Organoids, Volume 2, Issue 4 (December 2023) – 7 articles

Organoids have emerged as a powerful tool for studying organ development, disease modeling, and drug discovery due to their ability to mimic the in vivo structure and function of organs in a three-dimensional in vitro model. During in vivo organ maturation, the process of vascularization is crucial for the provision of nutrients and oxygen to cells and the removal of waste products as the organ increases in size. Similarly, organoids can grow to sizes greater than the millimeter scale, yet transport of oxygen and nutrients to the center becomes increasingly difficult, often resulting in the formation of a necrotic core. Herein, we provide a concise summary of the recent development of methods to initiate and maintain vascularization of organoids. Broadly, vascularization of organoids has been achieved primarily by two means: generating organoids that contain endothelial cells or employing the secretion of vascular growth factors to promote vascularization. Growth factors play a fundamental role in regulating blood vessel formation through chemical signals that cause changes in the cell–cell adhesions and ultimately the migration of endothelial cells. Furthermore, models with perfusable systems demonstrate that through the application of growth factors and cells, the vascular network in vascularization-based organoids can administer biological substances to the interior of the organoid, opening up new possibilities for long-term organoid culture in vitro. This goal is being realized through the development of bioengineering tools, such as vascularized organoids on a chip, which are currently tested for various organ systems, including the lung, brain, kidney, and tumors, with applications in cancer angiogenesis and metastasis research. Taken together, our review underlines the vast potential of vascularized organoids to improve the understanding of organ development, while also proposing exciting avenues of organoid-on-a-chip and disease modeling. Full article

While research on stem cell-derived tissues and organoids is rapidly expanding, the technically related creation of complex embryoids has recently excited a vivid discussion since it raises ethical questions about individuation and the possible gain of viability. The present study focuses on the onset of organismic development and the proposed biological and legal definitions for the terms embryo, embryoid, and organoid. It is concluded that such considerations have become important for investigators’ choices of the appropriate in vitro model systems, allowing the formation of organoids vs. complex embryoids. Full article

Meningiomas are the most common tumours of the central nervous system. According to the World Health Organization (WHO), this disease is classified into three different grades: 80% of meningioma patients present with benign grade I tumours, while less than 2% present with malignant grade III meningiomas. Despite affecting thousands of people worldwide, much remains unknown about this disease, and the development of systemic treatments is still far behind in comparison to other types of tumours. Therefore, forming 3D structures (spheroids and organoids) could facilitate research on the mechanisms of formation, proliferation, migration, and invasion of these, for the most part, benign tumours, while also helping in the process of drug development. To date, there are three published methods for the formation of meningioma organoids primarily derived from patient tissue samples. Organoids offer many advantages in the development of treatments because they recapitulate the cellular complexity within tumours. These new methodological advances could open a substantial number of possibilities for the further characterisation and treatment of meningiomas. This review includes an overview of the disease and a description and comparison of established protocols for meningioma organoid formation. Full article

A three-dimensional cell culture in hydrogel beads can support cell growth and differentiation into multi-cellular structures, and these gel beads could be used as building blocks for more complex three-dimensional assemblies. This requires hydrogel beads that are robust enough to sort via FACS yet can be degraded by cell-secreted enzymes. Collagen polymers form hydrogels that are excellent cell growth substrates; however, collagen-containing hydrogel beads typically include additional polymers that limit their degradation. Here, we introduce a simple microfluidic method to generate robust, sortable, cell-laden collagen hydrogel beads. We use on-device pH control to trigger collagen gelation without exposing cells to low pH, ensuring high cell viability. We fabricate microfluidic devices to generate droplets with a wide size range, as demonstrated by production of both small (~55 µm diameter) and large (~300 µm diameter) collagen gels. All hydrogels are sufficiently robust to allow for sorting using FACS. Moreover, high cell viability is maintained throughout the process. Full article

The generation of gastrointestinal tissues from human pluripotent stem cells has provided unprecedented insight into the molecular mechanisms that drive the patterning of the primitive gut tube. Previous work has identified bone-morphogenetic-protein (BMP) signaling as an important mediator of mid/hindgut versus foregut and hindgut versus midgut cell fate choice. Inhibition of BMP signaling during gut tube morphogenesis inhibits the expression of the pan-intestinal transcription factor CDX2. Treatment of CDX2+ mid/hindgut cultures with BMP patterns them into hindgut, which gives rise to colonic organoids (HCOs). While the role for BMP signaling is clear, the molecular mechanisms through which BMP signaling patterns the mid/hindgut and colon remain unclear. BMPs bind to BMP receptors, activating a signaling cascade that results in the activation of SMADs, which function as transcription factors. We hypothesized that one of these factors, SMAD1, would be necessary for establishing the CDX2 domain and the colon domain. Unexpectedly, endoderm derived from SMAD1-deficient induced pluripotent stem cells was capable of inducing CDX2 in response to WNT and FGF signaling. In addition, CDX2+ gut tube cultures could activate posterior HOX genes in response to BMP. However, examination of HCOs following cytodifferentiation revealed that SMAD1-deficient HCOs ectopically expressed small-intestinal markers despite expressing posterior HOX genes. These results indicate that there is redundancy of SMADs during early hindgut patterning but that SMAD1 is required for the inhibition of small-intestinal gene expression in HCOs. Full article

Ensuring drug safety for patients with specific neurological disorders is of paramount importance. For instance, certain antiepileptic drugs (AEDs) are contraindicated in Dravet Syndrome (DS), which is characterized by a deficiency in Na⁺ channel function. Constructing in vitro assessment methods capable of detecting contraindicated drug responses and medication effects on neurons derived from DS patients is highly anticipated for drug safety assessment and therapeutic innovation. This study used micro electrode array (MEA) measurements with low-frequency analysis on human iPSC-derived DS organoids to investigate AED responses. When exposed to the contraindicated drugs carbamazepine and phenytoin, the number of network oscillations increased in DS organoids while maintaining oscillation intensity. Furthermore, carbamazepine administration appeared to enhance activities beyond oscillations which is partially consistent with findings in the DS mouse model. Conversely, treatment with the therapeutic drug sodium valproate resulted in a similar decrease in activity both in healthy and DS organoids. The frequency characteristics of spontaneous firings and AEDs responsiveness in DS organoids demonstrated partial correlation with typical electroencephalography patterns observed in vivo. In conclusion, this study, employing MEA measurements with low-frequency analysis, revealed contraindicated drug responses and disease-specific functional characteristics in DS organoids, effective for DS patient safety assessment, precision medicine, and antiepileptic drug screening. Full article

Human cerebral organoids are readily generated from human embryonic stem cells and human induced pluripotent stem cells and are useful in studying human neurodevelopment. Recent work with human cerebral organoids have explored the creation of different brain regions and the impacts of soluble and mechanical cues. Matrigel is a gelatinous, heterogenous mixture of extracellular matrix proteins, morphogens, and growth factors secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is a core component of almost all cerebral organoid protocols, generally supporting neuroepithelial budding and tissue polarization; yet, its roles and effects beyond its general requirement in organoid protocols are not well understood, and its mode of delivery is variable, including the embedding of organoids within it or its delivery in soluble form. Given its widespread usage, we asked how H9 stem cell-derived hCO development and composition are affected by Matrigel dosage and delivery method. We found Matrigel exposure influences organoid size, morphology, and cell type composition. We also showed that greater amounts of Matrigel promote an increase in the number of choroid plexus (ChP) cells, and this increase is regulated by the BMP4 pathway. These results illuminate the effects of Matrigel on human cerebral organoid development and the importance of delivery mode and amount on organoid phenotype and composition. Full article