Chromatin remodeler CHD7 regulates the stem cell identity of human neural progenitors.

Multiple congenital disorders often present complex phenotypes, but how the mutation of individual genetic factors can lead to multiple defects remains poorly understood. In the present study, we used human neuroepithelial (NE) cells and CHARGE patient-derived cells as an in vitro model system to identify the function of chromodomain helicase DNA-binding 7 (CHD7) in NE-neural crest bifurcation, thus revealing an etiological link between the central nervous system (CNS) and craniofacial anomalies observed in CHARGE syndrome. We found that CHD7 is required for epigenetic activation of superenhancers and CNS-specific enhancers, which support the maintenance of the NE and CNS lineage identities. Furthermore, we found that BRN2 and SOX21 are downstream effectors of CHD7, which shapes cellular identities by enhancing a CNS-specific cellular program and indirectly repressing non-CNS-specific cellular programs. Based on our results, CHD7, through its interactions with superenhancer elements, acts as a regulatory hub in the orchestration of the spatiotemporal dynamics of transcription factors to regulate NE and CNS lineage identities.

Inflammatory myopathy associated with PD-1 inhibitors.

OBJECTIVE: To characterize the inflammatory myopathy associated with programmed cell death 1 inhibitors (PD-1 myopathy). METHODS: We studied 19 Japanese patients with PD-1 myopathy (13 men and 6 women, mean age 70 years), who were referred to Keio University. As control groups, we used 68 patients with anti-signal recognition particle antibodies, 51 patients with anti-aminoacyl transfer RNA synthetase antibodies and 460 healthy subjects. RESULTS: In regard to muscle-disease severity, 10 patients showed a mild form of disease and 9 patients showed a severe form. Non-small cell lung cancer was the most common underlying cancer. PD-1 inhibitor consisted of 11 nivolumab and 8 pembrolizumab. PD-1 myopathy occurred 29 days on average after the first administration of PD-1 inhibitor. The initial manifestation of muscle weakness was ptosis in 10 patients, 15 patients had ptosis, 13 diplopia, 8 facial muscle weakness, 10 bulbar symptoms, 13 limb weakness, 14 neck weakness, 4 cardiac involvement, 6 respiratory involvement and 16 myalgia. Ocular, facial, cardiac and respiratory involvement and myalgia were more frequently observed than controls. Serum creatine kinase was increased to 5247 IU/L on average. Autoantibodies related to inflammatory myopathy were negative, while anti-striational antibodies were found in 13 (68%) patients. HLA-C*12:02 alleles were more frequently detected than healthy controls. Muscle pathology was characterized by multifocal necrotic myofibers with endomysial inflammation and expression of MHC class I. Immunosuppressive therapy with corticosteroids was generally effective for muscle weakness. CONCLUSIONS: Based on our clinical, histological and immunological findings, PD-1 myopathy is a discrete subset of inflammatory myopathy.

[A case of recurrent Campylobacter fetus meningitis, occurring two months after the |initial infection was successfully treated].

A 43-year-old man was admitted to our department due to fever and headache. The cerebrospinal fluid analysis confirmed bacterial meningitis. Campylobacter species were isolated from blood cultures on the third day of admission. The patient was treated with meropenem (MEPM) and discharged on the 17th day. However, he experienced a recurrence of meningitis and was readmitted on the 68th day, initiating MEPM therapy. Campylobacter fetus was isolated from cerebrospinal fluid cultures on the 74th day. MEPM was continued until the 81st day, followed by one month of minocycline (MINO) therapy. The patient had an uneventful recovery without further recurrence. This case highlights the potential for recurrence of Campylobacter fetus meningitis approximately two months after the resolution of the initial infection. In addition to carbapenem therapy for at least two weeks, the adjunctive administration of MINO may be beneficial.

Mesenchymal properties of iPSC-derived neural progenitors that generate undesired grafts after transplantation.

Although neural stem/progenitor cells derived from human induced pluripotent stem cells (hiPSC-NS/PCs) are expected to be a cell source for cell-based therapy, tumorigenesis of hiPSC-NS/PCs is a potential problem for clinical applications. Therefore, to understand the mechanisms of tumorigenicity in NS/PCs, we clarified the cell populations of NS/PCs. We established single cell-derived NS/PC clones (scNS/PCs) from hiPSC-NS/PCs that generated undesired grafts. Additionally, we performed bioassays on scNS/PCs, which classified cell types within parental hiPSC-NS/PCs. Interestingly, we found unique subsets of scNS/PCs, which exhibited the transcriptome signature of mesenchymal lineages. Furthermore, these scNS/PCs expressed both neural (PSA-NCAM) and mesenchymal (CD73 and CD105) markers, and had an osteogenic differentiation capacity. Notably, eliminating CD73+ CD105+ cells from among parental hiPSC-NS/PCs ensured the quality of hiPSC-NS/PCs. Taken together, the existence of unexpected cell populations among NS/PCs may explain their tumorigenicity leading to potential safety issues of hiPSC-NS/PCs for future regenerative medicine.

Subretinal fluid accumulation in a patient with polycythemia vera after receiving a prostaglandin I2 analogue treatment.

Purpose: To report a case of polycythemia vera (PV) with subretinal fluid accumulation after the administration of prostaglandin I2 (PGI2) analogue. Observations: A 57-year-old woman diagnosed as having PV was referred to our department for the evaluation of severe metamorphopsia in the left eye, which gradually progressed after the initiation of oral administration of PGI2 mimetics. At the first visit, the patient's best-corrected visual acuities (BCVAs) were 20/20 OD and 20/30 OS. Fundus examination and optical coherence tomography revealed the presence of subretinal fluid (SRF) in the left eye and multiple serous pigment epithelial detachments (PEDs) in both eyes. Fluorescein angiography revealed central serous chorioretinopathy (CSC)-like lesions, consisting of dye pooling corresponding to the PEDs in both eyes and dye leakage in the left eye. Indocyanine green angiography and laser speckle flowgraphy revealed dilated choroidal veins and reduced choroidal blood flow, respectively. The central choroidal thickness (CCT) measured at the first visit showed a relatively thickened choroid in the left eye. Laboratory data showed mild pancytosis. The patient was diagnosed as having CSC associated with a background of PV, presumably triggered by the PGI2 analogue. One month after cessation of drug administration, the patient's BCVA improved, the CCT slightly decreased, and serous retinal detachment and PED disappeared in the left eye. Conclusions and importance: Our case of PV presenting with CSC-like lesions after PGI2 analogue administration indicates the possible risk of SRF accumulation by PGI2 analogues in patients with PV.

Chromatin remodeler CHD7 targets active enhancer region to regulate cell type-specific gene expression in human neural crest cells.

A mutation in the chromatin remodeler chromodomain helicase DNA-binding 7 (CHD7) gene causes the multiple congenital anomaly CHARGE syndrome. The craniofacial anomalies observed in CHARGE syndrome are caused by dysfunctions of neural crest cells (NCCs), which originate from the neural tube. However, the mechanism by which CHD7 regulates the function of human NCCs (hNCCs) remains unclear. We aimed to characterize the cis-regulatory elements governed by CHD7 in hNCCs by analyzing genome-wide ChIP-Seq data and identifying hNCC-specific CHD7-binding profiles. We compared CHD7-binding regions among cell types, including human induced pluripotent stem cells and human neuroepithelial cells, to determine the comprehensive properties of CHD7-binding in hNCCs. Importantly, analysis of the hNCC-specific CHD7-bound region revealed transcription factor AP-2α as a potential co-factor facilitating the cell type-specific transcriptional program in hNCCs. CHD7 was strongly associated with active enhancer regions, permitting the expression of hNCC-specific genes to sustain the function of hNCCs. Our findings reveal the regulatory mechanisms of CHD7 in hNCCs, thus providing additional information regarding the transcriptional programs in hNCCs.

MeCP2 controls neural stem cell fate specification through miR-199a-mediated inhibition of BMP-Smad signaling.

Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.

[A case of myasthenia gravis and myositis induced by pembrolizumab].

A 77-year-old woman with lung adenocarcinoma noticed bilateral ptosis 7 weeks after a first pembrolizumab infusion. Her symptoms rapidly progressed to generalized manifestations including limb and neck weakness, dyspnea, and dysphasia within the following two weeks. We diagnosed him with pembrolizumab-related myasthenia gravis and myositis based on clinical symptoms, elevation of muscle enzymes and anti-acetylcholine receptor antibodies, repetitive nerve stimulation and muscle biopsy. We commenced combination immunotherapy, including intravenous and oral steroid therapy, immune absorption therapy and plasma exchange therapy with noninvasive positive-pressure ventilation and tracheotomy positive pressure ventilation. She had gradual symptoms improvement and discharged after 209 days in a hospital. In this case, anti-titin antibodies, one of anti-striational antibodies, was positive and correlated with severity of myasthenia gravis. With the development of immune checkpoint inhibitors for various malignancies, clinicians should closely monitor patients for important immune-related adverse events and coordinate on early treatment.

Chemical decontamination of iPS cell-derived neural cell mixtures.

This report describes the design and evaluation of phosphorylated 7-ethyl-10-hydroxycamptothecin (SN38-P), which selectively eliminates tumor-forming proliferative stem cells, including human induced pluripotent stem cells (hiPSCs) and neural stem cells, from iPSC-derived neural cell mixtures. Results of the present study demonstrate that simple phosphorylation of an anticancer drug can provide a safe, cost-effective, and chemically-defined tool for decontaminating hiPSC-derived neuron.

T-type Calcium Channels Determine the Vulnerability of Dopaminergic Neurons to Mitochondrial Stress in Familial Parkinson Disease.

Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.

Differential X Chromosome Inactivation Patterns during the Propagation of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) represent a potentially useful tool for studying the molecular mechanisms of disease thanks to their ability to generate patient-specific hiPSC clones. However, previous studies have reported that DNA methylation profiles, including those for imprinted genes, may change during passaging of hiPSCs. This is particularly problematic for hiPSC models of X-linked disease, because unstable X chromosome inactivation status may affect the detection of phenotypes. In the present study, we examined the epigenetic status of hiPSCs derived from patients with Rett syndrome, an X-linked disease, during long-term culture. To analyze X chromosome inactivation, we used a methylation-specific polymerase chain reaction (MSP) to assay the human androgen receptor locus (HUMARA). We found that single cell-derived hiPSC clones exhibit various states of X chromosome inactivation immediately after clonal isolation, even when established simultaneously from a single donor. X chromosome inactivation states remain variable in hiPSC clones at early passages, and this variability may affect cellular phenotypes characteristic of X-linked diseases. Careful evaluation of X chromosome inactivation in hiPSC clones, particularly in early passages, by methods such as HUMARA-MSP, is therefore important when using patient-specific hiPSCs to model X-linked disease.

Gene Expression Profile of Inflammatory Myopathy with Malignancy is Similar to that of Dermatomyositis rather than Polymyositis.

Objective An association has been reported between inflammatory myopathies (IMs), which include polymyositis (PM) and dermatomyositis (DM), and malignancy, and the concept of cancer-associated myositis (CAM) was recently proposed. We herein attempted to determine the features and etiologies of these myopathies. Methods We analyzed the gene expression levels via microarray and real-time quantitative reverse transcription polymerase chain reaction analyses to identify genes that were specifically upregulated or downregulated with suspected inflammatory involvement and verified the microarray data via an immunohistochemical (IHC) analysis in additional cases. Patients We selected 14 patients with the following conditions: PM without malignancy (n=3), DM without malignancy (n=3), CAM (n=3), and Controls (no pathological changes or malignancy; n=5). Results PM was distinct from DM and CAM in a clustering analysis and exhibited the highest numbers of overexpressed genes and specific pathologies in a gene ontology analysis. The IHC analysis confirmed the gene expression results. Conclusion PM is associated with severe inflammatory pathological findings, primarily in the cell-mediated immune system. DM and CAM exhibit similarities in the gene expression and IHC results, which suggest that humoral immunity is the main etiology for both myopathies, indicating the importance of cancer screening in patients with IMs, particularly DM.

Modeling Rett Syndrome Using Human Induced Pluripotent Stem Cells.

Rett syndrome (RTT) is one of a group of neurodevelopmental disorders typically characterized by deficits in the X-linked gene MECP2 (methyl-CpG binding protein 2). The MECP2 gene encodes a multifunctional protein involved in transcriptional repression, transcriptional activation, chromatin remodeling, and RNA splicing. Genetic deletion of Mecp2 in mice revealed neuronal disabilities including RTT-like phenotypes and provided an excellent platform for understanding the pathogenesis of RTT. So far, there are no effective pharmacological treatments for RTT because the role of MECP2 in RTT is incompletely understood. Recently, human induced pluripotent stem cell (hiPSC) technologies have improved our knowledge of neurological and neurodevelopmental diseases including RTT because neurons derived from RTT-hiPSCs can be used for disease modeling to understand RTT phenotypes and to perform high throughput pharmaceutical drug screening. In this review, we provide an overview of RTT, including MeCP2 function and mouse models of RTT. In addition, we introduce recent advances in disease modeling of RTT using hiPSC-derived neural cells.

Distinct molecular mechanisms of HTRA1 mutants in manifesting heterozygotes with CARASIL.

OBJECTIVE: To elucidate the molecular mechanism of mutant HTRA1-dependent cerebral small vessel disease in heterozygous individuals. METHODS: We recruited 113 unrelated index patients with clinically diagnosed cerebral small vessel disease. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. RESULTS: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. The mean age at cognitive impairment onset was 51.1 years. Spondylosis deformans was observed in all cases, whereas alopecia was observed in 3 cases; an autopsied case with p.G283E showed arteriopathy in their cerebral small arteries. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (A252T and V297M) did not inhibit wild-type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer-associated HTRA1 activation. CONCLUSIONS: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.

Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling.

Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases.

Clinicopathological features of sarcoidosis manifesting as generalized chronic myopathy.

Although chronic myopathy has been reported to affect skeletal muscle in sarcoidosis, its clinicopathological features have not been fully elucidated. We characterized the clinical, histopathological, and prognostic features of eleven patients with sarcoidosis manifesting with chronically progressive, generalized myopathy that was confirmed with muscle biopsy. Generalized muscle weakness extending to the four extremities and trunk was the cardinal feature of these cases. Muscle atrophy was evident in nine patients, particularly in the lower limbs, whereas myalgia was reported in only two patients. Myopathy was the first manifestation of sarcoidosis in five patients. Only six patients showed elevated plasma creatine kinase levels. Using skeletal muscle computed tomography, the distribution of muscle atrophy was predominant in the hip adductors, knee flexors and ankle plantarflexors. Radiological assessments, including magnetic resonance imaging, gallium scintigraphy, and fluorodeoxyglucose positron emission tomography-computed tomography imaging, revealed findings suggestive of skeletal muscle inflammation in only half of the patients examined. However, in all patients, muscle biopsy specimens revealed an active inflammatory process, as observed by focal non-caseating epithelioid granuloma with predominant CD4-positive lymphocytic infiltration. Sarcolemmas were diffusely stained with HLA-ABC, HLA-DR and intercellular adhesion molecule-1 antibodies, suggesting diffuse and active antigen presentation. Functional improvement after immunomodulatory treatment was significantly better in patients with short disease durations (p < 0.05). The therapeutic response was poor in patients with long disease durations; thus, early diagnosis and early initiation of treatment are important.

Differentiation of multipotent neural stem cells derived from Rett syndrome patients is biased toward the astrocytic lineage.

BACKGROUND: Rett syndrome (RTT) is one of the most prevalent neurodevelopmental disorders in females, caused by de novo mutations in the X-linked methyl CpG-binding protein 2 gene, MECP2. Although abnormal regulation of neuronal genes due to mutant MeCP2 is thought to induce autistic behavior and impaired development in RTT patients, precise cellular mechanisms underlying the aberrant neural progression remain unclear. RESULTS: Two sets of isogenic pairs of either wild-type or mutant MECP2-expressing human induced pluripotent stem cell (hiPSC) lines were generated from a single pair of 10-year-old RTT-monozygotic (MZ) female twins. Mutant MeCP2-expressing hiPSC lines did not express detectable MeCP2 protein during any stage of differentiation. The lack of MeCP2 reflected altered gene expression patterns in differentiated neural cells rather than in undifferentiated hiPSCs, as assessed by microarray analysis. Furthermore, MeCP2 deficiency in the neural cell lineage increased astrocyte-specific differentiation from multipotent neural stem cells. Additionally, chromatin immunoprecipitation (ChIP) and bisulfite sequencing assays indicated that anomalous glial fibrillary acidic protein gene (GFAP) expression in the MeCP2-negative, differentiated neural cells resulted from the absence of MeCP2 binding to the GFAP gene. CONCLUSIONS: An isogenic RTT-hiPSC model demonstrated that MeCP2 participates in the differentiation of neural cells. Moreover, MeCP2 deficiency triggers perturbation of astrocytic gene expression, yielding accelerated astrocyte formation from RTT-hiPSC-derived neural stem cells. These findings are likely to shed new light on astrocytic abnormalities in RTT, and suggest that astrocytes, which are required for neuronal homeostasis and function, might be a new target of RTT therapy.

Demographic features of Japanese patients with sporadic inclusion body myositis: a single-center referral experience.

OBJECTIVE: The incidence of sporadic inclusion body myositis (sIBM) in the Japanese population has increased, and some researchers have suggested that race and genetic background may influence the clinical features of the disease. The aim of this study was to clarify the demographic features of Japanese patients with sIBM. METHODS: We retrospectively evaluated the demographic features of consecutive patients who were referred to our institution between 1995 and 2011 for diagnostic muscle biopsies and who subsequently were diagnosed to have sIBM. RESULTS: Seventy-three patients comprising 54 men and 19 women received a diagnosis of sIBM during the study period. The patients were divided into two groups based on the date of diagnosis (before and including 2002, and after 2002). The annual number of patients who received a diagnosis of sIBM increased significantly from 3.6±1.6 (mean ± SD) before and including 2002 to 4.9±3.1 (mean ± SD) after 2002 (p<0.05), whereas the annual number of patients who received a diagnosis of polymyositis (PM) or dermatomyositis (DM) remained consistent from 1995 to 2011. The ratio of PM and DM to sIBM was 7.6 during the period from 1995 to 2002 and 5.5 during the period from 2003 to 2011. However, the age-adjusted annual number of patients newly diagnosed with sIBM did not increase significantly after 2002. CONCLUSION: The number of Japanese patients with sIBM appears to have increased in recent years; however, the characteristics of the patients have not changed. Considering the increased size of the elderly population, prolonged lifespans could explain the demographic movement of sIBM in Japan.