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.

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.

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.

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids.

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

Viscoelasticity of Hyaluronic Acid Hydrogels Regulates Human Pluripotent Stem Cell-derived Spinal Cord Organoid Patterning and Vascularization.

Recently, it has been recognized that natural extracellular matrix (ECM) and tissues are viscoelastic, while only elastic properties have been investigated in the past. How the viscoelastic matrix regulates stem cell patterning is critical for cell-ECM mechano-transduction. Here, this study fabricated different methacrylated hyaluronic acid (HA) hydrogels using covalent cross-linking, consisting of two gels with similar elasticity (stiffness) but different viscoelasticity, and two gels with similar viscoelasticity but different elasticity (stiffness). Meanwhile, a second set of dual network hydrogels are fabricated containing both covalent and coordinated cross-links. Human spinal cord organoid (hSCO) patterning in HA hydrogels and co-culture with isogenic human blood vessel organoids (hBVOs) are investigated. The viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. More viscoelastic hydrogels can promote dorsal marker expression, while softer hydrogels result in higher interneuron marker expression. The effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects in the co-culture of hSCOs and hBVOs. In addition, more viscoelastic hydrogels can lead to more Yes-associated protein nuclear translocation, revealing the mechanism of cell-ECM mechano-transduction. This research provides insights into viscoelastic behaviors of the hydrogels during human organoid patterning with ECM-mimicking in vitro microenvironments for applications in regenerative medicine.

Antitumor effect of polyphyllin I (PPI) on colorectal cancer: Evidence from patient-derived organoids and Notch signaling suppression.

Colorectal cancer (CRC) is a malignant tumor with a high incidence, ranking first among gastrointestinal malignancies. We investigated the impact of polyphyllin I (PPI), a natural compound found in Paris polyphylla, on CRC. PPI has been documented to exhibit anticancer activity against various tumors. This study aimed to assess the effects of PPI on colorectal cancer and explore its potential mechanisms. Our research demonstrated that PPI inhibited proliferation, promoted apoptosis, and induced G2 cell-cycle arrest in a dose-dependent manner. Additionally, our results indicated that PPI suppressed Notch signaling by downregulating the Notch1 receptor, its ligand Jagged1, and the downstream target Hes1 expression. Furthermore, we confirmed the antitumor effect of PPI on patient-derived organoids. In conclusion, our study indicates that PPI impedes the growth of colon cancer by suppressing the Notch signaling pathway.

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.

Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

Microsatellite stable (MSS) colorectal cancers (CRCs) are often resistant to anti-programmed death-1 (PD-1) therapy. Here, we show that a CRC pathogen, Fusobacterium nucleatum (Fn), paradoxically sensitizes MSS CRC to anti-PD-1. Fecal microbiota transplantation (FMT) from patients with Fn-high MSS CRC to germ-free mice bearing MSS CRC confers sensitivity to anti-PD-1 compared to FMT from Fn-low counterparts. Single Fn administration also potentiates anti-PD-1 efficacy in murine allografts and CD34+-humanized mice bearing MSS CRC. Mechanistically, we demonstrate that intratumoral Fn generates abundant butyric acid, which inhibits histone deacetylase (HDAC) 3/8 in CD8+ T cells, inducing Tbx21 promoter H3K27 acetylation and expression. TBX21 transcriptionally represses PD-1, alleviating CD8+ T cell exhaustion and promoting effector function. Supporting this notion, knockout of a butyric acid-producing gene in Fn abolishes its anti-PD-1 boosting effect. In patients with MSS CRC, high intratumoral Fn predicts favorable response to anti-PD-1 therapy, indicating Fn as a potential biomarker of immunotherapy response in MSS CRC.

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.

ESM1 facilitates the EGFR/HER3-triggered epithelial-to-mesenchymal transition and progression of gastric cancer via modulating interplay between Akt and angiopoietin-2 signaling.

Gastric cancer (GC) poses global challenges due to its difficult early diagnosis and drug resistance, necessitating the identification of early detection markers and understanding of oncogenic pathways for effective GC therapy. Endothelial cell-specific molecule 1 (ESM1), a secreted glycoprotein, is elevated in various cancers, but its role in GC remains controversial. In our study, ESM1 was elevated in GC tissues, and its concentration was correlated with progression and poorer patient prognosis in independent cohorts. Functionally, ESM1 expression promoted proliferation, anoikis resistance, and motility of GC cells, as well as tumor growth in PDOs and in GC xenograft models. Mechanistically, ESM1 expression triggered the epithelial-to-mesenchymal transition (EMT) of GC cells by enhancing epidermal growth factor receptor (EGFR)/human EGFR 3 (HER3) association and activating the EGFR/HER3-Akt pathway. Additionally, angiopoietin-2 (ANGPT2) was found to be highly correlated with ESM1 and interplayed with Akt to induce the EMT and cancer progression. Use of a signal peptide deletion mutant (ESM1-19del) showed that the secreted form of ESM1 is crucial for its protumorigenic effects by activating the EGFR/HER3-Akt/ANGPT2 pathway to promote the EMT. Patients with high levels of both ESM1 and ANGPT2 had the poorest prognoses. Furthermore, therapeutic peptides successfully inhibited ESM1's induction of the aforementioned signals and motility of GC cells. ESM1's oncogenic role in GC involves activating the EGFR/HER3-Akt/ANGPT2 pathway, presenting a potential therapeutic target for GC.

Ophthalmologic manifestations of organoid nevus syndrome: A series of 13 cases.

Background: The organoid nevus syndrome is a rare neurocutaneous syndrome typified by cutaneous sebaceous nevus, seizures and epibulbar choristomas. The condition is associated with multiple ocular abnormalities. Herein, the authors aim to study and report the ophthalmic features of cases diagnosed with organoid nevus syndrome. Methods: The authors retrospectively evaluated the records of patients with the organoid nevus syndrome who had presented to a tertiary care eye hospital in northern India. The ocular features were studied and entered in MS excel and the data were evaluated. Results: Data of 13 patients with the organoid nevus syndrome were found. All 13 patients had cutaneous features in the form of Sebaceous nevus of Jadasson, 8 patients had alopecia of the scalp area, 2 had history seizures and 10 had arachnoid cysts on neuroimaging of the head. All 13 patients had a complex choristoma involving the ocular surface. Conclusions: We conclude that the most common ophthalmologic features associated with organoid nevus are complex choristoma of the bulbar surface, scleral coat calcification and upper eyelid coloboma.

A reference for selecting an appropriate method for generating glioblastoma organoids from the application perspective.

Glioblastoma organoids (GBOs) serve as a powerful and reliable tool to study glioblastoma stem cells (GSCs) and glioblastoma (GBM). GBOs can be derived from different materials using different methods. To identify the predominant generation methods and the most applications of GBOs, we searched four databases (PubMed, Embase, Web of Science, and Wiley Online Laboratory) from August 2021 to August 2023. After screening, 42 out of 295 articles were included and analyzed. GBOs in these articles were generated using only one material, such as tumor tissues, tumor cells, and gene-edited multifunctional stem cells, or simultaneously using two materials, such as tumor cells and normal organoids. Methodologically, direct cultivation of GBM cells or tissues was the most commonly used method to generate GBOs. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were the frequently used multifunctional stem cells to generate GBOs by simultaneously silencing P53, NF1, and PTEN using CRISPR/Cas9. In terms of applications, GBOs generated by direct cultivation of GBM tissue had the most applications, including molecular mechanisms, therapy, and culture technique. This review provides a theoretical reference for selecting an appropriate method to generate GBOs when studying GSCs and GBM.

Unveiling senescence-associated ocular pathogenesis via lacrimal gland organoid magnetic bioassembly platform and HMGB1-Box A gene therapy.

Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability. Yet, an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis, due to limited in vitro translational lacrimal gland (LG) models. Consequently, this remains a major roadblock to discover effective therapies for the restoration of tear film secretion. Herein, the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states. Using a proof-of-concept approach, porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform. This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity). DNA damage-induced senescence and cell death was induced with etoposide, and LG organoid hypofunction and senescence-associated pathogenesis were observed. To confer DNA protection against aging, a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group, was applied here to prevent LG cellular senescence for the first time. HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic, metabolomic and proteomic levels. Thus, M3DB platforms could generate functional and DNA damage-induced senescence LG organoids, and this latter damage could be prevented with HMGB1-Box A gene therapy.

GelMA loaded with exosomes from human minor salivary gland organoids enhances wound healing by inducing macrophage polarization.

Non-healing skin wounds pose significant clinical challenges, with biologic products like exosomes showing promise for wound healing. Saliva and saliva-derived exosomes, known to accelerate wound repair, yet their extraction is difficult due to the complex environment of oral cavity. In this study, as a viable alternative, we established human minor salivary gland organoids (hMSG-ORG) to produce exosomes (MsOrg-Exo). In vitro, MsOrg-Exo significantly enhanced cell proliferation, migration, and angiogenesis. When incorporated into a GelMA-based controlled-release system, MsOrg-Exo demonstrated controlled release, effectively improving wound closure, collagen synthesis, angiogenesis, and cellular proliferation in a murine skin wound model. Further molecular analyses revealed that MsOrg-Exo promotes proliferation, angiogenesis and the secretion of growth factors in wound sites. Proteomic profiling showed that MsOrg-Exo's protein composition is similar to human saliva and enriched in proteins essential for wound repair, immune modulation, and coagulation. Additionally, MsOrg-Exo was found to modulate macrophage polarization, inducing a shift towards M1 and M2 phenotypes in vitro within 48 h and predominantly towards the M2 phenotype in vivo after 15 days. In conclusion, our study successfully extracted MsOrg-Exo from hMSG-ORGs, confirmed the effectiveness of the controlled-release system combining MsOrg-Exo with GelMA in promoting skin wound healing, and explored the potential role of macrophages in this action.

Protective Effect of Rosavin Against Intestinal Epithelial Injury in Colitis Mice and Intestinal Organoids.

Introduction: Rhodiola species have been utilized as functional foods in Asia and Europe for promoting health. Research has demonstrated that Rhodiola has the potential to alleviate inflammatory bowel disease (IBD) in animal models. However, the specific active components and the underlying mechanism for ameliorating intestinal damage remain unclear. This study aims to explore the relieving effect of Rosavin (Rov), a known active constituent of Rhodiola, in IBD and the regulatory mechanisms. Methods: The therapeutic effect of Rov was evaluated using a murine model of acute colitis induced by dextran sulfate sodium salt (DSS). Inflammatory cytokines and neutrophil activation markers were measured by corresponding kits. Immunohistochemistry, immunofluorescence, TUNEL, and EdU assays were applied to investigate the tight conjunction proteins expression, epithelial marker expression, number of apoptotic cells, and epithelial proliferation, respectively. The protection effect of Rov on gut epithelial injury was assessed using TNF-α-induced intestinal organoids. Additinally, RNA sequencing was applied to observe the genetic alteration profile in these intestinal organoids. Results: Oral administration of Rov significantly attenuated weight loss and restored colon length in mice. Notably, Rov treatment led to decreased levels of pro-inflammatory cytokines and neutrophil activation markers while increasing anti-inflammatory factors. Importantly, Rov restored intestinal despair by increasing the number of Lgr5+ stem cells, Lyz1+ Paneth cells and Muc2+ goblet cells in intestines of colitis mice, displaying reduced epithelial apoptosis and recovered barrier function. In TNF-α-induced intestinal organoids, Rov facilitated epithelial cell differentiation and protected against TNF-α-induced damage. RNA sequencing revealed upregulation in the gene expression associated with epithelial cells (including Lgr5+, Lyz1+ and Muc2+ cells) proliferation and defensin secretion, unveiling the protective mechanisms of Rov on the intestinal epithelial barrier. Discussion: Rov holds potential as a natural prophylactic agent against IBD, with its protective action on the intestinal epithelium being crucial for its therapeutic efficacy.

Navigating the Intersection of Technology and Depression Precision Medicine.

This chapter primarily focuses on the progress in depression precision medicine with specific emphasis on the integrative approaches that include artificial intelligence and other data, tools, and technologies. After the description of the concept of precision medicine and a comparative introduction to depression precision medicine with cancer and epilepsy, new avenues of depression precision medicine derived from integrated artificial intelligence and other sources will be presented. Additionally, less advanced areas, such as comorbidity between depression and cancer, will be examined.

The WFS1-ZnT3-Zn2+ Axis Regulates the Vicious Cycle of Obesity and Depression.

Obesity, a growing global health concern, is closely linked to depression. However, the neural mechanism of association between obesity and depression remains poorly understood. In this study, neural-specific WFS1 deficiency exacerbates the vicious cycle of obesity and depression in mice fed a high-fat diet (HFD), positioning WFS1 as a crucial factor in this cycle. Through human pluripotent stem cells (hESCs) neural differentiation, it is demonstrated that WFS1 regulates Zn2+ homeostasis and the apoptosis of neural progenitor cells (NPCs) and cerebral organoids by inhibiting the zinc transporter ZnT3 under the situation of dysregulated lipid metabolism. Notably, riluzole regulates ZnT3 expression to maintain zinc homeostasis and protect NPCs from lipotoxicity-induced cell death. Importantly, riluzole, a therapeutic molecule targeting the nervous system, in vivo administration prevents HFD-induced obesity and associated depression. Thus, a WFS1-ZnT3-Zn2+ axis critical is demonstrated for the vicious cycle of obesity and depression and that riluzole may have the potential to reverse this process against obesity and depression.

Limosilactobacillus fermentum SLAM 216- Derived Extracellular Vesicles Promote Intestinal Maturation in Mouse Organoid Models.

Probiotics, when consumed in adequate amounts, can promote the health of the host and beneficially modulate the host's immunity. Particularly during the host's early life, the gut intestine undergoes a period of epithelial maturation in which epithelial cells organize into specific crypt and villus structures. This process can be mediated by the gut microbiota. Recent studies have reported that the administration of probiotics can further promote intestinal maturation in the neonatal intestine. Therefore, in this study, we investigated the effects of extracellular vesicles derived from the Limosilactobacillus fermentum SLAM 216 strain, which is an established probiotic with known immune and anti-aging effects on intestinal epithelial maturation and homeostasis, using mouse small intestinal organoids. As per our findings, treatment with L. fermentum SLAM 216-derived LF216EV (LF216EV) has significantly increased the bud number and size of organoid buds. Furthermore, extracellular vesicle (EV) treatment upregulated the expression of maturation-related genes, including Ascl2, Ephb2, Lgr5, and Sox9. Tight junctions are known to have an important role in the intestinal immune barrier, and EV treatment has significantly increased the expression of genes associated with tight junctions, such as Claudin, Muc2, Occludin, and Zo-1, indicating that it can promote intestinal development. This was supported by RNA sequencing, which revealed the upregulation of genes associated with cAMP-mediated signaling, which is known to regulate cellular processes including cell differentiation. Additionally, organoids exposed to LF216EV exhibited upregulation of genes associated with maintaining brain memory and neurotransmission, suggesting possible future functional implications.