Novel Organoid Culture System for Improved Safety Assessment of Nanomaterials.

Over the past few decades, the increased application of nanomaterials has raised questions regarding their safety and possible toxic effects. Organoids have been suggested as promising tools, offering efficient assays for nanomaterial-induced toxicity evaluation. However, organoid systems have some limitations, such as size heterogeneity and poor penetration of nanoparticles because of the extracellular matrix, which is necessary for organoid culture. Here, we developed a novel system for the improved safety assessment of nanomaterials by establishing a 3D floating organoid paradigm. In addition to overcoming the limitations of two-dimensional systems including the lack of in vitro-in vivo cross-talk, our method provides multiple benefits as compared with conventional organoid systems that rely on an extracellular matrix for culture. Organoids cultured using our method exhibited relatively uniform sizing and structural integrity and were more conducive to the internalization of nanoparticles. Our floating culture system will accelerate the research and development of safe nanomaterials.

Combination therapy of niclosamide with gemcitabine inhibited cell proliferation and apoptosis via Wnt/ß-catenin/c-Myc signaling pathway by inducing ß-catenin ubiquitination in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with high morbidity and mortality rates worldwide. Owing to a lack of therapeutic options, the overall survival rate of patients with pancreatic cancer is low. Gemcitabine has been mainly used to treat patients with pancreatic cancer, but its efficacy is limited by chemoresistance. Therefore, a novel therapeutic agent for PDAC therapy is urgently needed. An anthelminthic drug, niclosamide, has already been researched in breast, lung, colon, and pancreatic cancer as an anti-cancer purpose by re-positioning its original purpose. However, combination therapy of gemcitabine and niclosamide was not informed yet. Here, we found that niclosamide co-administered with gemcitabine significantly inhibited tumorigenesis of pancreatic cancer compared to gemcitabine alone. Further, combining niclosamide and gemcitabine inhibited cell proliferation and induced apoptosis. Niclosamide induced cell cycle arrest at the G1 phase, and the levels of CDK4/6 and cyclin D1 were lowered after gemcitabine treatment. In addition, the combination of these chemical compounds more effectively increased the binding level of activated ß-catenin destruction complex and ß-catenin to enable phosphorylation, compared to gemcitabine alone. After phosphorylation, niclosamide - gemcitabine upregulated the ubiquitin level, which caused phosphorylated ß-catenin to undergo proteasomal degradation; the combination was more potent than gemcitabine alone. Finally, the combination more effectively suppressed tumor growth in vivo, compared to gemcitabine alone. Altogether, our results indicate that niclosamide synergistically enhances the antitumor effect of gemcitabine in pancreatic cancer, by inducing the degradation of ß-catenin with ubiquitination. Therefore, this drug combination can potentially be used in PDAC therapy.

Prostaglandin F2α analogue, bimatoprost ameliorates colistin-induced nephrotoxicity.

Colistin (polymyxin E) is an antibiotic that is effective against multidrug-resistant gram-negative bacteria. However, the high incidence of nephrotoxicity caused by colistin limits its clinical use. To identify compounds that might ameliorate colistin-induced nephrotoxicity, we obtained 1707 compounds from the Korea Chemical Bank and used a high-content screening (HCS) imaging-based assay. In this way, we found that bimatoprost (one of prostaglandin F2α analogue) ameliorated colistin-induced nephrotoxicity. To further assess the effects of bimatoprost on colistin-induced nephrotoxicity, we used in vitro and in vivo models. In cultured human proximal tubular cells (HK-2), colistin induced dose-dependent cytotoxicity. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, indicative of apoptosis, was higher in colistin-treated cells, but this effect of colistin was ameliorated by cotreatment with bimatoprost. The generation of reactive oxygen species, assessed using 2,7-dichlorodihydrofluorescein diacetate, was less marked in cells treated with both colistin and bimatoprost than in those treated with colistin alone. Female C57BL/6 mice (n = 10 per group) that were intraperitoneally injected with colistin (10 mg/kg/12 hr) for 14 days showed high blood urea nitrogen and serum creatinine concentrations that were reduced by the coadministration of bimatoprost (0.5 mg/kg/12 hr). In addition, kidney injury molecule-1 (KIM1) and Neutrophil gelatinase-associated lipocalin (NGAL) expression also reduced by bimatoprost administration. Further investigation in tubuloid and kidney organoids also showed that bimatoprost attenuated the nephrotoxicity by colistin, showing dose-dependent reducing effect of KIM1 expression. In this study, we have identified bimatoprost, prostaglandin F2α analogue as a drug that ameliorates colistin-induced nephrotoxicity.

Current status and prospects of organoid-based regenerative medicine.

Organoids derived from stem cells or organ-specific progenitors are self-organizable, self-renewable, and multicellular threedimensional (3D) structures that can mimic the function and structure of the derived tissue. Due to such characteristics, organoids are attracting attention as an excellent ex vivo model for drug screening at the stage of drug development. In addition, since the applicability of organoids as therapeutics for tissue regeneration has been embossed, the development of various organoids-based regenerative medicine has been rapidly progressing, reaching the clinical trial stage. In this review, we give a general overview of organoids and describe current status and prospects of organoid-based regenerative medicine, focusing on organoid-based regenerative therapeutics currently under development including clinical trials. [BMB Reports 2023; 56(1): 10-14].

Effects of Sevoflurane Exposure on Fetal Brain Development Using Cerebral Organoids.

Sevoflurane is a safe and well-known inhaled anesthetic. Given that sevoflurane can be delivered to developing fetuses through the mother, it is critical to determine whether this agent affects fetal neurodevelopment. Recent research has sought to determine whether sevoflurane affects fetal brain development when the mother is exposed during the second to third trimester of pregnancy, considered to be the crucial period for the development of nervous system. However, even though the first trimester is a critical period for fetal organogenesis and the most susceptible time to teratogen exposure, research regarding the effects of sevoflurane on organogenesis, especially on brain development, is insufficient. In the present study, human embryonic stem cells (hESC)-derived cerebral organoids were exposed to sevoflurane during the time corresponding to the first trimester to investigate the effect of early sevoflurane exposure on fetal brain development, specifically the processes of neuronal differentiation and maturation. Organoid size exposed to the intermediate concentration of sevoflurane did not differ from control, immunofluorescence demonstrated that sevoflurane temporarily decreased the size of SOX2 + /N-cad + ventricular zone structures only during the mid-time point, and upregulated expression of TUJ1 and MAP2 only during the early time point. However, all markers returned to normal levels, and organoids formed normal cortical structures at the late time point. Our results suggest that maternal sevoflurane exposure during the first trimester of pregnancy can cause abnormal neuronal differentiation in the fetal brain. However, considering the recovery observed in later periods, sevoflurane exposure might not have lasting impacts on fetal brain development.

Human Palatine Tonsils Are Linked to Alzheimer's Disease through Function of Reservoir of Amyloid Beta Protein Associated with Bacterial Infection.

Amyloid-ß (Aß)-peptide production or deposition in the neuropathology of Alzheimer's disease (AD) was shown to be caused by chronic inflammation that may be induced by infection, but the role of pathogenic-bacteria-related AD-associated Aß is not yet clearly understood. In this study, we validated the hypothesis that there is a correlation between the Aß-protein load and bacterial infection and that there are effects of bacteria, Staphylococcus aureus (S. aureus), on the Aß load in the inflammatory environment of human tonsils. Here, we detected Aß-peptide deposits in human tonsil tissue as well as tissue similar to tonsilloliths found in the olfactory cleft. Interestingly, we demonstrated for the first time the presence of Staphylococcus aureus (S. aureus) clustered around or embedded in the Aß deposits. Notably, we showed that treatment with S. aureus upregulated the Aß-protein load in cultures of human tonsil organoids and brain organoids, showing the new role of S. aureus in Aß-protein aggregation. These findings suggest that a reservoir of Aß and pathogenic bacteria may be a possible therapeutic target in human tonsils, supporting the treatment of antibiotics to prevent the deposition of Aß peptides via the removal of pathogens in the intervention of AD pathogenesis.

Alternative splicing variant of NRP/B promotes tumorigenesis of gastric cancer.

Gastrointestinal cancer is associated with a high mortality rate. Here, we report that the splice variant of NRP/B contributes to tumorigenic activity in highly malignant gastric cancer through dissociation from the tumor repressor, HDAC5. NRP/B mRNA expression is significantly higher in the human gastric cancer tissues than in the normal tissues. Further, high levels of both the NRP/B splice variant and Lgr5, but not the full-length protein, are found in highly tumorigenic gastric tumor cells, but not in non-tumorigenic cells. The loss of NRP/B markedly inhibits cell migration and invasion, which reduces tumor formation in vivo. Importantly, the inhibition of alternative splicing increases the levels of NRP/B-1 mRNA and protein in AGS cells. The ectopic expression of full-length NRP/B exhibits tumor-suppressive activity, whereas NRP/B-2 induces the noninvasive human gastric cancer cells tumorigenesis. The splice variant NRP/B-2 which loses the capacity to interact with tumor repressors promoted oncogenic activity, suggesting that the BTB/POZ domain in the N-terminus has a crucial role in the suppression of gastric cancer. Therefore, the regulation of alternative splicing of the NRP/B gene is a potential novel target for the treatment of gastrointestinal cancer. [BMB Reports 2022; 55(7): 348-353].

Modeling Pancreatic Cancer with Patient-Derived Organoids Integrating Cancer-Associated Fibroblasts.

Pancreatic cancer is a devastating disease and is highly resistant to anticancer drugs because of its complex microenvironment. Cancer-associated fibroblasts (CAFs) are an important source of extracellular matrix (ECM) components, which alter the physical and chemical properties of pancreatic tissue, thus impairing effective intratumoral drug delivery and resulting in resistance to conventional chemotherapy. The objective of this study was to develop a new cancer organoid model, including a fibrous tumor microenvironment (TME) using CAFs. The CAF-integrated pancreatic cancer organoid (CIPCO) model developed in this study histologically mimicked human pancreatic cancer and included ECM production by CAFs. The cancer cell-CAF interaction in the CIPCO promoted epithelial-mesenchymal transition of cancer cells, which was reversed by CAF inhibition using all-trans retinoic acid. Deposition of newly synthesized collagen I in the CIPCO disturbed the delivery of gemcitabine to cancer cells, and treatment with collagenase increased the cytotoxic effect of gemcitabine. This model may lead to the development of next-generation cancer organoid models recapitulating the fibrous TME.

Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection.

The palatine tonsils (hereinafter referred to as "tonsils") serve as a reservoir for viral infections and play roles in the immune system's first line of defense. The aims of this study were to establish tonsil epithelial cell-derived organoids and examine their feasibility as an ex vivo model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The tonsil organoids successfully recapitulated the key characteristics of the tonsil epithelium, including cellular composition, histologic properties, and biomarker distribution. Notably, the basal layer cells of the organoids express molecules essential for SARS-CoV-2 entry, such as angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin, being susceptible to the viral infection. Changes in the gene expression profile in tonsil organoids revealed that 395 genes associated with oncostatin M signaling and lipid metabolism were highly upregulated within 72 h after SARS-CoV-2 infection. Notably, remdesivir suppressed the viral RNA copy number in organoid culture supernatants and intracellular viral protein levels in a dose-dependent manner. Here, we suggest that tonsil epithelial organoids could provide a preclinical and translational research platform for investigating SARS-CoV-2 infectivity and transmissibility or for evaluating antiviral candidates.

Recapitulation of First Pass Metabolism Using 3D Printed Microfluidic Chip and Organoid.

The low bioavailability of oral drugs due to first pass metabolism is a major obstacle in drug development. With significant developments in the field of in vitro organ modeling and microfluidic chip three-dimensional (3D) printing, the challenge is to apply these for the production and evaluation of new drug candidates. This study aimed to produce a microfluidic chip to recapitulate and assess the feasibility of the first pass metabolism. The infill condition of the polycarbonate transparent filament and layer height was optimized to visualize and maintain the organoid or spheroid on the chip. Next, the chip was fabricated using a 3D printer after a computer-aided design (CAD). The chip consisted of three wells of different heights. The small intestinal (SI) organoid and colorectal adenocarcinoma spheroids were placed on the second and third wells, respectively. No additional equipment was assembled, and the tilted tunnel was connected to each well to transport the material by gradient force. The chip was fabricated using 50% and 0.1 um thickness. Among the three different prototypes of chip (chips 1, 2, and 3), the highest distribution of plasmids in the Matrigel of the second well was observed in Chip 2 at 48 h. The effect of first pass metabolism was analyzed using docetaxel. In the chip without an SI organoid, there was a marked decrease in the viability of colorectal adenocarcinoma spheroids due to drug efficacy. However, in the chip with the SI organoid, no significant change in viability was observed because of first pass metabolism. In conclusion, we presented a simple, fast, and low-cost microfluidic chip to analyze the efficacy change of candidate drug by the first pass metabolism.

Crown-Ether-Modified SBA-15 for the Adsorption of Cr(VI) and Zn(II) from Water.

Recently, the release of some metal ions to the environment has been observed to cause serious damages to human health and the environment. Herein, a chromium(VI)- and zinc(II)-selective adsorbent (CB18crown6/SBA-15) was successfully fabricated through the covalent attachment of 4'-carboxybenzo-18-crown-6 (CB18crown6) as a ligand on mesoporous silica support (SBA-15). The CB18crown6/SBA-15 adsorbent was characterized by Fourier-transform infrared (FTIR) spectrometry, X-ray diffraction (XRD), N2 adsorption-desorption, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). To evaluate its ability to selectively capture Cr(VI) and Zn(II), adsorption experiments were conducted. The influences of pH, initial concentration of metal ions, and coexisting metal ions on the adsorption process were examined. The CB18crown6/SBA-15 selectively adsorbed Cr(VI) at pH 2 and Zn(II) at pH 5, respectively, from the mixed aqueous solutions of chromium, zinc, lithium, cadmium, cobalt, strontium, and cesium ions. The data for the adsorption of Cr(VI) onto the CB18crown6/SBA-15 were well explained by the Langmuir adsorption isotherm. In addition, the recycling and reuse of CB18crown6/SBA-15 was successfully achieved, and 71 and 76% reuse efficiency of Cr(VI) and Zn(II), respectively, was obtained after five cycles. This study suggests that the use of the CB18crown6/SBA-15 can be a feasible approach for the selective remediation of Cr(VI) and Zn(II) contamination.

Functional recovery by colon organoid transplantation in a mouse model of radiation proctitis.

Radiation proctitis is the collateral damage that occurs to healthy cells during radiation treatment of pelvic malignancies. Conservative treatment of radiation proctitis can mitigate inflammatory symptoms, but, to date, no therapeutic options are available for direct recovery of the damaged colonic epithelium. The present study assessed the ability of colon organoid-based regeneration to treat radiation proctitis. Radiation proctitis was induced in mice by irradiating their recta, followed by enema-based transplantation of mouse colon organoids. The transplanted colon organoids were found to successfully engraft onto the damaged rectal mucosa of the irradiated mice, reconstituting epithelial structure and integrity. Lgr5+ stem cells were shown to be pivotal to colon organoid mediated regeneration. Endoscopic examination showed the efficacy of localized transplantation of colon organoids with fibrin glue to irradiated sites. These findings provide useful insights into the use of colon organoid-based regenerative therapy for the treatment of radiation proctitis.

Establishment of functional epithelial organoids from human lacrimal glands.

BACKGROUND: Tear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics. METHODS: Digested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed  through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED. RESULTS: The histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ ions and ß-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren's syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation. CONCLUSION: This study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

Synthesis of size-controlled and highly monodispersed silica nanoparticles using a short alkyl-chain fluorinated surfactant.

Highly monodispersed silica nanoparticles (SiNPs) were synthesised using a fluorinated surfactant, HOCH2CH(CF3)CO2H, and its efficiency was compared with efficiencies of five other surfactants. The size of the SiNPs (∼50-200 nm) was controlled by controlling the surfactant amount. The short alkyl-chain fluoro surfactant was found to be more efficient at producing monodispersed SiNPs than its long alkyl-chain fluoro or non-fluorinated surfactant counterparts.

The epidermal growth factor receptor variant type III mutation frequently found in gliomas induces astrogenesis in human cerebral organoids.

OBJECTIVES: The epidermal growth factor receptor variant type III (EGFRvIII) is the most common mutation of EGFR in glioblastoma multiforme (GBM) and is found in approximately 25% of all GBMs. Intriguingly, EGFRvIII is mostly found in GFAP+ astrocytic tumour cells in the brain, suggesting connection of EGFRvIII to astrogenesis. In this study, we explored whether EGFRvIII mutation facilitates astrogenesis in human development setting. MATERIALS AND METHODS: Using CRISPR-Cas9, we generated EGFRvIII mutations in H9-hESCs. Wild type (wt) H9-hESCs were used as an isogenic control. Next, we generated cerebral organoids using the wt and EGFRvIII-hESCs and examined the astrogenic differentiation of the brain organoids. RESULTS: EGFRvIII-organoids showed abundant astrocytes (GFAP+ , S100ß+ ), while no astrocytes were detected in wt hESC-derived organoids at day 49. On the contrary, TUJ1+ neurons were more abundant in the wt-organoids than the EGFRvIII-organoids. This result suggested that constitutively active EGFRvIII promoted astrogenesis at the expense of neurogenesis. In addition, the EGFRvIII-organoids were larger in size and retained more Ki67+ cells than wt-organoids, indicating enhanced cell proliferation by the mutation. The EGFRvIII-organoids displayed massive apoptotic cell death after treatment with temozolomide and hence, could be used for evaluation of anti-GBM drugs. CONCLUSIONS: EGFRvIII mutation-induced astrogenesis and massive cell proliferation in a human brain development model. These results provide us new insights into the mechanisms relating EGFRvIII mutation-mediated gliogenesis and gliomagenesis.

Chronically infused angiotensin II induces depressive-like behavior via microglia activation.

Brain inflammation is one of hypotheses explaining complex pathomechanisms of depression. Angiotensin II (ANGII), which is associated with hypertension, also induces brain inflammation. However, there is no animal study showing the direct relationship between ANGII and depression. To address this issue, ANGII-containing osmotic pumps were implanted into adult male C57BL/6 mice subcutaneously for subacute (7 days) and chronic (at least 21 days) periods and behavioral and molecular analyses were conducted. Chronic infusion of ANGII into mice induced depressive-like behaviors, including the tail suspension test and forced swimming test, which were reversed by imipramine. Chronic infusion of ANGII also induced microglial activation in the hippocampus with increase of Il-1ß mRNA and decrease of Arg1 mRNA. In addition, chronic ANGII infusion activated the hypothalamic-pituitary-adrenal axis (HPA axis) and resulted in decreased hippocampal glucocorticoid receptor level. However, subacute ANGII infusion did not induce significant molecular and behavioral changes in mice compared to that of control. The molecular and behavioral changes by chronic ANGII infusion were reversed by co-treatment of minocycline or telmisartan. In addition, ANGII treatment also induced the pro-inflammatory changes in BV-2 microglial cells. Our results indicate that ANGII can induce depressive-like behaviors via microglial activation in the hippocampus and HPA axis hyperactivation in mice. These might suggest possible mechanism on depressive symptom in chronic hypertensive state.

Epitaxially Strained CeO2 /Mn3 O4 Nanocrystals as an Enhanced Antioxidant for Radioprotection.

Nanomaterials with antioxidant properties are promising for treating reactive oxygen species (ROS)-related diseases. However, maintaining efficacy at low doses to minimize toxicity is a critical for clinical applications. Tuning the surface strain of metallic nanoparticles can enhance catalytic reactivity, which has rarely been demonstrated in metal oxide nanomaterials. Here, it is shown that inducing surface strains of CeO2 /Mn3 O4 nanocrystals produces highly catalytic antioxidants that can protect tissue-resident stem cells from irradiation-induced ROS damage. Manganese ions deposited on the surface of cerium oxide (CeO2 ) nanocrystals form strained layers of manganese oxide (Mn3 O4 ) islands, increasing the number of oxygen vacancies. CeO2 /Mn3 O4 nanocrystals show better catalytic activity than CeO2 or Mn3 O4 alone and can protect the regenerative capabilities of intestinal stem cells in an organoid model after a lethal dose of irradiation. A small amount of the nanocrystals prevents acute radiation syndrome and increases the survival rate of mice treated with a lethal dose of total body irradiation.

Human umbilical cord-derived mesenchymal stem cells alleviate schizophrenia-relevant behaviors in amphetamine-sensitized mice by inhibiting neuroinflammation.

At present, therapeutic options available for treating schizophrenia are limited to monoamine-based antipsychotic drugs. Recent genome wide association study (GWAS) indicated a close relationship between immune system and schizophrenia. To leverage the GWAS finding for therapeutic strategy, we conducted a mechanism and effect study on application of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with potent immune-modulatory effect in an animal model useful for the study of schizophrenia. Schizophrenia-relevant behaviors were induced by amphetamine administration (amphetamine-sensitized mice) and the effect of a single intravenous administration of hUC-MSC was examined in the amphetamine-sensitized mice. Schizophrenia-relevant behaviors were assessed by open field test, light/dark box, social interaction test, latent inhibition, prepulse inhibition, tail suspension test, and forced swimming test. Our results indicated that neuroinflammation along with peripheral TNF-α elevation is associated with schizophrenia-relevant behaviors in amphetamine-sensitized mice. In addition, hUC-MSC inhibited schizophrenia-relevant and the neuroinflammatory changes. The main mechanism of hUC-MSC was associated with the induction of Treg and production of the anti-inflammatory cytokine, IL-10 in periphery. In vitro study revealed that amphetamine did not directly induce a neuroinflammatory reaction, while recombinant TNF-α (rTNF-α) increased mRNA expression of TNF-α, KMO, and IL-1ß in several microglial cell lines. Moreover, recombinant IL-10 (rIL-10) and MSC conditioned media inhibited the inflammatory response in rTNF-α-treated microglial cells. Assuming that hUC-MSCs rarely reach the CNS and do not remain in the body for an extended time, these findings suggest that a single hUC-MSC infusion have long-term beneficial effect via regulatory T cell induction and secretion of IL-10 in amphetamine-sensitized mice.

Epidemiological Characterization of a Directed and Weighted Disease Network Using Data From a Cohort of One Million Patients: Network Analysis.

BACKGROUND: In the past 20 years, various methods have been introduced to construct disease networks. However, established disease networks have not been clinically useful to date because of differences among demographic factors, as well as the temporal order and intensity among disease-disease associations. OBJECTIVE: This study sought to investigate the overall patterns of the associations among diseases; network properties, such as clustering, degree, and strength; and the relationship between the structure of disease networks and demographic factors. METHODS: We used National Health Insurance Service-National Sample Cohort (NHIS-NSC) data from the Republic of Korea, which included the time series insurance information of 1 million out of 50 million Korean (approximately 2%) patients obtained between 2002 and 2013. After setting the observation and outcome periods, we selected only 520 common Korean Classification of Disease, sixth revision codes that were the most prevalent diagnoses, making up approximately 80% of the cases, for statistical validity. Using these data, we constructed a directional and weighted temporal network that considered both demographic factors and network properties. RESULTS: Our disease network contained 294 nodes and 3085 edges, a relative risk value of more than 4, and a false discovery rate-adjusted P value of <.001. Interestingly, our network presented four large clusters. Analysis of the network topology revealed a stronger correlation between in-strength and out-strength than between in-degree and out-degree. Further, the mean age of each disease population was related to the position along the regression line of the out/in-strength plot. Conversely, clustering analysis suggested that our network boasted four large clusters with different sex, age, and disease categories. CONCLUSIONS: We constructed a directional and weighted disease network visualizing demographic factors. Our proposed disease network model is expected to be a valuable tool for use by early clinical researchers seeking to explore the relationships among diseases in the future.

Fabrication of Dentin-Pulp-Like Organoids Using Dental-Pulp Stem Cells.

We developed a novel dentin-pulp-like organoid. It has both stem-cell and odontoblast characteristics using a mesenchymal cell lineage of human dental-pulp stem cells (hDPSCs). The mixture of hDPSCs and Matrigel was transferred into the maintenance medium (MM) and divided into four different groups according to how long they were maintained in the odontogenic differentiation medium (ODM). All organoids were harvested at 21 days and analyzed to find the optimal differentiation condition. To assess the re-fabrication of dentin-pulp-like organoid, after dissociation of the organoids, it was successfully regenerated. Additionally, its biological activity was confirmed by analyzing changes of relevant gene expression and performing a histology analysis after adding Biodentine® into the ODM. The organoid was cultured for 11 days in the ODM (ODM 11) had the most features of both stem cells and differentiated cells (odontoblasts) as confirmed by relevant gene expression and histology analyses. Micro-computed tomography and an electron microscope also showed mineralization and odontoblastic differentiation. Finally, ODM 11 demonstrated a biologically active response to Biodentine® treatment. In conclusion, for the first time, we report the fabrication of a dentin-pulp-like organoid using mesenchymal stem cells. This organoid has potential as a future therapeutic strategy for tooth regeneration.