Objective Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease. Although heterozygous germline gain-of-function NLRP3 mutations are a known cause of this disease, conventional genetic analyses fail to detect disease-causing mutations in ~40% of patients. Since somatic NLRP3 mosaicism has been detected in several mutation-negative NOMID/CINCA syndrome patients, we undertook this study to determine the precise contribution of somatic NLRP3 mosaicism to the etiology of NOMID/CINCA syndrome. Methods An international case–control study was performed to detect somatic NLRP3 mosaicism in NOMID/CINCA syndrome patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID/CINCA syndrome patients and their healthy relatives. Clinical features were analyzed to identify potential genotype–phenotype associations. Results Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18 of 26 patients versus 0 of 19 relatives; P < 0.0001). In vitro functional assays indicated that the detected somatic NLRP3 mutations had disease-causing functional effects. No differences in NLRP3 mosaicism were detected between different cell lineages. Among nondescript clinical features, a lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurologic symptoms. Conclusion Somatic NLRP3 mutations were identified in 69.2% of patients with mutation-negative NOMID/CINCA syndrome. This indicates that somatic NLRP3 mosaicism is a major cause of NOMID/CINCA syndrome.

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70–80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.

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The discovery of somatic cell nuclear transfer proved that somatic cells can carry the same genetic code as the zygote, and that activating parts of this code are sufficient to reprogram the cell to an early developmental state. The discovery of induced pluripotent stem cells (iPSCs) nearly half a century later provided a molecular mechanism for the reprogramming. The initial creation of iPSCs was accomplished by the ectopic expression of four specific genes (OCT4, KLF4, SOX2, and c-Myc; OSKM). iPSCs have since been acquired from a wide range of cell types and a wide range of species, suggesting a universal molecular mechanism. Furthermore, cells have been reprogrammed to iPSCs using a myriad of methods, although OSKM remains the gold standard. The sources for iPSCs are abundant compared with those for other pluripotent stem cells; thus the use of iPSCs to model the development of tissues, organs, and other systems of the body is increasing. iPSCs also, through the reprogramming of patient samples, are being used to model diseases. Moreover, in the 10 years since the first report, human iPSCs are already the basis for new cell therapies and drug discovery that have reached clinical application. In this review, we examine the generation of iPSCs and their application to disease and development.

Objective. Blau syndrome and its sporadic counterpart, early-onset sarcoidosis (EOS), share a phenotype featuring the symptom triad of skin rash, arthritis, and uveitis. This systemic inflammatory granulomatosis is associated with mutations in the NOD2 gene. The aim of this study was to describe the clinical manifestations of Blau syndrome/EOS in Japanese patients and to determine whether the NOD2 genotype and its associated basal NF-B activity predict the Blau syndrome/ EOS clinical phenotype.Methods. Twenty Japanese patients with Blau syndrome/EOS and NOD2 mutations were recruited. Mutated NOD2 was categorized based on its basal NF-B activity, which was defined as the ratio of NF-B activity without a NOD2 ligand, muramyldipeptide, to NF-B activity with muramyldipeptide.Results. All 9 mutations, including E383G, a novel mutation that was identified in 20 patients with Blau syndrome/EOS, were detected in the centrally located NOD region and were associated with ligandindependent NF-B activation. The median age of the patients at disease onset was 14 months, although in 2 patients in Blau syndrome families (with mutations R334W and E383G, respectively) the age at onset was 5 years or older. Most patients with Blau syndrome/EOS had the triad of skin, joint, and ocular symptoms, the onset of which was in this order. Clinical manifestations varied even among familial cases and patients with the same mutations. There was no clear relationship between the clinical phenotype and basal NF-B activity due to mutated NOD2. However, when attention was focused on the 2 most frequent mutations, R334W and R334Q, R334W tended to cause more obvious visual impairment.

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×106±0.3×106 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

Urticarial rash observed in cryopyrin-associated periodic syndrome (CAPS) caused by nucleotide-binding oligomerization domain–leucine-rich repeats containing pyrin domain 3 (NLRP3) mutations is effectively suppressed by anti–interleukin (IL)-1 treatment, suggesting a pathophysiological role of IL-1β in the skin. However, the cellular mechanisms regulating IL-1β production in the skin of CAPS patients remain unclear. We identified mast cells (MCs) as the main cell population responsible for IL-1β production in the skin of CAPS patients. Unlike normal MCs that required stimulation with proinflammatory stimuli for IL-1β production, resident MCs from CAPS patients constitutively produced IL-1β. Primary MCs expressed inflammasome components and secreted IL-1β via NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain when stimulated with microbial stimuli known to activate caspase-1. Furthermore, MCs expressing disease-associated but not wild-type NLRP3 secreted IL-1β and induced neutrophil migration and vascular leakage, the histological hallmarks of urticarial rash, when transplanted into mouse skin. Our findings implicate MCs as IL-1β producers in the skin and mediators of histamine-independent urticaria through the NLRP3 inflammasome.

Cryopyrin-associated periodic syndrome (CAPS) is a spectrum of systemic autoinflammatory disorders in which the majority of patients have mutations in the coldinduced autoinflammatory syndrome (CIAS)1 gene. Despite having indistinguishable clinical features, some patients lack CIAS1 mutations by conventional nucleotide sequencing. We recently reported a CAPS patient with mosaicism of mutant CIAS1, and raised the possibility that CIAS1 mutations were overlooked in "mutation-negative" patients, due to a low frequency of mosaicism. To determine whether there were latent mutant cells in "mutation-negative" patients, we sought to identify mutation-associated biologic phenotypes of patients' monocytes. We found that lipopolysaccharide selectively induced necrosis-like cell death in monocytes bearing CIAS1 mutations. Monocyte death correlated with CIAS1 up-regulation, was dependent on cathepsin B, and was independent of caspase-1. Cell death was intrinsic to CIAS1-mutated monocytes, was not mediated by the inflammatory milieu, and was independent of disease severity or anti-IL-1 therapy. By collecting dying monocytes after lipopolysaccharide treatment, we succeeded in enriching CIAS1-mutant monocytes and identifying low-level CIAS1-mosaicism in 3 of 4 "mutationnegative" CAPS patients. Our findings reveal a novel effect of CIAS1 mutations in promoting necrosis-like cell death, and demonstrate that CIAS1 mosaicism plays an important role in mutation-negative CAPS patients. (Blood. 2008;111: 2132-2141)