• International Journal of Stem Cells
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• v.17 no.2
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• pp.113-119
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• 2024

This report presents guidelines for the systematic management of packaging, storage, transportation, and traceability of source cells used for organoid research. Given the important role of source cells in organoid studies, it is important to ensure the preservation of their quality and integrity throughout transportation and distribution processes. The proposed guidelines, therefore, call for a cohesive strategy through these stages to minimize the risks of contamination, deterioration, and loss-threats that significantly compromise the safety, efficacy, and efficiency of source cells. Central to these guidelines is the quality control measures that include roles and responsibilities across the entire supply chain, with recommendations specific to packaging materials, transportation facilities, and storage management. Furthermore, the need for an integrated management system is emphasized, spanning from source cell collection to the final application. This system is crucial for maintaining the traceability and accountability of source cells, facilitating the sharing, distribution, and utilization on a global scale, and supporting to advance organoid research and development.

• Journal of Microbiology and Biotechnology
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• v.34 no.8
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• pp.1711-1717
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• 2024

This study evaluates the efficacy of a decellularized intestine tissue-derived extracellular matrix (Intestine ECM) as a scaffold for culturing colorectal cancer (CRC) organoids and establishing cell-derived xenograft (CDX) models, comparing its performance to traditional Matrigel. Intestine ECM demonstrates comparable support for organoid formation and cellular function, highlighting its potential as a more physiologically relevant and reproducible platform. Our findings suggest that Intestine ECM enhances the mimetic environment for colon epithelium, supporting comparable growth and improved differentiation compared to Matrigel. Moreover, when used as a delivery carrier, Intestine ECM significantly increases the growth rate of CDX models using patient-derived primary colorectal cancer cells. This enhancement demonstrates Intestine ECM's role not only as a scaffold but also as a vital component of the tumor microenvironment, facilitating more robust tumorigenesis. These findings advocate for the broader application of Intestine ECM in cancer model systems, potentially leading to more accurate preclinical evaluations and the development of targeted cancer therapies.

• Development and Reproduction
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• v.27 no.2
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• pp.57-65
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• 2023

Kidney disease affects a significant portion of the global population, yet effective therapies are lacking despite advancements in identifying genetic causes. This limitation can be attributed to the absence of adequate in vitro models that accurately mimic human kidney disease, hindering targeted therapeutic development. However, the emergence of human induced pluripotent stem cells (PSCs) and the development of organoids using them have opened up a way to model kidney development and disease in humans, as well as validate the effects of new drugs. To fully leverage their capabilities in these fields, it is crucial for kidney organoids to closely resemble the structure and functionality of adult human kidneys. In this review, we aim to discuss the potential of using human PSCs or adult kidney stem cell-derived kidney organoids to model genetic kidney disease and renal cancer.

• Annals of Hepato-Biliary-Pancreatic Surgery
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• v.27 no.4
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• pp.342-349
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• 2023

Backgrounds/Aims: Liver organoids have emerged as a powerful tool for studying liver biology and disease and for developing new therapies and regenerative medicine approaches. For organoid culture, Matrigel, a type of extracellular matrix, is the most commonly used material. However, Matrigel cannot be used for clinical applications due to the presence of unknown proteins that can cause immune rejection, batch-to-batch variability, and angiogenesis. Methods: To obtain human primary hepatocytes (hPHs), we performed 2 steps collagenase liver perfusion protocol. We treated three small molecules cocktails (A83-01, CHIR99021, and HGF) for reprogramming the hPHs into human chemically derived hepatic progenitors (hCdHs) and used hCdHs to generate liver organoids. Results: In this study, we report the generation of liver organoids in a collagen scaffold using hCdHs. In comparison with adult liver (or primary hepatocyte)-derived organoids with collagen scaffold (hALO_C), hCdH-derived organoids in a collagen scaffold (hCdHO_C) showed a 10-fold increase in organoid generation efficiency with higher expression of liver- or liver progenitor-specific markers. Moreover, we demonstrated that hCdHO_C could differentiate into hepatic organoids (hCdHO_C_DM), indicating the potential of these organoids as a platform for drug screening. Conclusions: Overall, our study highlights the potential of hCdHO_C as a tool for liver research and presents a new approach for generating liver organoids using hCdHs with a collagen scaffold.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.269-280
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• 2023

Background and Objectives: The colonic epithelial layer is a complex structure consisting of multiple cell types that regulate various aspects of colonic physiology, yet the mechanisms underlying epithelial cell differentiation during development remain unclear. Organoids have emerged as a promising model for investigating organogenesis, but achieving organ-like cell configurations within colonic organoids is challenging. Here, we investigated the biological significance of peripheral neurons in the formation of colonic organoids. Methods and Results: Colonic organoids were co-cultured with human embryonic stem cell (hESC)-derived peripheral neurons, resulting in the morphological maturation of columnar epithelial cells, as well as the presence of enterochromaffin cells. Substance P released from immature peripheral neurons played a critical role in the development of colonic epithelial cells. These findings highlight the vital role of inter-organ interactions in organoid development and provide insights into colonic epithelial cell differentiation mechanisms. Conclusions: Our results suggest that the peripheral nervous system may have a significant role in the development of colonic epithelial cells, which could have important implications for future studies of organogenesis and disease modeling.

• International Journal of Stem Cells
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• v.15 no.1
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• pp.85-94
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• 2022

Background and Objectives: Brain organoids have the potential to improve our understanding of brain development and neurological disease. Despite the importance of brain organoids, the effect of vascularization on brain organoids is largely unknown. The objective of this study is to develop vascularized organoids by assembling vascular spheroids with cerebral organoids. Methods and Results: In this study, vascularized spheroids were generated from non-adherent microwell culture system of human umbilical vein endothelial cells, human dermal fibroblasts and human umbilical cord blood derived mesenchymal stem cells. These vascular spheroids were used for fusion with iPSCs induced cerebral organoids. Immunostaining studies of vascularized organoids demonstrated well organized vascular structures and reduced apoptosis. We showed that the vascularization in cerebral organoids up-regulated the Wnt/β-catenin signaling. Conclusions: We developed vascularized cerebral organoids through assembly of brain organoids with vascular spheroids. This method could not only provide a model to study human cortical development but also represent an opportunity to explore neurological disease.

• International Journal of Stem Cells
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• v.16 no.4
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• pp.385-393
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• 2023

In vertebrates, the entire central nervous system is derived from the neural tube, which is formed through a conserved early developmental morphogenetic process called neurulation. Although the perturbations in neurulation caused by genetic or environmental factors lead to neural tube defects (NTDs), the most common congenital malformation and the precise molecular pathological cascades mediating NTDs are not well understood. Recently, we have developed human spinal cord organoids (hSCOs) that recapitulate some aspects of human neurulation and observed that valproic acid (VPA) could cause neurulation defects in an organoid model. In this study, we identified and verified the significant changes in cell-cell junctional genes/proteins in VPA-treated organoids using transcriptomic and immunostaining analysis. Furthermore, VPA-treated mouse embryos exhibited impaired gene expression and NTD phenotypes, similar to those observed in the hSCO model. Collectively, our data demonstrate that hSCOs provide a valuable biological resource for dissecting the molecular pathways underlying the currently unknown human neurulation process using destructive biological analysis tools.

• BMB Reports
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• v.57 no.7
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• pp.311-317
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• 2024

Brain organoid is a three-dimensional (3D) tissue derived from stem cells such as induced pluripotent stem cells (iPSCs) embryonic stem cells (ESCs) that reflect real human brain structure. It replicates the complexity and development of the human brain, enabling studies of the human brain in vitro. With emerging technologies, its application is various, including disease modeling and drug screening. A variety of experimental methods have been used to study structural and molecular characteristics of brain organoids. However, electrophysiological analysis is necessary to understand their functional characteristics and complexity. Although electrophysiological approaches have rapidly advanced for monolayered cells, there are some limitations in studying electrophysiological and neural network characteristics due to the lack of 3D characteristics. Herein, electrophysiological measurement and analytical methods related to neural complexity and 3D characteristics of brain organoids are reviewed. Overall, electrophysiological understanding of brain organoids allows us to overcome limitations of monolayer in vitro cell culture models, providing deep insights into the neural network complex of the real human brain and new ways of disease modeling.

• Molecules and Cells
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• v.41 no.4
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• pp.290-300
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• 2018

Using an in vitro model of intestinal organoids derived from intestinal crypts, we examined effects of indole-3-carbinol (I3C), a phytochemical that has anticancer and aryl hydrocarbon receptor (AhR)-activating abilities and thus is sold as a dietary supplement, on the development of intestinal organoids and investigated the underlying mechanisms. I3C inhibited the in vitro development of mouse intestinal organoids. Addition of ${\alpha}$-naphthoflavone, an AhR antagonist or AhR siRNA transfection, suppressed I3C function, suggesting that I3C-mediated interference with organoid development is AhR-dependent. I3C increased the expression of Muc2 and lysozyme, lineage-specific genes for goblet cells and Paneth cells, respectively, but inhibits the expression of IAP, a marker gene for enterocytes. In the intestines of mice treated with I3C, the number of goblet cells was reduced, but the number of Paneth cells and the depth and length of crypts and villi were not changed. I3C increased the level of active nonphosphorylated ${\beta}$-catenin, but suppressed the Notch signal. As a result, expression of Hes1, a Notch target gene and a transcriptional repressor that plays a key role in enterocyte differentiation, was reduced, whereas expression of Math1, involved in the differentiation of secretory lineages, was increased. These results provide direct evidence for the role of AhR in the regulation of the development of intestinal stem cells and indicate that such regulation is likely mediated by regulation of Wnt and Notch signals.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.136-143
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• 2022

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.