Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

Replacing Animal Testing with Stem Cell-Organoids : Advantages and Limitations.

Various groups including animal protection organizations, medical organizations, research centers, and even federal agencies such as the U.S. Food and Drug Administration, are working to minimize animal use in scientific experiments. This movement primarily stems from animal welfare and ethical concerns. However, recent advances in technology and new studies in medicine have contributed to an increase in animal experiments throughout the years. With the rapid increase in animal testing, concerns arise including ethical issues, high cost, complex procedures, and potential inaccuracies.Alternative solutions have recently been investigated to address the problems of animal testing. Some of these technologies are related to stem cell technologies, such as organ-on-a-chip, organoids, and induced pluripotent stem cell models. The aim of the review is to focus on stem cell related methodologies, such as organoids, that can serve as an alternative to animal testing and discuss its advantages and limitations, alongside regulatory considerations.Although stem cell related methodologies has shortcomings, it has potential to replace animal testing. Achieving this requires further research on stem cells, with potential societal and technological benefits.

Impaired Osteogenesis in Human Induced Pluripotent Stem Cells with Acetaldehyde Dehydrogenase 2 Mutations.

Acetaldehyde dehydrogenase 2 (ALDH2) is the second enzyme involved in the breakdown of acetaldehyde into acetic acid during the process of alcohol metabolism. Roughly 40% of East Asians carry one or two ALDH2*2 alleles, and the presence of ALDH2 genetic mutations in individuals may affect the bone remodeling cycle owing to accumulation of acetaldehyde in the body. In this study, we investigated the effects of ALDH2 mutations on bone remodeling. In this study, we examined the effects of ALDH2 polymorphisms on in vitro osteogensis using human induced pluripotent stem cells (hiPSCs). We differentiated wild-type (ALDH2*1/*1-) and ALDH2*1/*2-genotyped hiPSCs into osteoblasts (OBs) and confirmed their OB characteristics. Acetaldehyde was administered to confirm the impact caused by the mutation during OB differentiation. Calcium deposits formed during osteogenesis were significantly decreased in ALDH2*1/*2 OBs. The expression of osteogenic markers were also decreased in acetaldehyde-treated OBs differentiated from the ALDH2*1/*2 hiPSCs. Furthermore, the impact of ALDH2 polymorphism and acetaldehyde-induced stress on inflammatory factors such as 4-hydroxynonenal and tumor necrosis factor α was confirmed. Our findings suggest that individuals with ALDH2 deficiency may face challenges in acetaldehyde breakdown, rendering them susceptible to disturbances in normal bone remodeling therefore, caution should be exercised regarding alcohol consumption. In this proof-of-concept study, we were able to suggest these findings as a result of a disease-in-a-dish concept using hiPSCs derived from individuals bearing a certain mutation. This study also shows the potential of patient-derived hiPSCs for disease modeling with a specific condition.

Effects of stepwise administration of osteoprotegerin and parathyroid hormone-related peptide DNA vectors on bone formation in ovariectomized rat model.

Osteoporosis is a metabolic bone disease that impairs bone mineral density, microarchitecture, and strength. It requires continuous management, and further research into new treatment options is necessary. Osteoprotegerin (OPG) inhibits bone resorption and osteoclast activity. The objective of this study was to investigate the effects of stepwise administration of OPG-encoded minicircles (mcOPG) and a bone formation regulator, parathyroid hormone-related peptide (PTHrP)-encoded minicircles (mcPTHrP) in osteoporosis. The combined treatment with mcOPG and mcPTHrP significantly increased osteogenic marker expression in osteoblast differentiation compared with the single treatment groups. A model of postmenopausal osteoporosis was established in 12-week-old female rats through ovariectomy (OVX). After 8 weeks of OVX, mcOPG (80 µg/kg) was administered via intravenous injection. After 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for 3 weeks. The bone microstructure in the femur was evaluated 24 weeks after OVX using micro-CT. In a proof-of-concept study, stepwise treatment with mcOPG and mcPTHrP on an OVX rat model significantly improved bone microstructure compared to treatment with mcOPG or mcPTHrP alone. These results suggest that stepwise treatment with mcOPG and mcPTHrP may be a potential treatment for osteoporosis.

Intranasal administration of induced pluripotent stem cell-derived cortical neural stem cell-secretome as a treatment option for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, resulting in gradual destruction of cognitive abilities. Research on the development of various AD treatments is underway; however, no definitive treatment has been developed yet. Herein, we present induced pluripotent stem cell (iPSC)-derived cortical neural stem cell secretome (CNSC-SE) as a new treatment candidate for AD and explore its efficacy. METHODS: We first assessed the effects of CNSC-SE treatment on neural maturation and electromagnetic signal during cortical nerve cell differentiation. Then to confirm the efficacy in vivo, CNSC-SE was administered to the 5×FAD mouse model through the nasal cavity (5 µg/g, once a week, 4 weeks). The cell-mediated effects on nerve recovery, amyloid beta (Aß) plaque aggregation, microglial and astrocyte detection in the brain, and neuroinflammatory responses were investigated. Metabolomics analysis of iPSC-derived CNSC-SE revealed that it contained components that could exert neuro-protective effects or amplify cognitive restorative effects. RESULTS: Human iPSC-derived CNSC-SE increased neuronal proliferation and dendritic structure formation in vitro. Furthermore, CNSC-SE-treated iPSC-derived cortical neurons acquired electrical network activity and action potential bursts. The 5×FAD mice treated with CNSC-SE showed memory restoration and reduced Aß plaque accumulation. CONCLUSIONS: Our findings suggest that the iPSC-derived CNSC-SE may serve as a potential, non-invasive therapeutic option for AD in reducing amyloid infiltration and restoring memory.

Prochondrogenic effect of decellularized extracellular matrix secreted from human induced pluripotent stem cell-derived chondrocytes.

Cartilage is mainly composed of chondrocytes and the extracellular matrix (ECM), which transmits important biochemical and biomechanical signals necessary for differentiation and homeostasis. Human articular cartilage has a low ability for regeneration because it lacks blood vessels, nerves, and lymphatic vessels. Currently, cell therapeutics, including stem cells, provide a promising strategy for cartilage regeneration and treatment; however, there are various hurdles to overcome, such as immune rejection and teratoma formation. In this study, we assessed the applicability of stem cell-derived chondrocyte ECM for cartilage regeneration. Human induced pluripotent stem cell (hiPSC)-derived chondrocytes (iChondrocytes) were differentiated, and decellularized ECM (dECM) was successfully isolated from cultured chondrocytes. Isolated dECM enhanced the in vitro chondrogenesis of iPSCs when recellularized. Implanted dECM also restored osteochondral defects in a rat osteoarthritis model. A possible association with the glycogen synthase kinase-3 beta (GSK3ß) pathway demonstrated the fate-determining importance of dECM in regulating cell differentiation. Collectively, we suggest the prochondrogenic effect of hiPSC-derived cartilage-like dECM and offer a promising approach of a noncellular therapeutic for articular cartilage reconstruction without cell transplantation. STATEMENT OF SIGNIFICANCE: Human articular cartilage has low ability for regeneration and cell culture-based therapeutics could aid cartilage regeneration. Yet, the applicability of human induced pluripotent stem cell-derived chondrocyte (iChondrocyte) extracellular matrix (ECM) has not been elucidated. Therefore, we first differentiated iChondrocytes and isolated the secreted ECM by decellularization. Recellularization was performed to confirm the pro-chondrogenic effect of the decellularized ECM (dECM). In addition, we confirmed the possibility of cartilage repair by transplanting the dECM into the cartilage defect in osteochondral defect rat knee joint. We believe that our proof-of-concept study will serve as a basis for investigating the potential of dECM obtained from iPSC-derived differentiated cells as a non-cellular resource for tissue regeneration and other future applications.

Recent updates of stem cell-based erythropoiesis.

Blood transfusions are now an essential part of modern medicine. Transfusable red blood cells (RBCs) are employed in various therapeutic strategies; however, the processes of blood donation, collection, and administration still involve many limitations. Notably, a lack of donors, the risk of transfusion-transmitted disease, and recent pandemics such as COVID-19 have prompted us to search for alternative therapeutics to replace this resource. Originally, RBC production was attempted via the ex vivo differentiation of stem cells. However, a more approachable and effective cell source is now required for broader applications. As a viable alternative, pluripotent stem cells have been actively used in recent research. In this review, we discuss the basic concepts related to erythropoiesis, as well as early research using hematopoietic stem cells ex vivo, and discuss the current trend of in vitro erythropoiesis using human-induced pluripotent stem cells.

Anti-fibrotic effect of a selective estrogen receptor modulator in systemic sclerosis.

BACKGROUND: The rarity of systemic sclerosis (SSc) has hampered the development of therapies for this intractable autoimmune disease. Induced pluripotent stem cell (iPSC) can be differentiated into the key disease-affected cells in vitro. The generation of patient-derived iPSCs has opened up possibilities for rare disease modeling. Since these cells can recapitulate the disease phenotypes of the cell in question, they are useful high-throughput platforms for screening for drugs that can reverse these abnormal phenotypes. METHODS: SSc iPSC was generated from PBMC by Sendai virus. Human iPSC lines from SSc patients were differentiated into dermal fibroblasts and keratinocytes. The iPSC-derived differentiated cells from the SSc patients were used on high-throughput platforms to screen for FDA-approved drugs that could be effective treatments for SSc. RESULTS: Skin organoids were generated from these cells exhibited fibrosis that resembled SSc skin. Screening of the 770-FDA-approved drug library showed that the anti-osteoporotic drug raloxifene reduced SSc iPSC-derived fibroblast proliferation and extracellular matrix production and skin fibrosis in organoids and bleomycin-induced SSc-model mice. CONCLUSIONS: This study reveals that a disease model of systemic sclerosis generated using iPSCs-derived skin organoid is a novel tool for in vitro and in vivo dermatologic research. Since raloxifene and bazedoxifene are well-tolerated anti-osteoporotic drugs, our findings suggest that selective estrogen receptor modulator (SERM)-class drugs could treat SSc fibrosis.

Preferential stimulation of melanocytes by M2 macrophages to produce melanin through vascular endothelial growth factor.

Post-inflammatory hyperpigmentation is a skin discoloration process that occurs following an inflammatory response or wound. As the skin begins to heal, macrophages first exhibit a proinflammatory phenotype (M1) during the early stages of tissue repair and then transition to a pro-healing, anti-inflammatory phenotype (M2) in later stages. During this process, M1 macrophages remove invading bacteria and M2 macrophages remodel surrounding tissue; however, the relationship between macrophages and pigmentation is unclear. In this study, we examined the effect of macrophages on melanin pigmentation using human induced pluripotent stem cells. Functional melanocytes were differentiated from human induced pluripotent stem cells and named as hiMels. The generated hiMels were then individually cocultured with M1 and M2 macrophages. Melanin synthesis decreased in hiMels cocultured with M1 macrophages but significantly increased in hiMels cocultured with M2 macrophages. Moreover, the expression of vascular endothelial growth factor was increased in M2 cocultured media. Our findings suggest that M2 macrophages, and not M1 macrophages, induce hyperpigmentation in scarred areas of the skin during tissue repair.

Mitigating Effect of Estrogen in Alzheimer's Disease-Mimicking Cerebral Organoid.

Alzheimer's disease (AD) is the most common condition in patients with dementia and affects a large population worldwide. The incidence of AD is expected to increase in future owing to the rapid expansion of the aged population globally. Researchers have shown that women are twice more likely to be affected by AD than men. This phenomenon has been attributed to the postmenopausal state, during which the level of estrogen declines significantly. Estrogen is known to alleviate neurotoxicity in the brain and protect neurons. While the effects of estrogen have been investigated in AD models, to our knowledge, they have not been investigated in a stem cell-based three-dimensional in vitro system. Here, we designed a new model for AD using induced pluripotent stem cells (iPSCs) in a three-dimensional, in vitro culture system. We used 5xFAD mice to confirm the potential of estrogen in alleviating the effects of AD pathogenesis. Next, we confirmed a similar trend in an AD model developed using iPSC-derived cerebral organoids, in which the key characteristics of AD were recapitulated. The findings emphasized the potential of estrogen as a treatment agent for AD and also showed the suitability of AD-recapitulating cerebral organoids as a reliable platform for disease modeling and drug screening.

The Role of Transforming Growth Factor Beta in Joint Homeostasis and Cartilage Regeneration.

Transforming growth factor-beta (TGF-ß) is an important regulator of joint homeostasis, of which dysregulation is closely associated with the development of osteoarthritis (OA). In normal conditions, its biological functions in a joint environment are joint protective, but it can be dramatically altered in different contexts, making its therapeutic application a challenge. However, with the deeper insights into the TGF-ß functions, it has been proven that TGF-ß augments cartilage regeneration by chondrocytes, and differentiates both the precursor cells of chondrocytes and stem cells into cartilage-generating chondrocytes. Following documentation of the therapeutic efficacy of chondrocytes augmented by TGF-ß in the last decade, there is an ongoing phase III clinical trial examining the therapeutic efficacy of a mixture of allogeneic chondrocytes and TGF-ß-overexpressing cells. To prepare cartilage-restoring chondrocytes from induced pluripotent stem cells (iPSCs), the stem cells are differentiated mainly using TGF-ß with some other growth factors. Of note, clinical trials evaluating the therapeutic efficacy of iPSCs for OA are scheduled this year. Mesenchymal stromal stem cells (MSCs) have inherent limitations in that they differentiate into the osteochondral pathway, resulting in the production of poor-quality cartilage. Despite the established essential role of TGF-ß in chondrogenic differentiation of MSCs, whether the coordinated use of TGF-ß in MSC-based therapy for degenerated cartilage is effective is unknown. We herein reviewed the general characteristics and mechanism of action of TGF-ß in a joint environment. Furthermore, we discussed the core interaction of TGF-ß with principal cells of OA cell-based therapies, the chondrocytes, MSCs, and iPSCs. Impact Statement Transforming growth factor-beta (TGF-ß) has been widely used as a core regulator to improve or formulate therapeutic regenerative cells for degenerative joints. It differentiates stem cells into chondrocytes and improves the chondrogenic potential of differentiated chondrocytes. Herein, we discussed the overall characteristics of TGF-ß and reviewed the comprehension and utilization of TGF-ß in cell-based therapy for degenerative joint disease.

Review of the Current Trends in Clinical Trials Involving Induced Pluripotent Stem Cells.

In 2006, the induced pluripotent stem cell (iPSC) was presented to the world, paving the way for the development of a magnitude of novel therapeutic alternatives, addressing a diverse range of diseases. However, despite the immense cell therapy potential, relatively few clinical trials evaluating iPSC-technology have actually translated into interventional, clinically applied treatment regimens. Herein, our aim was to determine trends in globally conducted clinical trials involving iPSCs. Data were derived both from well-known registries recording clinical trials from across the globe, and databases from individual countries. Comparisons were firstly drawn between observational and interventional studies before the latter was further analyzed in terms of therapeutic and nontherapeutic trials. Our main observations included global distribution, purpose, target size, and types of disorder relevant to evaluated trials. In terms of nontherapeutic trials, the USA conducted the majority, a large average number of participants-187-was included in the trials, and studies on circulatory system disorders comprised a slightly higher proportion of total studies. Conversely, Japan was the frontrunner in terms of conducting therapeutic trials, and the average number of participants was much lower, at roughly 29. Disorders of the circulatory, as well as nervous and visual systems, were all studied in equal measure. This review highlights the impact that iPSC-based cell therapies can have, should development thereof gain more traction. We lastly considered a few companies that are actively utilizing iPSCs in the development of therapies for various diseases, for whom the global trends in clinical trials could become increasingly important.

Lupus Heart Disease Modeling with Combination of Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Lupus Patient Serum.

Background and Objectives: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease mainly affecting young women of childbearing age. SLE affects the skin, joints, muscles, kidneys, lungs, and heart. Cardiovascular complications are common causes of death in patients with SLE. However, the complexity of the cardiovascular system and the rarity of SLE make it difficult to investigate these morbidities. Patient-derived induced pluripotent stem cells (iPSCs) serve as a novel tool for drug screening and pathophysiological studies in the absence of patient samples. Methods and Results: We differentiated CMs from HC- and SLE-iPSCs using 2D culture platforms. SLE-CMs showed decreased proliferation and increased levels of fibrosis and hypertrophy marker expression; however, HC-and SLE-monolayer CMs reacted differently to SLE serum treatment. HC-iPSCs were also differentiated into CMs using 3D spheroid culture and anti-Ro autoantibody was treated along with SLE serum. 3D-HC-CMs generated more mature CMs compared to the CMs generated using 2D culture. The treatment of anti-Ro autoantibody rapidly increased the gene expression of fibrosis, hypertrophy, and apoptosis markers, and altered the calcium signaling in the CMs. Conclusions: iPSC derived cardiomyocytes with patient-derived serum, and anti-Ro antibody treatment could serve in effective autoimmune disease modeling including SLE. We believe that the present study might briefly provide possibilities on the application of a combination of patient-derived materials and iPSCs in disease modeling of autoimmune diseases.

Application of Induced Pluripotent Stem Cells for Disease Modeling and 3D Model Construction: Focus on Osteoarthritis.

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have shown promising potential, specifically because of their accessibility and plasticity. Hence, the clinical applicability of iPSCs was investigated in various fields of research. However, only a few iPSC studies pertaining to osteoarthritis (OA) have been performed so far, despite the high prevalence rate of degenerative joint disease. In this review, we discuss some of the most recent applications of iPSCs in disease modeling and the construction of 3D models in various fields, specifically focusing on osteoarthritis and OA-related conditions. Notably, we comprehensively reviewed the successful results of iPSC-derived disease models in recapitulating OA phenotypes for both OA and early-onset OA to encompass their broad etiology. Moreover, the latest publications with protocols that have used iPSCs to construct 3D models in recapitulating various conditions, particularly the OA environment, were further discussed. With the overall optimistic results seen in both fields, iPSCs are expected to be more widely used for OA disease modeling and 3D model construction, which could further expand OA drug screening, risk assessment, and therapeutic capabilities.

Increased Potential of Bone Formation with the Intravenous Injection of a Parathyroid Hormone-Related Protein Minicircle DNA Vector.

Osteoporosis is commonly treated via the long-term usage of anti-osteoporotic agents; however, poor drug compliance and undesirable side effects limit their treatment efficacy. The parathyroid hormone-related protein (PTHrP) is essential for normal bone formation and remodeling; thus, may be used as an anti-osteoporotic agent. Here, we developed a platform for the delivery of a single peptide composed of two regions of the PTHrP protein (1-34 and 107-139); mcPTHrP 1-34+107-139 using a minicircle vector. We also transfected mcPTHrP 1-34+107-139 into human mesenchymal stem cells (MSCs) and generated Thru 1-34+107-139-producing engineered MSCs (eMSCs) as an alternative delivery system. Osteoporosis was induced in 12-week-old C57BL/6 female mice via ovariectomy. The ovariectomized (OVX) mice were then treated with the two systems; (1) mcPTHrP 1-34+107-139 was intravenously administered three times (once per week); (2) eMSCs were intraperitoneally administered twice (on weeks four and six). Compared with the control OVX mice, the mcPTHrP 1-34+107-139-treated group showed better trabecular bone structure quality, increased bone formation, and decreased bone resorption. Similar results were observed in the eMSCs-treated OVX mice. Altogether, these results provide experimental evidence to support the potential of delivering PTHrP 1-34+107-139 using the minicircle technology for the treatment of osteoporosis.

Promoting Long-Term Cultivation of Motor Neurons for 3D Neuromuscular Junction Formation of 3D In Vitro Using Central-Nervous-Tissue-Derived Bioink.

3D cell printing technology is in the spotlight for producing 3D tissue or organ constructs useful for various medical applications. In printing of neuromuscular tissue, a bioink satisfying all the requirements is a challenging issue. Gel integrity and motor neuron activity are two major characters because a harmonious combination of extracellular materials essential to motor neuron activity consists of disadvantages in mechanical properties. Here, a method for fabrication of 3D neuromuscular tissue is presented using a porcine central nervous system tissue decellularized extracellular matrix (CNSdECM) bioink. CNSdECM retains CNS tissue-specific extracellular molecules, provides rheological properties crucial for extrusion-based 3D cell printing, and reveals positive effects on the growth and maturity of axons of motor neurons compared with Matrigel. It also allows long-term cultivation of human-induced-pluripotent-stem-cell-derived lower motor neurons and sufficiently supports their cellular behavior to carry motor signals to muscle fibers. CNSdECM bioink holds great promise for producing a tissue-engineered motor system using 3D cell printing.

Perichondrium-inspired permeable nanofibrous tube well promoting differentiation of hiPSC-derived pellet toward hyaline-like cartilage pellet.

The pellet formation has been regarded as a golden standard forin vitrochondrogenic differentiation. However, a spatially inhomogeneous chondrogenic microenvironment around a pellet resulted from the use of a traditional impermeable narrow tube, such as the conical tube, undermines the differentiation performance and therapeutic potential of differentiated cartilage pellet in defective articular cartilage treatment. To address this drawback, a perichondrium-inspired permeable nanofibrous tube (PINaT) well with a nanofibrous wall permeable to gas and soluble molecules is proposed. The PINaT well was fabricated with a micro deep drawing process where a flat thin nanofibrous membrane was transformed to a 3.5 mm deep tube well with a ∼50µm thick nanofibrous wall. Similar toin vivoperichondrium, the PINaT well was found to allow oxygen and growth factor diffusion required for chondrogenic differentiation across the entire nanofibrous wall. Analyses of gene expressions (COL2A1, COL10A1, ACAN, and SOX9), proteins (type II and X collagen), and glycosaminoglycans contents were conducted to assess the differentiation performance and clinical efficacy of differentiated cartilage pellet. The regulated spatially homogeneous chondrogenic microenvironment around the human induced pluripotent stem cell-derived pellet (3 × 105cells per pellet) in the PINaT well remarkably improved the quality of the differentiated pellet toward a more hyaline-like cartilage pellet. Furthermore, an accelerated chondrogenic differentiation process of the pellet produced by the PINaT well was achieved for 14 days, demonstrating a hyaline cartilage-specific marker similar to the control pellet differentiated for 20 days. Finally, the enhanced clinical efficacy of the hyaline-like cartilage pellet was confirmed using an osteochondral defect rat model, with the repaired tissue resembling hyaline cartilage rather than fibrous cartilage after 8 weeks of regeneration.

Pathogenic Role of Circulating Citrullinated Antigens and Anti-Cyclic Monoclonal Citrullinated Peptide Antibodies in Rheumatoid Arthritis.

We examined whether it is possible to directly detect citrullinated antigens in the serum of rheumatoid arthritis (RA) patients using a monoclonal antibody (mAb) designed to be specific for citrullinated peptides. In order to confirm the potential of the mAb as a direct arthritis-inducing substance through experimental model of RA, a monoclonal antibody (mAb) 12G1 was generated using by immunization of mice with a challenging cyclic citrullinated peptide. Immunohistochemical analysis of RA-affected synovial tissue showed that our mAb 12G1 could indeed detect citrullinated proteins in target tissues. Subsequently, serum levels of citrullinated type II collagen and filaggrin were measured in healthy volunteers, patients with RA, ankylosing spondylitis (AS), and systemic lupus erythematosus (SLE) using a 12G1-based sandwich ELISA. This showed that citrullinated filaggrin showed 78.9% sensitivity and 85.9% specificity for RA diagnosis with a cutoff optical density (OD) value of 1.013, comparable with the results from a second-generation anti-citrullinated protein antibody (ACPA) test. Circulating citrullinated collagen and filaggrin were detected even in sera of RA patients who were negative for both rheumatoid factor (RF) and ACPA. ELISA results also showed that RF and ACPA titers showed significantly positive correlation with both citrullinated collagen and filaggrin OD values in sera of RA patients. 12G1 challenging aggravated the severity of murine arthritis. In summary, mAb 12G1 can directly detect citrullinated proteins in RA target tissue and in sera of RA patients and 12G1 showed direct arthritogenic potential in vivo. This, 12G1 might be useful for diagnosis of RA including seronegative RA and may help to elucidate the pathophysiological role of citrullination in RA.

Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

Minicircles for Investigating and Treating Arthritic Diseases.

Gene delivery systems have become an essential component of research and the development of therapeutics for various diseases. Minicircles are non-viral vectors with promising characteristics for application in a variety of fields. With their minimal size, minicircles exhibit relatively high safety and efficient delivery of genes of interest into cells. Cartilage tissue lacks the natural ability to heal, making it difficult to treat osteoarthritis (OA) and rheumatoid arthritis (RA), which are the two main types of joint-related disease. Although both OA and RA affect the joint, RA is an autoimmune disease, while OA is a degenerative joint condition. Gene transfer using minicircles has also been used in many studies regarding cartilage and its diseased conditions. In this review, we summarize the cartilage-, OA-, and RA-based studies that have used minicircles as the gene delivery system.