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Abstract Delirium is risky and indicates poor outcomes for patients. Therefore, it is crucial to create an effective delirium detection method. However, the epigenetic pathophysiology of delirium remains largely unknown. We aimed to discover reliable and replicable epigenetic (DNA methylation: DNAm) markers that are associated with delirium including post-operative delirium (POD) in blood obtained from patients among four independent cohorts. Blood DNA from four independent cohorts (two inpatient cohorts and two surgery cohorts; 16 to 88 patients each) were analyzed using the Illumina EPIC array platform for genome-wide DNAm analysis. We examined DNAm differences in blood between patients with and without delirium including POD. When we compared top CpG sites previously identified from the initial inpatient cohort with three additional cohorts (one inpatient and two surgery cohorts), 11 of the top 13 CpG sites showed statistically significant differences in DNAm values between the delirium group and non-delirium group in the same directions as found in the initial cohort. This study demonstrated the potential value of epigenetic biomarkers as future diagnostic tools. Furthermore, our findings provide additional evidence of the potential role of epigenetics in the pathophysiology of delirium including POD.

Abstract Background Janus kinase (JAK) inhibitors, such as baricitinib, are widely used to treat rheumatoid arthritis (RA). Clinical studies show that baricitinib is more effective at reducing pain than other similar drugs. Here, we aimed to elucidate the molecular mechanisms underlying the pain relief conferred by baricitinib, using a mouse model of arthritis. Methods We treated collagen antibody-induced arthritis (CAIA) model mice with baricitinib, celecoxib, or vehicle, and evaluated the severity of arthritis, histological findings of the spinal cord, and pain-related behaviours. We also conducted RNA sequencing (RNA-seq) to identify alterations in gene expression in the dorsal root ganglion (DRG) following baricitinib treatment. Finally, we conducted in vitro experiments to investigate the direct effects of baricitinib on neuronal cells. Results Both baricitinib and celecoxib significantly decreased CAIA and improved arthritis-dependent grip-strength deficit, while only baricitinib notably suppressed residual tactile allodynia as determined by the von Frey test. CAIA induction of inflammatory cytokines in ankle synovium, including interleukin (IL)-1β and IL-6, was suppressed by treatment with either baricitinib or celecoxib. In contrast, RNA-seq analysis of the DRG revealed that baricitinib, but not celecoxib, restored gene expression alterations induced by CAIA to the control condition. Among many pathways changed by CAIA and baricitinib treatment, the interferon-alpha/gamma, JAK-signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) pathways were considerably decreased in the baricitinib group compared with the celecoxib group. Notably, only baricitinib decreased the expression of colony-stimulating factor 1 (CSF-1), a potent cytokine that causes neuropathic pain through activation of the microglia–astrocyte axis in the spinal cord. Accordingly, baricitinib prevented increases in microglia and astrocytes caused by CAIA. Baricitinib also suppressed JAK/STAT3 pathway activity and Csf1 expression in cultured neuronal cells. Conclusions Our findings demonstrate the effects baricitinib has on the DRG in relation to ameliorating both inflammatory and neuropathic pain.

Abstract Aim Work engagement is critical in both occupational and mental health contexts. However, no studies have compared the usefulness of the nine‐, three‐, and two‐item measures from the Utrecht Work Engagement Scale (UWES). Therefore, this study aimed to examine the internal consistency and convergent validity of the two‐item measure and compare its usefulness with the nine‐item and three‐item versions for assessing engagement at work among Japan Self‐Defense Forces (JSDF) personnel. Methods This cross‐sectional study included 229,383 participants who underwent an annual mental health check between October 19 and December 17, 2021. To test the internal consistency of the scales, Cronbach's alpha was used. To test the convergent validity, Pearson's correlation coefficients were examined for each item corresponding to job resources, job satisfaction, stress reactions, and job demands assessed by the Brief Job Stress Questionnaire. Results Most participants were men (89.8%). Cronbach's alpha coefficients for the nine‐, three‐, and two‐item scales were 0.95, 0.85, and 0.80, respectively. All three versions showed significant and positive correlations with each of the items corresponding to job resources and job satisfaction. The correlation coefficients of the two‐item scale were not inferior to those of the nine‐item and three‐item scales for job resources and job satisfaction items. Conclusion Our results showed the internal consistency and convergent validity for the two‐item measure of engagement at work among JSDF personnel. The two‐item measure may be useful for briefly and efficiently assessing the actual state of workers' engagement at work.

Summary:. Symptomatic neuroma represents a debilitating complication after major limb amputation. The regenerative peripheral nerve interface (RPNI) has emerged as a reproducible and practical surgery aimed at mitigating the formation of painful neuroma. Although previous animal studies revealed axonal sprouting, elongation, and synaptogenesis of proximal nerve stump within the muscle graft in RPNI, there is a lack of reports confirming these physiological reactions at the histopathological level in human samples. This report presents a case of below-knee amputation with RPNI due to foot gangrene resulting from polyarteritis nodosa. Subsequently, an above-knee amputation was necessitated due to the exacerbation of polyarteritis nodosa, providing the opportunity for histopathological examination of the RPNI site. The examination revealed sprouting, elongation, and existence of neuromuscular junction of the tibial nerve within the grafted muscle. To the best of our knowledge, this is the first report demonstrating axonal sprouting, elongation, and possibility of synaptogenesis of the nerve stump within the grafted muscle in a human sample.

Abstract Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate, where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Although nutritional status is a known regulator of long bone growth, it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2Cre ERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2Cre ERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.

Abstract The molecular pathophysiology underlying lumbar spondylosis development remains unclear. To identify genetic factors associated with lumbar spondylosis, we conducted a genome-wide association study using 83 severe lumbar spondylosis cases and 182 healthy controls and identified 65 candidate disease-associated single nucleotide polymorphisms (SNPs). Replication analysis in 510 case and 911 control subjects from five independent Japanese cohorts identified rs2054564, located in intron 7 of ADAMTS17, as a disease-associated SNP with a genome-wide significance threshold (P = 1.17 × 10–11, odds ratio = 1.92). This association was significant even after adjustment of age, sex, and body mass index (P = 7.52 × 10–11). A replication study in a Korean cohort, including 123 case and 319 control subjects, also verified the significant association of this SNP with severe lumbar spondylosis. Immunohistochemistry revealed that fibrillin-1 (FBN1) and ADAMTS17 were co-expressed in the annulus fibrosus of intervertebral discs (IVDs). ADAMTS17 overexpression in MG63 cells promoted extracellular microfibrils biogenesis, suggesting the potential role of ADAMTS17 in IVD function through interaction with fibrillin fibers. Finally, we provided evidence of FBN1 involvement in IVD function by showing that lumbar IVDs in patients with Marfan syndrome, caused by heterozygous FBN1 gene mutation, were significantly more degenerated. We identified a common SNP variant, located in ADAMTS17, associated with susceptibility to lumbar spondylosis and demonstrated the potential role of the ADAMTS17-fibrillin network in IVDs in lumbar spondylosis development.

Burnout and secondary traumatic stress (STS), also referred to as compassion fatigue, are undeniable negative consequences experienced by healthcare professionals when working with patients. As frontline healthcare professionals are essential to communities, it is crucial to understand their mental health and how they cope with negative psychological responses. This study investigated the relationships between burnout, STS, compassion satisfaction, dispositional empathy, and stress management among Japanese healthcare professionals and students taking care of patients in clinical practice. The participants were 506 Japanese healthcare professionals and students (doctors, nurses, medical students, and nursing students) affiliated with Japanese Ministry of Defense Hospitals. The data were collected from March 2020 to May 2021. We assessed burnout, STS, and compassion satisfaction using the Professional Quality of Life Scale, dispositional empathy using the Interpersonal Reactivity Index, and coping with stress using the Coping Orientation to Problems Experienced Inventory (Brief-COPE). Exploratory factor analysis of the Brief-COPE yielded three factors: active coping; support-seeking; and indirect coping. Personal distress, a self-oriented emotional empathy index, was related to higher burnout and STS scores and lower compassion satisfaction. Empathic concern, an other-oriented emotional empathy index, was associated with lower burnout and higher compassion satisfaction. Active coping strategies were associated with lower burnout and higher compassion satisfaction, whereas indirect coping strategies were associated with higher burnout and STS scores. In a comparison of empathy in professional categories, nurses presented higher personal distress than nursing students, and medical doctors showed lower fantasy tendencies than medical students. These results imply the complex relationships between empathy, coping strategies, and psychological responses among healthcare professionals. Further longitudinal study is needed to explore these complex relationships and to develop more precise and effective psycho-educational interventions to prevent burnout and STS.

Adipose-derived stem cells (ASCs) are an attractive cell source for cell therapy. Despite the increasing number of clinical applications, the methodology for ASC isolation is not optimized for every individual. In this study, we developed an effective material to stabilize explant cultures from small-fragment adipose tissues. Methods: Polypropylene/polyethylene nonwoven sheets were coated with hydroxyapatite (HA) particles. Adipose fragments were then placed on these sheets, and their ability to trap tissue was monitored during explant culture. The yield and properties of the cells were compared to those of cells isolated by conventional collagenase digestion. Results: Hydroxyapatite-coated nonwovens immediately trapped adipose fragments when placed on the sheets. The adhesion was stable even in culture media, leading to cell migration and proliferation from the tissue along with the nonwoven fibers. A higher fiber density further enhanced cell growth. Although cells on nonwoven explants could not be fully collected with cell dissociation enzymes, the cell yield was significantly higher than that of conventional monolayer culture without impacting stem cell properties. Conclusions: Hydroxyapatite-coated nonwovens are useful for the effective primary explant culture of connective tissues without enzymatic cell dissociation.

Possible distinct contributions of Runx 2 and Runx3 in osteoarthritis have not been clarified. Nagata et al. show that Runx3 protects adult articular cartilage by extracellular matrix protein production in normal conditions, while Runx2 exerts both catabolic and anabolic effects during inflammation.

Introduction: Cell therapy using adipose-derived mesenchymal stem cells (ASCs) is a promising avenue of regenerative medicine for the treatment of various diseases. It has been considered that ASCs exert their therapeutic effects through the secretion of multiple factors that are critical for tissue remodeling or the suppression of inflammation. Recently, conditioned medium (CM) from ASCs that contains a complex of secreted factors has received attention as a cost-effective alternative to cell therapy. Methods: We investigated the effects of CM obtained from ASCs (ASCs-CM) using human dermal fibroblasts (hDFs) and human epidermal keratinocytes with or without interleukin (IL)-1β and examined mRNA levels of marker genes. We also examined alterations in cell proliferation and morphology of hDFs following treatment with ASCs-CM. We further investigated the effects of ASCs-CM treatment on prevention of skin inflammation using a mouse model. Results: In hDFs and human epidermal keratinocytes, the ASCs-CM treatment suppressed pro-inflammatory factors and enhanced regenerative and remodeling factors with or without interleukin (IL)-1β exposure. The ASCs-CM treatment also enhanced cell proliferation of hDFs and prevented morphological changes in response to IL-1β exposure. Furthermore, in a mouse model of skin inflammation, treatment with ASCs-CM reduced the inflammatory reactions, including redness and thickness. Conclusions: CM from ASCs may represent a potential alternative to ASC therapy for the treatment of inflammatory skin conditions.

Abstract The superficial zone of articular cartilage contributes to smooth joint motion through the production of proteoglycan 4 (PRG4), also known as lubricin. Recent studies indicate novel effects of PRG4 as a signaling molecule, other than a simple extracellular matrix protein. Additionally, the accumulating evidence displays that various molecules and signaling pathways are involved in regulating the superficial zone and PRG4 expression. In addition, Prg4-expressing cells include a progenitor population of articular chondrocytes. Several non-clinical and clinical studies have shown that PRG4 and related molecules are promising candidates for disease-modifying drugs for treating osteoarthritis. Since PRG4 is also expressed in the synovium, tendons, and ligaments, further studies of PRG4-related pathways and PRG4-positive cells may elucidate the mechanisms underlying joint homeostasis.

Abstract Background Somatic stem cell transplantation has been performed for cartilage injury, but the reparative mechanisms are still conflicting. The chondrogenic potential of stem cells are thought as promising features for cartilage therapy; however, the correlation between their potential for chondrogenesis in vitro and in vivo remains undefined. The purpose of this study was to investigate the intrinsic chondrogenic condition depends on cell types and explore an indicator to select useful stem cells for cartilage regeneration. Methods The chondrogenic potential of two different stem cell types derived from adipose tissue (ASCs) and synovium (SSCs) of mice and humans was assessed using bone morphogenic protein-2 (BMP2) and transforming growth factor-β1 (TGFβ1). Their in vivo chondrogenic potential was validated through transplantation into a mouse osteochondral defect model. Results All cell types showed apparent chondrogenesis under the combination of BMP2 and TGFβ1 in vitro, as assessed by the formation of proteoglycan- and type 2 collagen (COL2)-rich tissues. However, our results vastly differed with those observed following single stimulation among species and cell types; apparent chondrogenesis of mouse SSCs was observed with supplementation of BMP2 or TGFβ1, whereas chondrogenesis of mouse ASCs and human SSCs was observed with supplementation of BMP2 not TGFβ1. Human ASCs showed no obvious chondrogenesis following single stimulation. Mouse SSCs showed the formation of hyaline-like cartilage which had less fibrous components (COL1/3) with supplementation of TGFβ1. However, human cells developed COL1/3+ tissues with all treatments. Transcriptomic analysis for TGFβ receptors and ligands of cells prior to chondrogenic induction did not indicate their distinct reactivity to the TGFβ1 or BMP2. In the transplanted site in vivo, mouse SSCs formed hyaline-like cartilage (proteoglycan+/COL2+/COL1−/COL3−) but other cell types mainly formed COL1/3-positive fibrous tissues in line with in vitro reactivity to TGFβ1. Conclusion Optimal chondrogenic factors driving chondrogenesis from somatic stem cells are intrinsically distinct among cell types and species. Among them, the response to TGFβ1 may possibly represent the fate of stem cells when locally transplanted into cartilage defects.

Abstract In this research, we propose a new index of emotional arousal level using sound pressure change acceleration, called the emotional arousal level voice index (EALVI), and investigate the relationship between this index and depression severity. First, EALVI values were calculated from various speech recordings in the interactive emotional dyadic motion capture database, and the correlation with the emotional arousal level of each voice was examined. The resulting correlation coefficient was 0.52 (n = 10,039, p

Summary: Single-cell RNA sequencing (scRNAseq) has been used to assess the intra-tumor heterogeneity and microenvironment of pancreatic ductal adenocarcinoma (PDAC). However, previous knowledge is not fully universalized. Here, we built a single cell atlas of PDAC from six datasets containing over 70 samples and >130,000 cells, and demonstrated its application to the reanalysis of the previous bulk transcriptomic cohorts and inferring cell–cell communications. The cell decomposition of bulk transcriptomics using scRNAseq data showed the cellular heterogeneity of PDAC; moreover, high levels of tumor cells and fibroblasts were indicative of poor-prognosis. Refined tumor subtypes signature indicated the tumor cell dynamics in intra-tumor and their specific regulatory network. We further identified functionally distinct tumor clusters that had close interaction with fibroblast subtypes via different signaling pathways dependent on subtypes. Our analysis provided a reference dataset for PDAC and showed its utility in research on the microenvironment of intra-tumor heterogeneity.

ABSTRACT Bone turnover is finely tuned by cells in the bone milieu, including osteoblasts, osteoclasts, and osteocytes. Osteoclasts are multinucleated giant cells with a bone‐resorbing function that play a critical role in regulating skeletal homeostasis. Osteoclast differentiation is characterized by dramatic changes in morphology and gene expression following receptor activator of nuclear factor‐kappa‐Β ligand (RANKL) stimulation. We performed single‐cell RNA‐sequencing analyses of human and murine osteoclast‐lineage cells (OLCs) and found that OLCs in the mitotic phase do not differentiate into mature osteoclasts. We also identified a guanosine triphosphatase (GTPase) family member, RAB38, as a highly expressed molecule in both human and murine osteoclast clusters; RAB38 gene expression is associated with dynamic changes in histone modification and transcriptional regulation. Silencing Rab38 expression by using short hairpin RNA (shRNA) inhibited osteoclast differentiation and maturation. In summary, we established an integrated fate map of human and murine osteoclastogenesis; this will help identify therapeutic targets in bone diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ABSTRACT We investigated the role of hematopoietically expressed homeobox protein (Hhex) in osteoclast development. Trimethylation of lysine 27 of histone H3 at the cis‐regulatory element of Hhex was maintained and that of lysine 4 was reduced during receptor activator of nuclear factor κB ligand (RANKL)‐induced osteoclastogenesis, which was associated with a reduction of Hhex expression. Overexpression of Hhex in bone marrow–derived macrophages inhibited, whereas Hhex suppression promoted, RANKL‐induced osteoclastogenesis in vitro. Conditional deletion of Hhex in osteoclast‐lineage cells promoted osteoclastogenesis and reduced cancellous bone volume in mice, confirming the negative regulatory role of Hhex in osteoclast differentiation. Expression of cyclin‐dependent kinase inhibitors such as Cdkn2a and Cdkn1b in osteoclast precursors was negatively regulated by Hhex, and Hhex deletion increased the ratio of cells at the G1 phase of the cell cycle. In conclusion, Hhex is an inhibitor of osteoclast differentiation that is regulated in an epigenetic manner and regulates the cell cycle of osteoclast precursors and the skeletal homeostasis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Introduction: Clinical studies of intra-articular injection of mesenchymal stem cells for osteoarthritis (OA) indicate its efficacy. Here, we retrospectively investigated the associations of pretherapeutic magnetic resonance imaging (MRI) findings with the clinical outcomes up to 6 months, after intra-articular administration of adipose-derived stem cells (ASCs) to knee OA patients. Methods: We first analyzed alterations of the visual analog scale (VAS) and knee injury and osteoarthritis outcome score (KOOS) in 57 knees of 34 patients from whom clinical scores were obtained before ASC therapy, and at 1, 3, and 6 months. Among the patients, we further examined MRI findings of 34 knees of 19 patients whose pretherapeutic MRI data were available. Results: The mean improvement of VAS and KOOS-total during 6 months was 2.6 ± 4.0 (from 6.1 ± 2.5 to 3.5 ± 2.9, P

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

Abstract Purpose Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946. Methods Articular chondrocytes were isolated from mini pig knees and expanded in 2-dimensional cell culture and subsequently used in the experiments. 3-D pellets were then cultured for two weeks. Cells were also cultured in a type I collagen scaffolds for four weeks. Specimens were cultured with TD198946, BMP-2, or both in combination. Outcomes were determined by gene expression levels of RUNX1, SOX9, ACAN, COL1A1, COL2A1 and COL10A1, the glycosaminoglycan content, and characteristics of histology and immunohistochemistry. Furthermore, the maturity of the engineered cartilage cultured for two weeks was evaluated through subcutaneous implantation in nude mice for four weeks. Results Addition of TD198946 demonstrated the upregulation of gene expression level except for ACAN, type II collagen and glycosaminoglycan synthesis in both pellet and 3D scaffold cultures. TD198946 and BMP-2 combination cultures showed higher chondrogenic differentiation than TD198946 or BMP-2 alone. The engineered cartilage maintained its extracellular matrices for four weeks post implantation. In contrast, engineered cartilage treated with either TD198946 or BMP-2 alone was mostly absorbed. Conclusions Our results indicate that TD198946 could improve quality of engineered cartilage by redifferentiation of dedifferentiated chondrocytes pre-implantation and promoting collagen and glycosaminoglycan synthesis.

Abstract Background Both loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage. Methods Wnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-Cre ERT2 ;Ctnnb1 fl/fl and Prg4-Cre ERT2 ;Ctnnb1-ex3 fl/wt mice for loss- and gain-of-function, respectively, of Wnt/β-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/β-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/β-catenin signaling in SFZ cells. Results Wnt/β-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific β-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific β-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by β-catenin knockout, while it was upregulated by β-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/β-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. Conclusions We demonstrated that Wnt/β-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

Summary: A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5–9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation. : Saito and colleagues show that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, a GSK3 inhibitor and an RAR agonist, within 5–9 days. Genome-wide analysis of RAR and β-catenin association with DNA for sequential samples at each protocol stage demonstrates that RA and Wnt/β-catenin signaling is collaboratively involved in direct regulation of chondrocyte differentiation. Keywords: pluripotent stem cells, cartilage, chondrocyte, chondrocyte differentiation, regenerative medicine, small-molecule compound

Excessive mechanical stress promotes the development of osteoarthritis. Here Chang et al. identify gremlin-1 as a factor expressed in chondrocytes in response to mechanical stress, and contributing to osteoarthritis via activation of the NF-κB pathway.

Abstract Intervertebral disc degeneration (IDD) is the main contributor to low back pain, which is a leading cause of disability worldwide. Although substantial progress has been made in elucidating the molecular mechanisms of IDD, fundamental and long‐lasting treatments for IDD are still lacking. With increased understanding of the complex pathomechanism of IDD, alternative strategies for treating IDD can be discovered. A brief overview of the prevalence and epidemiologic risk factors of IDD is provided in this review, followed by the descriptions of anatomic, cellular, and molecular structure of the intervertebral disc as well as the molecular pathophysiology of IDD. Finally, the recent findings of intervertebral disc progenitors are reviewed and the future perspectives are discussed.

Abstract Background Disaster workers suffer from psychological distress not only through the direct experience of traumatic situations but also through the indirect process of aiding disaster victims. This distress, called secondary traumatic stress, is linked to dispositional empathy, which is the tendency for individuals to imagine and experience the feelings and experiences of others. However, the association between secondary traumatic stress and dispositional empathy remains understudied. Methods To examine the relationship between dispositional empathy and mental health among disaster workers, we collected data from 227 Japan Ground Self-Defense Force personnel who engaged in international disaster relief activities in the Philippines following Typhoon Yolanda in 2013. The Impact of Event Scale-Revised and the Kessler Psychological Distress Scale were used to evaluate posttraumatic stress responses (PTSR) and general psychological distress (GPD), respectively. Dispositional empathy was evaluated through the Interpersonal Reactivity Index, which consists of four subscales: Perspective Taking, Fantasy, Empathic Concern, and Personal Distress. Hierarchial linear regression analyses were performed to identify the variables related to PTSR and GPD. Results High PTSR was significantly associated with high Fantasy (identification tendency, β = 0.21, p

Abstract Osteoarthritis (OA) is a multi-factorial and highly prevalent joint disorder worldwide. Since the establishment of murine surgical knee OA models in 2005, many of the key molecules and signalling pathways responsible for OA development have been identified. Here we review the roles of two multi-functional signalling pathways in OA development: Notch and nuclear factor kappa-light-chain-enhancer of activated B cells. Previous studies have identified various aspects of articular chondrocyte regulation by these pathways. However, comprehensive understanding of the molecular networks regulating articular cartilage homeostasis and OA pathogenesis is needed.

A 34-year-old woman was diagnosed with acute promyelocytic leukemia. Chemotherapy was administered following the JALSG APL204 protocol. Induction therapy with all-trans retinoic acid resulted in complete remission on day 49. She developed coccygeal pain from day 18, which spread to the spine and cheekbones and lasted 5 weeks. She had similar bone pain on days 7–10 of the first consolidation therapy and on days 4–12 of the second consolidation therapy. Oral loxoprofen was prescribed for pain relief. On day 33 of the third consolidation, white blood cell and neutrophil counts were 320/μL and 20/μL, respectively. After she developed epigastralgia and hematemesis, she developed septic shock. Gastroendoscopy revealed markedly thickened folds and diffusely damaged mucosa with blood oozing. Computed tomography revealed thickened walls of the antrum and the pylorus. Despite emergency treatments, she died. Bacterial culture of the gastric fluid yielded Enterobacter cloacae and enterococci growth. Collectively, she was diagnosed with phlegmonous gastritis. Retrospective examination of serial bone marrow biopsy specimens demonstrated progressive bone marrow fibrosis, which may have caused prolonged myelosuppression. Thus, evaluation of bone marrow fibrosis by bone marrow biopsy after each treatment cycle might serve as a predictor of persistent myelosuppression induced by chemotherapy.

Rela is a transcription factor shown to have seemingly contradictory roles in anabolism and catabolism of cartilage. Here the authors find that Rela prevents chondrocyte apoptosis and that homozygous knockout causes accelerated osteoarthritis in adults, whereas heterozygous knockout suppresses osteoarthritis by maintaining wild-type effects on apoptosis but inhibiting catabolic gene expression.

Recently, the relationship between emotional arousal and depression has been studied. Focusing on this relationship, we first developed an arousal level voice index (ALVI) to measure arousal levels using the Interactive Emotional Dyadic Motion Capture database. Then, we calculated ALVI from the voices of depressed patients from two hospitals (Ginza Taimei Clinic (H1) and National Defense Medical College hospital (H2)) and compared them with the severity of depression as measured by the Hamilton Rating Scale for Depression (HAM-D). Depending on the HAM-D score, the datasets were classified into a no depression (HAM-D < 8) and a depression group (HAM-D ≥ 8) for each hospital. A comparison of the mean ALVI between the groups was performed using the Wilcoxon rank-sum test and a significant difference at the level of 10% (p = 0.094) at H1 and 1% (p = 0.0038) at H2 was determined. The area under the curve (AUC) of the receiver operating characteristic was 0.66 when categorizing between the two groups for H1, and the AUC for H2 was 0.70. The relationship between arousal level and depression severity was indirectly suggested via the ALVI.

Purpose: Joint pain is the most common symptom of osteoarthritis (OA); however, its mechanism remains unclarified. The present study investigated hindlimb motion during locomotion on the treadmill using a three-dimensional (3D) motion analysis system with high-speed cameras to evaluate whether this method can be used as an indication of joint pain in a mouse model of surgically induced OA. Methods: We resected the medial meniscus and medial collateral ligament in 8-week old C57BL/6 male mice and performed locomotion recording 6 months post-operatively. Additionally, we performed the same recording after oral administration of the selective cyclooxygenase-2 inhibitor to determine whether alteration of the parameters were associated with joint pain. Results: OA development, characterized by cartilage degeneration and osteophyte formation, was markedly enhanced in the OA group. There was no significant difference between the sham and OA groups in basic gait parameters, including stance duration, swing duration and gait cycle. However, when we divided the gait cycle into four phases and calculated the joint ranges of motion in each phase, the range of motion of the knee joint during the stepping-in phase and the swing duration were significantly decreased in the OA group. These significant differences between the sham and OA groups were diminished by the oral administration of a selective cyclooxygenase-2 inhibitor to the OA group. Conclusion: The present method may be useful to evaluate joint pain in experimental mice and contribute to elucidating the molecular mechanisms of pain in the OA knee joint in combination with genetically modified mice.

We examined the individual and combined effects of teriparatide and anti-RANKL (receptor activator of nuclear factor κB ligand) monoclonal antibody in ovariectomized mice. Three-month-old female C57BL/6 mice were ovariectomized (OVX) or sham operated. Four weeks after OVX, they were assigned to 3 different groups to receive anti-RANKL monoclonal antibody (Ab) alone (5 mg/kg single injection at 4 weeks after OVX, Ab group), teriparatide alone (80 μg/kg daily injection for 4 weeks from 4 weeks after OVX, PTH group), or mAb plus teriparatide (Ab + PTH group). Mice were sacrificed 8 weeks after OVX. Bone mineral density (BMD) was measured at the femur and lumbar spine. Hind limbs were subjected to histological and histomorphometric analysis. Serum osteocalcin and CTX-I levels were measured to investigate the bone turnover. Compared with Ab group, Ab + PTH group showed a significant increase in BMD at distal femur and femoral shaft. Cortical bone volume was significantly increased in PTH and Ab + PTH groups compared with Ab group. Bone turnover in Ab + PTH group was suppressed to the same degree as in Ab group. The number of TRAP-positive multinucleated cells was markedly reduced in Ab and Ab + PTH groups. These results suggest that combined treatment of teriparatide with anti-RANKL antibody has additive effects on BMD in OVX mice compared with individual treatment.

Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6). We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

Schwann cells are an important cell source for regenerative therapy for neural disorders. We investigated the role of the transcription factor sex determining region Y (SRY)-box 10 (SOX10) in the proliferation and myelination of Schwann cells. SOX10 is predominantly expressed in rat sciatic nerve-derived Schwann cells and is induced shortly after birth. Among transcription factors known to be important for the differentiation of Schwann cells, SOX10 potently transactivates the S100B promoter. In cultures of Schwann cells, overexpressing SOX10 dramatically induces S100B expression, while knocking down SOX10 with shRNA suppresses S100B expression. Here, we identify three core response elements of SOX10 in the S100B promoter and intron 1 with a putative SOX motif. Knockdown of either SOX10 or S100B enhances the proliferation of Schwann cells. In addition, using dissociated cultures of dorsal root ganglia, we demonstrate that suppressing S100B with shRNA impairs myelination of Schwann cells. These results suggest that the SOX10-S100B signaling axis critically regulates Schwann cell proliferation and myelination, and therefore is a putative therapeutic target for neuronal disorders.

Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC) marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

BACKGROUND: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) in chondrocytes during endochondral ossification. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryos with homozygous deficiency in C/EBPbeta (C/EBPbeta-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPbeta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPbeta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPbeta. In fact, a DNA cell cycle histogram revealed that the C/EBPbeta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2) as the transcriptional target of C/EBPbeta. p57(Kip2) was co-localized with C/EBPbeta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPbeta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPbeta in the p57(Kip2) promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2) by the siRNA inhibited the C/EBPbeta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPbeta+/- littermates, the C/EBPbeta insufficiency caused resistance to joint cartilage destruction. CONCLUSIONS/SIGNIFICANCE: C/EBPbeta transactivates p57(Kip2) to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPbeta-p57(Kip2) signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.