Publications (14)63.09 Total impact
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Article: Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions.
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ABSTRACT: 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 1.3×10(6)±0.3×10(6) 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.PLoS ONE 01/2013; 8(4):e59243. · 4.09 Impact Factor -
Article: Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery.
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ABSTRACT: Chronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1-driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.Blood 06/2012; 120(6):1299-308. · 9.90 Impact Factor -
Article: Clinical profile and genetic basis of Wiskott-Aldrich syndrome at Chandigarh, North India.
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ABSTRACT: The Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency disorder characterized by thrombocytopenia with small sized platelets, eczema, and recurrent infections. There is paucity of information on WAS from the Indian subcontinent. We describe the clinical and molecular profile of 8 patients with WAS as seen in the Pediatric Immunodeficiency Clinic at the Advanced Pediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India. A detailed analysis of the clinical profiles, investigations and outcome of the 8 children diagnosed with WAS during the period 2006- 2010 was performed. Confirmation of the genetic diagnosis was done at the Service d'Hématologie, d'Immunologie et de Cytogénétique, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France and the National Defense Medical College, Saitama, Japan. 8 patients were diagnosed as WAS in 5 years. The ages at diagnosis ranged from 13 weeks to 9 years while the mean age of onset of the symptoms was 117 days +/- 136 days. The diagnosis was established within a mean period of 31 months (ranging 1-108 months) from the onset of symptoms. Recurrent infections and diarrhea were seen in 6 and 7 out of the 8 patients, respectively, while eczema was variable. Autoimmunity manifestations were observed in 2 children. Thrombocytopenia and small platelet size was the hallmark of the disease and the main clinical clue to diagnosis in our patients. Mutations in the WASP gene were seen in 8 children, out of which 2 were novel mutations. While one child successfully underwent bone marrow transplantation, two children are doing well on immunoglobulin replacement and cotrimoxazole prophylaxis. Out of 8 children 4 children in our cohort died--all had high WAS scores and could not be offered hematopoietic stem cell transplantation. WAS should be suspected clinically in any male infant with persistent unexplained thrombocytopenia and especially if the platelet size is small. Clinical presentation can be very variable and it is therefore important to recognize the entire spectrum of the disease. Understanding the molecular basis has important implications for the diagnosis, treatment, and genetic counseling of patients with WAS.Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 03/2012; 30(1):71-8. · 0.65 Impact Factor -
Article: A Dual Reporter Splicing Assay Using HaloTag-containing Proteins.
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ABSTRACT: To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5'-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6.Current Chemical Genomics 01/2012; 6:27-37. -
Article: Novel mutations of MVK gene in Japanese family members affected with hyperimmunoglobulinemia D and periodic fever syndrome.
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ABSTRACT: Hyperimmunoglobulinemia D with periodic fever syndrome (HIDS) is a recessively inherited recurrent fever syndrome. We describe a family of eldest son and monozygotic twin younger sisters with characteristic syndrome of HIDS, but normal level of IgD. Mevalonate kinase (MK) activity was deficient in all of them, and analysis of the MVK gene revealed compound heterozygosity for 2 new mutations, one of which was the disease-causing splicing mutation and the other was a novel missense mutation. All the patients had the same compound heterozygous mutations c.227-1 G > A and c.833 T > C, which resulted in exon 4 skipping and p.Val278Ala. This is the first case in which exon skipping mutation of the MVK gene has been certainly identified at the genomic DNA level. In each case, in which HIDS is clinically suspected, despite normal IgD level, analysis of MK activity and the MVK gene should be performed.Rheumatology International 12/2011; · 1.88 Impact Factor -
Article: High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study.
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ABSTRACT: 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. 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. 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. 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.Arthritis & Rheumatism 06/2011; 63(11):3625-32. · 7.87 Impact Factor -
Article: Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein.
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ABSTRACT: Familial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4-deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.Blood 06/2011; 118(5):1225-30. · 9.90 Impact Factor -
Article: Quantification of κ-deleting recombination excision circles in Guthrie cards for the identification of early B-cell maturation defects.
The Journal of allergy and clinical immunology 03/2011; 128(1):223-225.e2. · 9.17 Impact Factor -
Article: A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.
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ABSTRACT: Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.PLoS ONE 01/2011; 6(7):e22261. · 4.09 Impact Factor -
Article: A case of familial Mediterranean fever associated with compound heterozygosity for the pyrin variant L110P-E148Q/M680I in Japan.
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ABSTRACT: Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent and self-limited fever attacks and serositis/arthritis. The M694V, M694I, M680I, V726A, and E148Q mutations in MEFV, the gene responsible for FMF, account for most FMF cases in Mediterranean populations. In Japan, M694I and E148Q are most frequently detected; M694V, M680I, and V726A have not been identified so far. We report the first case of FMF associated with M680I in Japan.Modern Rheumatology 12/2009; 20(2):193-5. · 1.58 Impact Factor -
Article: Hemophagocytosis after bone marrow transplantation for JAK3-deficient severe combined immunodeficiency.
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ABSTRACT: HSCT is the optimal treatment for patients with SCID. In particular, HSCT from a HLA-identical donor gives rise to successful engraftment with long survival. We report a six-month-old girl with JAK3-deficient SCID who developed hemophagocytosis after BMT without conditioning from her HLA-identical father. She had suffered from pneumonia and hepatitis before BMT. Prophylaxis for GVHD was short-term methotrexate and tacrolimus. On day 18 after BMT, the patient developed hemophagocytosis in bone marrow when donor lymphocytes were increasing in peripheral blood. Analysis of chimerism confirmed host origin of macrophages and donor origin of lymphocytes. Thus, host macrophage activation was presumably induced in response to donor lymphocytes through immunoreaction to infections and/or alloantigens. HSCT for SCID necessitates caution with respect to hemophagocytosis.Pediatric Transplantation 08/2009; 14(8):E105-9. · 1.48 Impact Factor -
Article: A case of a long-time survivor with chronic active Epstein-Barr virus infection.
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ABSTRACT: Epstein-Barr virus (EBV) is associated with hypersensitivity to mosquito bites (HMB) and fatal EBV-associated hemophagocytic syndrome (HPS). The prognosis of patients with chronic active EBV infection (CAEBV) is very poor. We report a rare case of an adult woman patient with a 28-yr history of HMB, who developed EBV-HPS. EBV genome was detected in the serum and peripheral blood lymphocytes. Clonal proliferation of EBV was demonstrated by Southern blot analysis using an EBV genome terminal-repeat probe. This is a very rare case of a long-term survivor with CAEBV. The patient was initially treated with immunochemotherapy and achieved complete remission. However, the patient immediately relapsed and underwent allogeneic bone marrow transplantation (BMT) from her HLA-matched brother. Peripheral blood cell recovered well, and EBV genome disappeared from the peripheral blood. Allogeneic BMT may be effective in eradicating EBV-HPS. Unfortunately, the patient died of graft vs. host disease on the 92nd day after BMT.European Journal Of Haematology 02/2004; 72(1):73-6. · 2.61 Impact Factor -
Article: Engraftment and dissemination of T lymphocytes from primary haemophagocytic lymphohistiocytosis in scid mice.
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ABSTRACT: Although primary haemophagocytic lymphohistiocytosis (HLH) is a genetic disorder of T lymphocytes, it remains unclear why T lymphocytes of primary HLH patients preferentially infiltrate the central nervous system and peripheral blood, in addition to the reticuloendothelial systems. We engrafted Herpesvirus saimiri (HVS)-immortalized T-lymphocyte lines established from primary HLH patients into severe combined immunodeficient (scid) mice and examined their capacity to infiltrate mouse organs. A diffuse infiltration of human T lymphocytes was detected in each organ of scid mice treated with 1 x 10(6) T lymphocytes from all four primary HLH patients assessed, whereas no infiltration of T lymphocytes from healthy individuals was observed in any organ. The infiltration of T lymphocytes was mainly observed in the lung, brain and peripheral blood, in association with haemophagocytosis. These cells were positive for HLA-DR, CD3 and either CD8 or CD4, but negative for CD68. Certain markers of proliferation and apoptotic activities were highly positive in these cells. There was no difference between the infiltration pattern of T lymphocytes of primary HLH patients with a perforin deficiency and those without. By Southern blot analysis, T lymphocytes infiltrating mouse organs were observed to be polyclonal. These findings suggest that our murine model implementing HVS-immortalized human T lymphocytes is suitable to clarify the pathogenesis of primary HLH.British Journal of Haematology 05/2003; 121(2):349-58. · 4.94 Impact Factor -
Article: Engraftment and dissemination of T lymphocytes from primary haemophagocytic lymphohistiocytosis in scid mice
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ABSTRACT: Although primary haemophagocytic lymphohistiocytosis (HLH) is a genetic disorder of T lymphocytes, it remains unclear why T lymphocytes of primary HLH patients preferentially infiltrate the central nervous system and peripheral blood, in addition to the reticuloendothelial systems. We engrafted Herpesvirus saimiri (HVS)-immortalized T-lymphocyte lines established from primary HLH patients into severe combined immunodeficient (scid) mice and examined their capacity to infiltrate mouse organs. A diffuse infiltration of human T lymphocytes was detected in each organ of scid mice treated with 1 × 106 T lymphocytes from all four primary HLH patients assessed, whereas no infiltration of T lymphocytes from healthy individuals was observed in any organ. The infiltration of T lymphocytes was mainly observed in the lung, brain and peripheral blood, in association with haemophagocytosis. These cells were positive for HLA-DR, CD3 and either CD8 or CD4, but negative for CD68. Certain markers of proliferation and apoptotic activities were highly positive in these cells. There was no difference between the infiltration pattern of T lymphocytes of primary HLH patients with a perforin deficiency and those without. By Southern blot analysis, T lymphocytes infiltrating mouse organs were observed to be polyclonal. These findings suggest that our murine model implementing HVS-immortalized human T lymphocytes is suitable to clarify the pathogenesis of primary HLH.British Journal of Haematology 04/2003; 121(2):349 - 358. · 4.94 Impact Factor
Top co-authors
Top Journals
Institutions
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2011–2013
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Kyoto University
- Center for iPS Cell Research and Application (CiRA)
Kyoto, Kyoto-fu, Japan
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2009–2012
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RIKEN
- Laboratory for Immunogenetics
Wako, Saitama-ken, Japan
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