the mutant otic progenitors suggests that CHD7 can regulate otic genes through a chromatin remodeling-independent mechanism. Results from transcriptome profiling of hair cells reveal disruption of deafness gene expression as a potential underlying mechanism of CHARGE-associated sensorineural hearing loss. Notably, co-differentiating CHD7 knockout and wild-type cells in chimeric organoids partially rescues mutant phenotypes by restoring otherwise severely dysregulated otic genes. Taken together, our results suggest that CHD7 plays a critical role in regulating human otic lineage specification and hair cell differentiation.

Human organoid models to study SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the deadliest pandemics in history. SARS-CoV-2 not only infects the respiratory tract, but also causes damage to many organs. Organoids, which can self-renew and recapitulate the various physiology of different organs, serve as powerful platforms to model COVID-19. In this Perspective, we overview the current effort to apply both human pluripotent stem cell-derived organoids and adult organoids to study SARS-CoV-2 tropism, host response and immune cell-mediated host damage, and perform drug discovery and vaccine development. We summarize the technologies used in organoid-based COVID-19 research, discuss the remaining challenges and provide future perspectives in the application of organoid models to study SARS-CoV-2 and future emerging viruses.

Human pluripotent stem cell-derived organoids and their potential to reduce the use of animal models in research

Efficient and reproducible generation of tissue-specific organoids from Human Pluripotent Stem Cells (hPSCs) represents one of the key tools for reducing the use of animals in research. STEMCELL Technologies is committed to optimizing workflows that efficiently support the generation and maintenance of multiple types of organoid cultures derived from hPSCs.

Development of a quantitative prediction algorithm for target organ-specific similarity of human pluripotent stem cell-derived organoids and cells

Human pluripotent stem cell (hPSC)-derived organoids and cells have similar characteristics to human organs and tissues. Thus, in vitro human organoids and cells serve as a superior alternative to conventional cell lines and animal models in drug development and regenerative medicine. For a simple and reproducible analysis of the quality of organoids and cells to compensate for the shortcomings of existing experimental validation studies, a quantitative evaluation method should be developed. Here, using the GTEx database, we construct a quantitative calculation system to assess similarity to the human organs. To evaluate our system, we generate hPSC-derived organoids and cells, and detected organ similarity. To facilitate the access of our system by researchers, we develop a web-based user interface presenting similarity to the appropriate organs as percentages. Thus, this program could provide valuable information for the generation of high-quality organoids and cells and a strategy to guide proper lineage-oriented differentiation.

Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids.

SummaryThe development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.

Human Pluripotent Stem Cell-Derived Organoids as a Model of Intestinal Xenobiotic Metabolism

Background: The human intestine is the site of absorption and first-pass metabolism for oral intake. Assessment of absorption, distribution, metabolism, excretion, and toxicity (ADMET) of xenobiotics has transformed the understanding of in vivo pharmacology. However, these processes are difficult torecapitulate in vitro. Objective: We have developed a simple protocol for the generation of mature functional intestinal organoids from human pluripotent stem cells (hPSCs)under xenogeneic-free conditions. We sought to characterize transcription level in drug transporters and metabolism and evaluate CYP3A4 catalytic function of the organoids. Methods: Human pluripotent stem cell-derived intestinal organoids were generated and evaluated the expression of drug transporters and metabolizing enzymes. We examined the induction of CYP3A4 and ABCB1 gene expression in the organoids. Furthermore, we analyzed the CYP3A4 enzyme activity of the organoids by the p450-Glo CYP3A4 assay kit with luciferin isopropyl acetal. Results: Stem cell-derived intestinal organoids had an outward polarized intestinal epithelial layer and showed similar expression levels of drug transporters and metabolism genes as the adult healthy intestine. They also exhibited CYP3A4 enzymatic function in vitro. Conclusion: This model provides a novel platform for pharmacological testing and can enhance human ADMET studies in drug development.

Rethinking organoid technology through bioengineering.

In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An important step in this direction has also been the derivation of organoids. This technology generally relies on traditional three-dimensional culture techniques that exploit cell-autonomous self-organization responses of hPSCs with minimal control over the external inputs supplied to the system. The convergence of stem cell biology and bioengineering offers the possibility to provide these stimuli in a controlled fashion, resulting in the development of naturally inspired approaches to overcome major limitations of this nascent technology. Based on the current developments, we emphasize the achievements and ongoing challenges of bringing together hPSC organoid differentiation, bioengineering and ethics. This Review underlines the need for providing engineering solutions to gain control of self-organization and functionality of hPSC-derived organoids. We expect that this knowledge will guide the community to generate higher-grade hPSC-derived organoids for further applications in developmental biology, drug screening, disease modelling and personalized medicine.

Covering the Cover

Covering the Cover

Modeling endodermal organ development and diseases using human pluripotent stem cell-derived organoids.

Recent advances in development of protocols for directed differentiation from human pluripotent stem cells (hPSCs) to defined lineages, in combination with 3D organoid technology, have facilitated the generation of various endoderm-derived organoids for in vitro modeling of human gastrointestinal development and associated diseases. In this review, we discuss current state-of-the-art strategies for generating hPSC-derived endodermal organoids including stomach, liver, pancreatic, small intestine, and colonic organoids. We also review the advantages of using this system to model various human diseases and evaluate the shortcomings of this technology. Finally, we emphasize how other technologies, such as genome editing and bioengineering, can be incorporated into the 3D hPSC-organoid models to generate even more robust and powerful platforms for understanding human organ development and disease modeling.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease

There are few invitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

Recapitulating human tissue damage, repair, and fibrosis with human pluripotent stem cell-derived organoids.

As new applications for human pluripotent stem cell-derived organoids in drug screenings and tissue replacement therapies emerge, there is a need to examine the mechanisms of tissue injury and repair recently reported for various organoid models. In most cases, organoids contain the main cell types and tissues present in human organs, spatially arranged in a manner that largely resembles the architecture of the organ. Depending on the differentiation protocol used, variations may exist in cell type ratios relative to the organ of reference, and certain tissues, including some parenchymal components and the endothelium, might be poorly represented, or lacking altogether. Despite those caveats, recent studies have shown that organoid tissue injury recapitulates major events and histopathological features of damaged human tissues. In particular, major mechanisms of parenchyma cell damage and interstitial fibrosis can be reproduced with remarkable faithfulness. Although further validation remains to be done in order to establish the relevance of using organoid for either mechanistic studies or drug assays, this technology is becoming a promising tool for the study of human tissue homeostasis, injury, and repair.