The Application of Organoids in the Study of Antiviral Innate Immunity.

Antiviral innate immunity plays a critical role in the defense against viral infections, yet its complex interactions with viruses have been challenging to study using traditional models. Organoids, three-dimensional (3D) tissue-like structures derived from stem cells, have emerged as powerful tools for modeling human tissues and studying the complex interactions between viruses and the host innate immune system. This chapter summarizes relevant applications of organoids in antiviral innate immunity studies and provides detailed information and experimental procedures for using organoids to study antiviral innate immunity.

Generation of Induced-Primary Retinal Pigment Epithelium from Human Retinal Organoids.

Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.

Generating ESC-Derived RGCs for Cell Replacement Therapy.

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.

Development of Liver Cancer Organoids: Reproducing Tumor Microenvironment and Advancing Research for Liver Cancer Treatment.

Liver cancer a leading cause of cancer-related deaths worldwide, yet understanding of its development mechanism remains limited, and treatment barriers present substantial challenges. Owing to the heterogeneity of tumors, traditional 2D culture models are inadequate for capturing the complexity and diversity of tumor biology and understanding of the disease. Organoids have garnered considerable attention because of their ability to self-renew and develop functional structures in vitro that closely resemble those of human organs. This review explores the history of liver organoids, their cellular origins, techniques of constructing tumor microenvironments that recapitulate liver cancer organoids, and the biological and clinical applications of liver and liver cancer organoids and explores the current challenges related to liver cancer organoid applications and potentially valuable solutions, with the aim of facilitating the construction of in vitro clinical models of liver cancer therapeutic research.

Oncolytic activity of a coxsackievirus B3 strain in patient-derived cervical squamous cell carcinoma organoids and synergistic effect with paclitaxel.

BACKGROUND: Cervical squamous cell carcinoma (CSCC) is a prevalent gynecological malignancy worldwide. Current treatments for CSCC can impact fertility and cause long-term complications, underscoring the need for new therapeutic strategies. Oncolytic virotherapy has emerged as a promising option for cancer treatment. Previous research has demonstrated the oncolytic activity of the coxsackievirus B3 strain 2035 A (CVB3/2035A) against various tumor types. This study aims to evaluate the clinical viability of CVB3/2035A for CSCC treatment, focusing on its oncolytic effect in patient-derived CSCC organoids. METHODS: The oncolytic effects of CVB3/2035A were investigated using human CSCC cell lines in vitro and mouse xenograft models in vivo. Preliminary tests for tumor-selectivity were conducted on patient-derived CSCC tissue samples and compared to normal cervical tissues ex vivo. Three patient-derived CSCC organoid lines were developed and treated with CVB3/2035A alone and in combination with paclitaxel. Both cytotoxicity and virus replication were evaluated in vitro. RESULTS: CVB3/2035A exhibited significant cytotoxic effects in human CSCC cell lines and xenograft mouse models. The virus selectively induced oncolysis in patient-derived CSCC tissue samples while sparing normal cervical tissues ex vivo. In patient-derived CSCC organoids, which retained the immunohistological characteristics of the original tumors, CVB3/2035A also demonstrated significant cytotoxic effects and efficient replication, as evidenced by increased viral titers and presence of viral nucleic acids and proteins. Notably, the combination of CVB3/2035A and paclitaxel resulted in enhanced cytotoxicity and viral replication. CONCLUSIONS: CVB3/2035A showed oncolytic activity in CSCC cell lines, xenografts, and patient-derived tissue cultures and organoids. Furthermore, the virus exhibited synergistic anti-tumor effects with paclitaxel against CSCC. These results suggest CVB3/2035A could serve as an alternative or adjunct to current CSCC chemotherapy regimens.

Development of an intestinal epithelial cell line and organoids derived from the same swine and characterization of their antiviral responses.

Intestinal homeostasis and integrity are important factors for maintaining host health. This study established intestinal epithelial cell lines and organoids from the same swine jejunal crypts to develop seamless swine intestinal in vitro evaluation systems. The study evaluated the proliferative capacity and tight junction formation of the epithelial cell line and characterized the cell differentiation potential of the intestinal organoids. The evaluation systems were subsequently exposed to the Toll-like receptor 3 (TLR3) agonist poly(I:C) to simulate viral infections and assess the antiviral responses. The results demonstrated no differences in the response to type I interferons. There were, however, significant differences in the expression of interferon-stimulated genes. This study collectively introduced a flexible evaluation system using cell lines and organoids and revealed notable differences in the expression of interferon-stimulated genes, highlighting the complexity of the immune responses in these in vitro systems and the importance of intestinal heterogeneity in assessing viral responses.

Embryonic development grand challenge: crosslinking advances.

Research on embryonic development is entering into a new era. As a traditionally descriptive discipline within anatomy, embryologists have formed international consortia and digitized important histological collections for preservation and open access. Embryonic development has recently received a wider attention in context with temporo-spatial transcriptomics at single cell level. These can be expected to fuel the realization of the transdisciplinary significance of efforts to decipher embryonic development. Addressing its complexities encompasses a wealth of challenges that intersect across the domains of science, society, and politics underlining its outstanding importance as well as its inherently interdisciplinary nature. The challenges of this field are by no means confined to understanding the intricate biological mechanisms but also have humanitarian implications. To fully appreciate the mechanisms underlying human development, principles of embryogenesis have predominantly been analyzed employing animal models which allow us to broaden our view on developmental processes. As a result of recent pioneering work and technical progress centered around stem cell-based 3D approaches, we are entering into a historical new phase of learning about mammalian embryonic development. In vertebrates, a growing concern now focuses the reduction of animal experimentation. This perspective article outlines the major challenges in this amazing field that offer an enormous potential for basic biomedical sciences as well as related translational approaches if they are tackled in a multidisciplinary discourse.

Establishment of goat mammary organoid cultures modeling the mammary gland development and lactation.

BACKGROUND: Although several cell culture systems have been developed to investigate the function of the mammary gland in dairy livestock, they have potential limitations, such as the loss of alveolar structure or genetic and phenotypic differences from their native counterparts. Overcoming these challenges is crucial for lactation research. Development of protocols to establish lactating organoid of livestock represents a promising goal for the future. In this study, we developed a protocol to establish a culture system for mammary organoids in dairy goats to model the mammary gland development and lactation process. RESULTS: The organoids cultured within an extracellular matrix gel maintained a bilayer structure that closely resembled the native architecture of mammary tissue. The expansion of mammary organoids was significantly promoted by growth factors containing epidermal growth factor and fibroblast growth factor 2 whereas the proliferative index of the organoids was significantly inhibited by the treatment with WNT inhibitors. Upon stimulation with a lactogenic medium containing prolactin, the mammary organoids exhibited efficient lactation, characterized by the accumulation of lipid droplets in the lumen space. The lactation could be sustained for more than 3 weeks. Importantly, the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in mammary tissues. These observations were confirmed by data from proteomic analysis that the bulk of milk proteins was produced in the lactating organoids. CONCLUSION: This study is the first to establish a mammary organoid culture system modeling the mammary gland development and lactation process in ruminants. The efficient induction of lactation in ruminant mammary organoids holds promises for advancing the field of cell-based milk bio-manufacture in the food industry.

RCAN1.4 regulates tumor cell engraftment and invasion in a thyroid cancer to lung metastasis-on-a-chip microphysiological system.

Progressive metastasis is the primary cause of cancer-related deaths. It has been recognized that many cancers are characterized by long periods of stability followed by subsequent progression. Genes termed metastasis progression suppressors (MPS) are functional gatekeepers of this process, and their loss leads to late-stage progression. Previously, we identified regulator of calcineurin 1, isoform 4 (RCAN1.4) as a functional MPS for several cancers, including thyroid cancer, a tumor type prone to metastatic dormancy. RCAN1.4 knockdown increases expression of the cancer-promoting transcription factor NFE2-like bZIP transcription factor (NFE2L3), and through this mechanism increases cancer cell proliferation and invasion in in vitro and in vivo and promotes metastatic potential to lungs in tail vein models. However, the mechanisms by which RCAN 1.4 regulates specific metastatic steps is incompletely characterized. Studies of the metastatic cascade are limited in mouse systems due to high cost and long duration. Here, we have shown the creation of a thyroid-to-lung metastasis-on-a-chip (MOC) model to address these limitations, allowing invasion analysis and quantification on a single cell level. We then deployed the platform to investigate RCAN1.4 knockdown in fluorescently tagged hTh74 and FTC236 thyroid cancer cell lines. Cells were circulated through microfluidic channels, running parallel to lung hydrogel constructs allowing tumor cell-lung tissue interactions. Similar to studies in mouse models, RCAN1.4 knockdown increased NFE2L3 expression, globally increased invasion distance into lung constructs and had cell line and clonally dependent variations on bulk metastatic burden. In line with previous in vivo observations, RCAN1.4 knockdown had a greater impact on hTh74 metastatic propensity than FTC236. In summary, we have developed and validated a novel MOC system evaluate and quantify RCAN1.4-regulated thyroid cancer cell lung adherence and invasion. This system creates opportunities for more detailed and rapid mechanistic studies the metastatic cascade and creates opportunities for translational assay development.

Transplantation of human pluripotent stem cell-derived retinal sheet in a primate model of macular hole.

Macular hole (MH) is a retinal break involving the fovea that causes impaired vision. Although advances in vitreoretinal surgical techniques achieve >90% MH closure rate, refractory cases still exist. For such cases, autologous retinal transplantation is an optional therapy showing good anatomic success, but visual improvement is limited and peripheral visual field defects are inevitable after graft harvesting. Here, using a non-human primate model, we evaluated whether human embryonic stem cell-derived retinal organoid (RO) sheet transplantation can be an effective option for treating MH. After transplantation, MH was successfully closed by continuous filling of the MH space with the RO sheet, resulting in improved visual function, although no host-graft synaptic connections were confirmed. Mild xeno-transplantation rejection was controlled by additional focal steroid injections and rod/cone photoreceptors developed in the graft. Overall, our findings suggest pluripotent stem cell-derived RO sheet transplantation as a practical option for refractory MH treatment.

A human organoid drug screen identifies α2-adrenergic receptor signaling as a therapeutic target for cartilage regeneration.

Directed differentiation of stem cells toward chondrogenesis in vitro and in situ to regenerate cartilage suffers from off-target differentiation and hypertrophic tendency. Here, we generated a cartilaginous organoid system from human expanded pluripotent stem cells (hEPSCs) carrying a COL2A1mCherry and COL10A1eGFP double reporter, enabling real-time monitoring of chondrogenesis and hypertrophy. After screening 2,040 FDA-approved drugs, we found that α-adrenergic receptor (α-AR) antagonists, especially phentolamine, stimulated chondrogenesis but repressed hypertrophy, while α2-AR agonists reduced chondrogenesis and induced hypertrophy. Phentolamine prevented cartilage degeneration in hEPSC cartilaginous organoid and human cartilage explant models and stimulated microfracture-activated endogenous skeletal stem cells toward hyaline-like cartilage regeneration without fibrotic degeneration in situ. Mechanistically, α2-AR signaling induced hypertrophic degeneration via cyclic guanosine monophosphate (cGMP)-dependent secretory leukocyte protease inhibitor (SLPI) production. SLPI-deleted cartilaginous organoid was degeneration resistant, facilitating large cartilage defect healing. Ultimately, targeting α2-AR/SLPI was a promising and clinically feasible strategy to regenerate cartilage via promoting chondrogenesis and repressing hypertrophy.

Bioreactor-produced iPSCs-derived dopaminergic neuron-containing neural microtissues innervate and normalize rotational bias in a dose-dependent manner in a Parkinson rat model.

A breadth of preclinical studies now support the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, most cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such a format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with cryopreserved single-cell DA progenitors. Here, we used a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabling the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods, including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner using a Parkinsonian rat model. The survival of the cells was confirmed by post-mortem histological analysis, characterised by the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson's disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks after transplantation using cryopreserved 3D format.

Beyond skin deep: Revealing the essence of iPS cell-generated skin organoids in regeneration.

Various methods have been used for in vivo and in vitro skin regeneration, including stem cell therapy, tissue engineering, 3D printing, and platelet-rich plasma (PRP) injection therapy. However, these approaches are rooted in the existing knowledge of skin structures, which overlook the normal physiological processes of skin development and fall short of replicating the skin's regenerative processes outside the body. This comprehensive review primarily focuses on skin organoids derived from human pluripotent stem cells, which have the capacity to regenerate human skin tissue by restoring the embryonic skin structure, thus offering a novel avenue for producing in vitro skin substitutes. Furthermore, they contribute to the repair of damaged skin lesions in patients with systemic sclerosis or severe burns. Particular emphasis will be placed on the origins, generations, and applications of skin organoids, especially in dermatology, and the challenges that must be addressed before clinical implementation.

Establishment of an Apical-Out Organoid Model for Directly Assessing the Function of Postbiotics.

In vitro organoids that mimic the physiological properties of in vivo organs based on threedimensional cell cultures overcome the limitations of two-dimensional culture systems. However, because the lumen of a typical intestinal organoid is internal, we used an apical-out intestinal organoid model in which the lumen that absorbs nutrients is outside to directly assess the function of postbiotics. A composite culture supernatant of Lactiplantibacillus plantarum KM2 and Bacillus velezensis KMU01 was used as a postbiotic treatment. Expression of COX-2 decreased in apical-out organoids co-treated with a lipopolysaccharide (LPS) and postbiotics. Expression of tight-junction markers such as ZO-1, claudin, and Occludin increased, and expression of mitochondrial homeostasis factors such as PINK1, parkin, and PGC1a also increased. As a result, small and large intestine organoids treated with postbiotics protected tight junctions from LPS-induced damage and maintained mitochondrial homeostasis through mitophagy and mitochondrial biogenesis. This suggests that an apical-out intestinal organoid model can confirm the function of food ingredients.

A novel combination therapy for ER+ breast cancer suppresses drug resistance via an evolutionary double-bind.

Chemotherapy remains a commonly used and important treatment option for metastatic breast cancer. A majority of ER+ metastatic breast cancer patients ultimately develop resistance to chemotherapy, resulting in disease progression. We hypothesized that an "evolutionary double-bind", where treatment with one drug improves the response to a different agent, would improve the effectiveness and durability of responses to chemotherapy. This approach exploits vulnerabilities in acquired resistance mechanisms. Evolutionary models can be used in refractory cancer to identify alternative treatment strategies that capitalize on acquired vulnerabilities and resistance traits for improved outcomes. To develop and test these models, ER+ breast cancer cell lineages sensitive and resistant to chemotherapy are grown in spheroids with varied initial population frequencies to measure cross-sensitivity and efficacy of chemotherapy and add-on treatments such as disulfiram combination treatment. Different treatment schedules then assessed the best strategy for reducing the selection of resistant populations. We developed and parameterized a game-theoretic mathematical model from this in vitro experimental data, and used it to predict the existence of a double-bind where selection for resistance to chemotherapy induces sensitivity to disulfiram. The model predicts a dose-dependent re-sensitization (a double-bind) to chemotherapy for monotherapy disulfiram.

Developing patient-derived organoids to demonstrate JX24120 inhibits SAMe synthesis in endometrial cancer by targeting MAT2B.

Endometrial cancer (EC) is one of the most common gynecologic malignancies, which lacking effective drugs for intractable conditions or patients unsuitable for surgeries. Recently, the patient-derived organoids (PDOs) are found feasible for cancer research and drug discoveries. Here, we have successfully established a panel of PDOs from EC and conducted drug repurposing screening and mechanism analysis for cancer treatment. We confirmed that the regulatory ß subunit of methionine adenosyltransferase (MAT2B) is highly correlated with malignant progression in endometrial cancer. Through drug screening on PDOs, we identify JX24120, chlorpromazine derivative, as a specific inhibitor for MAT2B, which directly binds to MAT2B (Kd = 4.724 µM) and inhibits the viability of EC PDOs and canonical cell lines. Correspondingly, gene editing assessment demonstrates that JX24120 suppresses tumor growth depending on the presence of MAT2B in vivo and in vitro. Mechanistically, JX24120 induces inhibition of S-adenosylmethionine (SAMe) synthesis, leading to suppressed mTORC1 signaling, abnormal energy metabolism and protein synthesis, and eventually apoptosis. Taken together, our study offers a novel approach for drug discovery and efficacy assessment by using the PDOs models. These findings suggest that JX24120 may be a potent MAT2B inhibitor and will hopefully serve as a prospective compound for endometrial cancer therapy.

Enhancing organoid culture: harnessing the potential of decellularized extracellular matrix hydrogels for mimicking microenvironments.

Over the past decade, organoids have emerged as a prevalent and promising research tool, mirroring the physiological architecture of the human body. However, as the field advances, the traditional use of animal or tumor-derived extracellular matrix (ECM) as scaffolds has become increasingly inadequate. This shift has led to a focus on developing synthetic scaffolds, particularly hydrogels, that more accurately mimic three-dimensional (3D) tissue structures and dynamics in vitro. The ECM-cell interaction is crucial for organoid growth, necessitating hydrogels that meet organoid-specific requirements through modifiable physical and compositional properties. Advanced composite hydrogels have been engineered to more effectively replicate in vivo conditions, offering a more accurate representation of human organs compared to traditional matrices. This review explores the evolution and current uses of decellularized ECM scaffolds, emphasizing the application of decellularized ECM hydrogels in organoid culture. It also explores the fabrication of composite hydrogels and the prospects for their future use in organoid systems.

Cell and gene therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disorder with rapidly progressive skeletal muscle weakness, which can also cause a variable cognitive deficit. Genetic causes are only identified in approximately 10% of all cases, with complex genotype-phenotype associations, making it challenging to identify treatment targets. What further hampers therapeutic development is a broad heterogeneity in mechanisms, possible targets, and disturbances across various cell types, aside from the cortical and spinal motor neurons that lie at the heart of the pathology of ALS. Over the last decade, significant progress in biotechnologic techniques, cell and ribonucleic acid (RNA) engineering, animal models, and patient-specific human stem cell and organoid models have accelerated both mechanistic and therapeutic discoveries. The growing number of clinical trials mirrors this. This chapter reviews the current state of human preclinical models supporting trial strategies as well as recent clinical cell and gene therapy approaches.

The use of organoids in creating immune microenvironments and treating gynecological tumors.

Owing to patient-derived tumor tissues and cells, significant advances have been made in personalized cancer treatment and precision medicine, with cancer stem cell-derived three-dimensional tumor organoids serving as crucial in vitro models that accurately replicate the structural, phenotypic, and genetic characteristics of tumors. However, despite their extensive use in drug testing, genome editing, and transplantation for facilitating personalized treatment approaches in clinical practice, the inadequate capacity of these organoids to effectively model immune cells and stromal components within the tumor microenvironment limits their potential. Additionally, effective clinical immunotherapy has led the tumor immune microenvironment to garner considerable attention, increasing the demand for simulating patient-specific tumor-immune interactions. Consequently, co-culture techniques integrating tumor organoids with immune cells and tumor microenvironment constituents have been developed to expand the possibilities for personalized drug response investigations, with recent advancements enhancing the understanding of the strengths, limitations, and applicability of the co-culture approach. Herein, the recent advancements in the field of tumor organoids have been comprehensively reviewed, specifically highlighting the tumor organoid co-culture-related developments with various immune cell models and their implications for clinical research. Furthermore, this review delineates the current state of research and application of organoid models regarding the therapeutic approaches and related challenges for gynecological tumors. This study may provide a theoretical basis for further research on the use of patient-derived organoids in tumor immunity, drug development, and precision medicine.

Human Multi-Lineage Liver Organoid Model Reveals Impairment of CYP3A4 Expression upon Repeated Exposure to Graphene Oxide.

Three-dimensional hepatic cell cultures can provide an important advancement in the toxicity assessment of nanomaterials with respect to 2D models. Here, we describe liver organoids (LOs) obtained by assembling multiple cell lineages in a fixed ratio 1:1:0.2. These are upcyte® human hepatocytes, UHHs, upcyte® liver sinusoidal endothelial cells, LSECs, and human bone marrow-derived mesenchymal stromal cells, hbmMSCs. The structural and functional analyses indicated that LOs reached size stability upon ca. 10 days of cultivation (organoid maturation), showing a surface area of approximately 10 mm2 and the hepatic cellular lineages, UHHs and LSECs, arranged to form both primitive biliary networks and sinusoid structures, alike in vivo. LOs did not show signs of cellular apoptosis, senescence, or alteration of hepatocellular functions (e.g., dis-regulation of CYP3A4 or aberrant production of Albumin) for the entire culture period (19 days since organoid maturation). After that, LOs were repeatedly exposed for 19 days to a single or repeated dose of graphene oxide (GO: 2-40 µg/mL). We observed that the treatment did not induce any macroscopic signs of tissue damage, apoptosis activation, and alteration of cell viability. However, in the repeated dose regimen, we observed a down-regulation of CYP3A4 gene expression. Notably, these findings are in line with recent in vivo data, which report a similar impact on CYP3A4 when mice were repeatedly exposed to GO. Taken together, these findings warn of the potential detrimental effects of GO in real-life exposure (e.g., occupational scenario), where its progressive accumulation is likely expected. More in general, this study highlights that LOs formed by many cell lineages can enable repeated exposure regimens (suitable to mimic accumulation); thus, they can be suitably considered alternative or complementary in vitro systems to animal models.