Efficient and reproducible generation of human induced pluripotent stem cell-derived expandable liver organoids for disease modeling.

Genetic liver disease modeling is difficult because it is challenging to access patient tissue samples and to develop practical and relevant model systems. Previously, we developed novel proliferative and functional liver organoids from pluripotent stem cells; however, the protocol requires improvement for standardization and reproducible mass production. Here, we improved the method such that it is suitable for scalable expansion and relatively homogenous production, resulting in an efficient and reproducible process. Moreover, three medium components critical for long-term expansion were defined. Detailed transcriptome analysis revealed that fibroblast growth factor signaling, the essential pathway for hepatocyte proliferation during liver regeneration, was mainly enriched in proliferative liver organoids. Short hairpin RNA-mediated knockdown of FGFR4 impaired the generation and proliferation of organoids. Finally, glycogen storage disease type Ia (GSD1a) patient-specific liver organoids were efficiently and reproducibly generated using the new protocol. They well maintained disease-specific phenotypes such as higher lipid and glycogen accumulation in the liver organoids and lactate secretion into the medium consistent with the main pathologic characteristics of patients with GSD1a. Therefore, our newly established liver organoid platform can provide scalable and practical personalized disease models and help to find new therapies for incurable liver diseases including genetic liver diseases.

A multicellular liver organoid model for investigating hepatitis C virus infection and nonalcoholic fatty liver disease progression.

BACKGROUND AND AIMS: HCV infection can be successfully managed with antiviral therapies; however, progression to chronic liver disease states, including NAFLD, is common. There is currently no reliable in vitro model for investigating host-viral interactions underlying the link between HCV and NAFLD; although liver organoids (LOs) show promise, they currently lack nonparenchymal cells, which are key to modeling disease progression. APPROACH AND RESULTS: Here, we present a novel, multicellular LO model using a coculture system of macrophages and LOs differentiated from the same human pluripotent stem cells (PSCs). The cocultured macrophages shifted toward a Kupffer-like cell type, the liver-resident macrophages present in vivo , providing a suitable model for investigating NAFLD pathogenesis. With this multicellular Kupffer-like cell-containing LO model, we found that HCV infection led to lipid accumulation in LOs by upregulating host lipogenesis, which was more marked with macrophage coculture. Reciprocally, long-term treatment of LOs with fatty acids upregulated HCV amplification and promoted inflammation and fibrosis. Notably, in our Kupffer-like cell-containing LO model, the effects of 3 drugs for NASH that have reached phase 3 clinical trials exhibited consistent results with the clinical outcomes. CONCLUSIONS: Taken together, we introduced a multicellular LO model consisting of hepatocytes, Kupffer-like cells, and HSCs, which recapitulated host-virus intercommunication and intercellular interactions. With this novel model, we present a physiologically relevant system for the investigation of NAFLD progression in patients with HCV.

Advances in liver organoids: model systems for liver disease.

Reliable in vitro models with human-derived cells that recapitulate in vivo-like physiologies are required for drug discovery and development to reduce the gap between the results of cell-based drug testing, animal testing, and human clinical trials. Liver organoid models have emerged as novel tools for hepatotoxicity evaluation, liver disease modeling, and drug screening. Liver organoids can be generated from biopsies of liver tissues or pluripotent stem cells and can be applied to various liver diseases, including metabolic associated fatty liver disease, infectious liver disease, genetic liver disease, and liver cancer. This review focuses on recent studies on organoids to model human liver diseases and discusses the advantages and limitations of current liver organoids for translational applications.

Generation of An Induced Pluripotent Stem Cell Line from Human Liver Fibroblasts from A Patient with Combined Hepatocellular-Cholangiocarcinoma.

Objective: Combined hepatocellular-cholangiocarcinoma (cHCC-CC) is a rare type of primary liver cancer with characteristics of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The pathogenesis of cHCCCC is poorly understood due to a shortage of suitable in vitro models. Due to scarce availability of human liver tissue, induced pluripotent stem cells (iPSCs) are a useful alternative source to produce renewable liver cells. For use in the development of liver pathology models, here we successfully developed and evaluated iPSCs from liver fibroblasts of a patient with cHCC-CC. Materials and Methods: In this experimental study, human liver fibroblasts (HLFs) were obtained from the liver biopsy of a 69-year-old male patient with cHCC-CC and transduced with a retroviral cocktail that included four factors - OCT4, SOX2, KLF4, and c-MYC (OSKM). Pluripotency of the iPSCs was determined by alkaline phosphatase (AP) staining, quantitative real-time polymerase chain reaction (PCR), and immunofluorescence. We induced in vitro embryoid body (EB) formation and performed an in vivo teratoma assay to confirm their differentiation capacity into the three germ layers. Results: HLF iPSCs derived from the cHCC-CC patient displayed typical iPSC-like morphology and pluripotency marker expression. The proficiency of the iPSCs to differentiate into three germ layers was assessed both in vitro and in vivo. Compared to normal control iPSCs, differentiated HLF iPSCs showed increased expressions of HCC markers alpha-fetoprotein (AFP) and Dickkopf-1 (DKK1) and the CC marker cytokeratin 7 (CK7), and a decreased expression of the CC tumour suppressor SRY-related HMG-box 17 (SOX17). Conclusion: We established HLF iPSCs using liver fibroblasts from a patient with cHCC-CC for the first time. The HLF iPSCs maintained marker expression in the patient when differentiated into EBs. Therefore, HLF iPSCs may be a sustainable cell source for modelling cHCC-CC and beneficial for understanding liver cancer pathology and developing therapies for cHCC-CC treatment.

Generation of human induced pluripotent stem cell line, KRIBBi003-A, from urinary cells of a patient with glycogen storage disease type IXa.

Glycogen storage disease type IXa (GSD IXa) is a rare genetic disorder characterized by phosphorylase kinase (PhK) deficiency, which leads to excessive glycogen accumulation in the liver. Urinary cells (UCs) were isolated from a GSD IXa patient and reprogrammed into induced pluripotent stem cells (iPSCs) using Sendai virus. The established iPSC line, KRIBBi003-A, exhibited pluripotency marker expression and a normal karyotype. The differentiation capacity of the cell line was confirmed by the differentiation of the three germ layers in vitro. The established iPSC line is a potential useful resource for disease modeling of GSD IXa.

Effect of Microbial Short-Chain Fatty Acids on CYP3A4-Mediated Metabolic Activation of Human Pluripotent Stem Cell-Derived Liver Organoids.

The early and accurate prediction of the hepatotoxicity of new drug targets during nonclinical drug development is important to avoid postmarketing drug withdrawals and late-stage failures. We previously established long-term expandable and functional human-induced pluripotent stem cell (iPSC)-derived liver organoids as an alternative source for primary human hepatocytes. However, PSC-derived organoids are known to present immature fetal characteristics. Here, we treated these liver organoids with microbial short-chain fatty acids (SCFAs) to improve metabolic maturation based on microenvironmental changes in the liver during postnatal development. The effects of the three main SCFA components (acetate, propionate, and butyrate) and their mixture on liver organoids were determined. Propionate (1 µM) significantly promoted the CYP3A4/CYP3A7 expression ratio, and acetate (1 µM), propionate (1 µM), and butyrate (1 µM) combination treatment, compared to no treatment (control), substantially increased CYP3A4 activity and albumin secretion, as well as gene expression. More importantly, mixed SCFA treatment accurately revealed troglitazone-induced hepatotoxicity, which was redeemed on a potent CYP3A4 inhibitor ketoconazole treatment. Overall, we determined, for the first time, that SCFA mixture treatment might contribute to the accurate evaluation of the CYP3A4-dependent drug toxicity by improving metabolic activation, including CYP3A4 expression, of liver organoids.

Accuracy of virtual 3-dimensional cephalometric images constructed with 2-dimensional cephalograms using the biplanar radiography principle.

PURPOSE: The purpose of this study was to evaluate the accuracy of virtual 3-dimensional (3D) cephalograms constructed using the principle of biplanar radiography by comparing them with cone-beam computed tomography(CBCT) images. MATERIALS AND METHODS: Thirty orthodontic patients were enrolled in this study. Frontal and lateral cephalograms were obtained with the use of a head posture aligner and reconstructed into 3D cephalograms using biplanar radiography software. Thirty-four measurements representing the height, width, depth, and oblique distance were computed in 3 dimensions, and compared with the measurements from the 3D images obtained by CBCT, using the paired t-test and Bland-Altman analysis. RESULTS: Comparison of height, width, depth, and oblique measurements showed no statistically significant differences between the measurements obtained from 3D cephalograms and those from CBCT images (P>0.05). Bland-Altman plots also showed high agreement between the 3D cephalograms and CBCT images. CONCLUSION: Accurate 3D cephalograms can be constructed using the principle of biplanar radiography if frontal and lateral cephalograms can be obtained with a head posture aligner. Three-dimensional cephalograms generated using biplanar radiography can replace CBCT images taken for diagnostic purposes.

Organ-Level Functional 3D Tissue Constructs with Complex Compartments and their Preclinical Applications.

There is an increasing interest in organ-level 3D tissue constructs, owing to their mirroring of in vivo-like features. This has resulted in a wide range of preclinical applications to obtain cell- or tissue-specific responses. Additionally, the development and improvement of sophisticated technologies, such as organoid generation, microfluidics, hydrogel engineering, and 3D printing, have enhanced 3D tissue constructs to become more elaborate. In particular, recent studies have focused on including complex compartments, i.e., vascular and innervation structured 3D tissue constructs, which mimic the nature of the human body in that all tissues/organs are interconnected and physiological phenomena are mediated through vascular and neural systems. Here, the strategies are categorized according to the number of dimensions (0D, 1D, 2D, and 3D) of the starting materials for scaling up, and novel approaches to introduce increased complexity in 3D tissue constructs are highlighted. Recent advances in preclinical applications are also investigated to gain insight into the future direction of 3D tissue construct research. Overcoming the challenges in improving organ-level functional 3D tissue constructs both in vitro and in vivo will ultimately become a life-saving tool in the biomedical field.

Integrating Organs-on-Chips: Multiplexing, Scaling, Vascularization, and Innervation.

Organs-on-chips (OoCs) have attracted significant attention because they can be designed to mimic in vivo environments. Beyond constructing a single OoC, recent efforts have tried to integrate multiple OoCs to broaden potential applications such as disease modeling and drug discoveries. However, various challenges remain for integrating OoCs towards in vivo-like operation, such as incorporating various connections for integrating multiple OoCs. We review multiplexed OoCs and challenges they face: scaling, vascularization, and innervation. In our opinion, future OoCs will be constructed to have increased predictive power for in vivo phenomena and will ultimately become a mainstream tool for high quality biomedical and pharmaceutical research.

In vivo-mimicking microfluidic perfusion culture of pancreatic islet spheroids.

Native pancreatic islets interact with neighboring cells by establishing three-dimensional (3D) structures, and are surrounded by perfusion at an interstitial flow level. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Dynamic culture, compared to static culture, enhanced islet health and maintenance of islet endothelial cells, reconstituting the main component of islet extracellular matrix within spheroids. Optimized flow condition allowed localization of secreted soluble factors near spheroids, facilitating diffusion-mediated paracrine interactions within islets, and enabled long-term maintenance of islet morphology and function for a month. The proposed model can aid islet preconditioning before transplantation and has potential applications as an in vitro model for diabetic drug testing.

Primordial odontogenic tumor: a case report and literature review.

BACKGROUND: A primordial odontogenic tumor (POT) is a rare, benign, mixed epithelial and mesenchymal odontogenic tumor that has been included as a new entity in the latest World Health Organization (WHO) classification (2017). POT consists of dental papilla-like myxoid connective tissue covered with a delicate membrane of ameloblastic epithelium. Only 15 cases have been documented worldwide, and here, we report the sixteenth case and the first one of South Korea. CASE PRESENTATION: An asymptomatic lesion was discovered as an incidental radiographic finding in a 10-year-old boy. The patient had no complaints about the lesion. Cone-beam computerized tomograms revealed a round cavity with a defined cortical border measuring approximately 5 × 5 × 5 mm in size. The lesion was a POT. The patient was treated with enucleation. The tumor showed no recurrence for one year. CONCLUSION: This is the first report of POT in South Korea using the novel diagnosis of POT after it was recognized and defined in the latest WHO classification. This novel diagnosis will be useful for pathologists and clinicians in diagnosing and differentiating this new and rare disease from other odontogenic tumors.

Benefits of lateral cephalogram during landmark identification on posteroanterior cephalograms.

OBJECTIVE: Precise identification of landmarks on posteroanterior (PA) cephalograms is necessary when evaluating lateral problems such as facial asymmetry. The aim of the present study was to investigate whether the use of lateral (LA) cephalograms can reduce errors in landmark identification on PA cephalograms. METHODS: Five examiners identified 16 landmarks (Cg, N, ANS, GT, Me, RO, Lo, FM, Z, Or, Zyg, Cd, NC, Ms, M, and Ag) on 32 PA cephalograms with and without LA cephalograms at the same time. The positions of the landmarks were recorded and saved in the horizontal and vertical direction. The mean errors and standard deviation of landmarks location according to the use of LA cephalograms were compared for each landmark. RESULTS: Relatively small errors were found for ANS, Me, Ms, and Ag, while relatively large errors were found for N, GT, Z, Or, and Cd. No significant difference was found between the horizontal and vertical errors for Z and Or, while large vertical errors were found for N, GT, and Cd. The value of identification error was lower when the landmarks were identified using LA cephalograms. Statistically significant error reductions were found at N and Cd with LA cephalograms, especially in the vertical direction. CONCLUSIONS: The use of LA cephalograms during identification of landmarks on PA cephalograms could help reduce identification errors.

Osteoporosis detection in panoramic radiographs using a deep convolutional neural network-based computer-assisted diagnosis system: a preliminary study.

OBJECTIVES: To evaluate the diagnostic performance of a deep convolutional neural network (DCNN)-based computer-assisted diagnosis (CAD) system in the detection of osteoporosis on panoramic radiographs, through a comparison with diagnoses made by oral and maxillofacial radiologists. METHODS: Oral and maxillofacial radiologists with >10 years of experience reviewed the panoramic radiographs of 1268 females {mean [± standard deviation (SD)] age: 52.5 ± 22.3 years} and made a diagnosis of osteoporosis when cortical erosion of the mandibular inferior cortex was observed. Among the females, 635 had no osteoporosis [mean (± SD) age: 32.8 ± SD 12.1 years] and 633 had osteoporosis (72.2 ± 8.5 years). All panoramic radiographs were analysed using three CAD systems, single-column DCNN (SC-DCNN), single-column with data augmentation DCNN (SC-DCNN Augment) and multicolumn DCNN (MC-DCNN). Among the radiographs, 200 panoramic radiographs [mean (± SD) patient age: 63.9 ± 10.7 years] were used for testing the performance of the DCNN in detecting osteoporosis in this study. The diagnostic performance of the DCNN-based CAD system was assessed by receiver operating characteristic (ROC) analysis. RESULTS: The area under the curve (AUC) values obtained using SC-DCNN, SC-DCNN (Augment) and MC-DCNN were 0.9763, 0.9991 and 0.9987, respectively. CONCLUSIONS: The DCNN-based CAD system showed high agreement with experienced oral and maxillofacial radiologists in detecting osteoporosis. A DCNN-based CAD system could provide information to dentists for the early detection of osteoporosis, and asymptomatic patients with osteoporosis can then be referred to the appropriate medical professionals.

Bonding Evaluation Between Nanotube by Anodic Oxidation and Machined Dental Titanium Implant in Beagle Dog.

This study is to evaluate the bonding test between nanotube surface and machined dental titanium implant during the clinical procedure in beagle dog. A total of 24 screw-shaped implants (4 mm in diameter and 8.5 mm in length) were used. The implants were classified into 2 groups (n = 12): machined surface (M group) and nano surface which is nanotube formation on the machined surface (MA group). Anodic oxidation was performed at a 20 V for 10 min with 1 M H3PO4 and 1.5 wt% HF solutions. The implant were installed on beagle dog's humerus. After 4 and 12 weeks later, the beagle dog is sacrificed and the implant were removed with torque force. The removed implant was evaluated by SEM and EDX to observe the implant surface and nanotube's deformation. By investigating removed implant surfaces using SEM and EDX, M group was partially covered with bone, and MA groups were completely covered with bone. Nanotube was not shown any deformity. It indicated that nanotube showed good bonding stability with dental titanium implant.

Accuracy of three-dimensional cephalograms generated using a biplanar imaging system.

OBJECTIVE: Biplanar imaging systems allow for simultaneous acquisition of lateral and frontal cephalograms. The purpose of this study was to compare measurements recorded on three-dimensional (3D) cephalograms constructed from two-dimensional conventional radiographs and biplanar radiographs generated using a new biplanar imaging system with those recorded on cone-beam computed tomography (CBCT)-generated cephalograms in order to evaluate the accuracy of the 3D cephalograms generated using the biplanar imaging system. METHODS: Three sets of lateral and frontal radiographs of 15 human dry skulls with prominent facial asymmetry were obtained using conventional radiography, the biplanar imaging system, and CBCT. To minimize errors in the construction of 3D cephalograms, fiducial markers were attached to anatomical landmarks prior to the acquisition of radiographs. Using the 3D Ceph™ program, 3D cephalograms were constructed from the images obtained using the biplanar imaging system (3D cephbiplanar), conventional radiography (3D cephconv), and CBCT (3D cephcbct). A total of 34 measurements were obtained compared among the three image sets using paired t-tests and Bland-Altman plotting. RESULTS: There were no statistically significant differences between the 3D cephbiplanar and 3D cephcbct measurements. In addition, with the exception of one measurement, there were no significant differences between the 3D cephcbct and 3D cephconv measurements. However, the values obtained from 3D cephconv showed larger deviations than those obtained from 3D cephbiplanar. CONCLUSIONS: The results of this study suggest that the new biplanar imaging system enables the construction of accurate 3D cephalograms and could be a useful alternative to conventional radiography.

Diagnostic accuracy and reliability of panoramic temporomandibular joint (TMJ) radiography to detect bony lesions in patients with TMJ osteoarthritis.

BACKGROUND/PURPOSE: The value of the temporomandibular joint (TMJ) projections of panoramic radiography for diagnosing TMJ osteoarthritis is not completely elucidated. This study aimed to assess the diagnostic accuracy and reliability of panoramic TMJ radiography to detect bony lesions in patients with TMJ osteoarthritis. MATERIALS AND METHODS: This study included 55 TMJs of 44 subjects who were diagnosed with TMJ osteoarthritis. They underwent panoramic radiography (PanRad), lateral (LatTMJ) and frontal (FrnTMJ) projection panoramic TMJ radiography, and cone-beam computed tomography (CBCT). All images were examined by two observers for flattening, erosion, and osteophytes on the condylar head and articular eminence of the TMJ. RESULTS: For detecting flattening lesions on the mandibular condyle, the sensitivities of PanRad, LatTMJ, and FrnTMJ were less than 67% and the combination of LatTMJ and FrnTMJ (ComTMJ) had the highest sensitivity for both observers (67.6% and 79.7%, respectively). For erosion lesions, the sensitivity of ComTMJ for observer 1 was the highest, at 84.3%, whereas the specificity of ComTMJ was the lowest, at 37.5%. The sensitivities of all four methods for observer 2 were less than 54% and the specificities ranged from 75.0% to 100%. The overall diagnostic accuracy was highest for ComTMJ (64.3%), followed by LatTMJ (59.5%). The intraobserver reliability was good for one observer and excellent for the other, and the interobserver reliability was fair or moderate. CONCLUSION: Panoramic TMJ radiography demonstrated limited diagnostic accuracy and acceptable reliability in detecting bony lesions of the TMJ, although it was better than conventional panoramic radiography.

Korean dentists' perceptions and attitudes regarding radiation safety and protection.

OBJECTIVES: To investigate dentists' perceptions and attitudes regarding radiation safety in dental practices in Korea. METHODS: 207 dentists participated in this study. An anonymous survey was conducted regarding the following areas: years of practice, characteristics of the radiography equipment, knowledge of diagnostic reference levels, prescription of dental radiographs, use of radiation protection procedures and participation in the radiation safety program. Data were evaluated according to frequency distribution; the χ test; and Fisher's exact test, which was used to investigate statistical significances (p < 0.05) between those with less than 10 years of experience in practice vs those with 10 years of experience or more. RESULTS: Approximately 83% of the dentists who responded to our survey participated in the radiation safety program, but only 58% were satisfied with it. There were statistically significant differences in the installation of X-ray units, learning pathway of diagnostic reference levels, prescription of dental radiography for new adult patients, use of personnel radiation monitoring systems and participation in the radiation safety program between those with less than 10 years of experience in practice vs those with 10 years of experience or more (p < 0.05). CONCLUSIONS: Dentists with less than 10 years of experience were less attentive to radiation protection procedures. Therefore, taking both the duration of experience of dentists and the installed X-ray equipment into consideration, continuous and efficient education for both dental workers and patients regarding radiation safety and protection is recommended.

Enhanced oxygen permeability in membrane-bottomed concave microwells for the formation of pancreatic islet spheroids.

Oxygen availability is a critical factor in regulating cell viability that ultimately contributes to the normal morphogenesis and functionality of human tissues. Among various cell culture platforms, construction of 3D multicellular spheroids based on microwell arrays has been extensively applied to reconstitute in vitro human tissue models due to its precise control of tissue culture conditions as well as simple fabrication processes. However, an adequate supply of oxygen into the spheroidal cellular aggregation still remains one of the main challenges to producing healthy in vitro spheroidal tissue models. Here, we present a novel design for controlling the oxygen distribution in concave microwell arrays. We show that oxygen permeability into the microwell is tightly regulated by varying the poly-dimethylsiloxane (PDMS) bottom thickness of the concave microwells. Moreover, we validate the enhanced performance of the engineered microwell arrays by culturing non-proliferated primary rat pancreatic islet spheroids on varying bottom thickness from 10 µm to 1050 µm. Morphological and functional analyses performed on the pancreatic islet spheroids grown for 14 days prove the long-term stability, enhanced viability, and increased hormone secretion under the sufficient oxygen delivery conditions. We expect our results could provide knowledge on oxygen distribution in 3-dimensional spheroidal cell structures and critical design concept for tissue engineering applications. STATEMENT OF SIGNIFICANCE: In this study, we present a noble design to control the oxygen distribution in concave microwell arrays for the formation of highly functional pancreatic islet spheroids by engineering the bottom of the microwells. Our new platform significantly enhanced oxygen permeability that turned out to improve cell viability and spheroidal functionality compared to the conventional thick-bottomed 3-D culture system. Therefore, we believe that this could be a promising medical biotechnology platform to further develop high-throughput tissue screening system as well as in vivo-mimicking customised 3-D tissue culture systems.

Recurrent osteochondroma of the mandibular condyle: A case report.

A 21-year-old woman presented with facial asymmetry. Crepitus and clicking of the temporomandibular joint were noted. The midline deviated 5.5 mm to the left, and secondary malocclusion was observed. Panoramic and cone-beam computed tomographic images showed an irregular and exophytic bony mass on the anteromedial surface of the right mandibular condyle. A 3-phase bone scan revealed increased tracer uptake on the affected side. The lesion was treated with excision and reshaping under the diagnosis of osteochondroma confirmed by a histopathological examination. The lesion recurred after 3 years, and the patient underwent condylectomy. Mandibular condylar osteochondroma is often resected because it causes functional and aesthetic problems, but it rarely recurs. To the best of our knowledge, only 2 cases of recurrent osteochondromas of the mandibular condyle have been reported previously. Surgical treatment of the osteochondroma should be performed considering the possibility of recurrence, and long-term follow-up is recommended.