Masatoshi Nishizawa

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

11C-Methionine PET/CT for multiple myeloma

A 57-year-old woman with 6-year history of IgA-k myeloma experienced an increase of serum IgA level from 331 to 683 mg/dl during maintenance therapy with thalidomide after autologous peripheral blood stem cell transplantation followed by allogeneic mini-transplantation. Bone marrow aspiration revealed no sign of relapse of the myeloma. We conducted positron emission tomography/computed tomography (PET/CT) scans using C-methionine (MET) and F-fluorodeoxyglucose (FDG). In MET-PET/CT, there were multiple abnormal hypermetabolic lesions, while FDG uptake in these lesions was faint (Fig. 1). After four courses of bortezomib therapy, MET-PET/CT revealed no abnormal uptake of MET, with decreased serum IgA level (25.3 mg/dl), indicating that MET uptake was correlated with the clinical course, as denoted by IgA level. C-Methionine is a radiolabelled PET tracer that is clinically used for brain tumor. An earlier study reported that MET-PET depicted active myeloma clearly, which might reflect the increased metabolism of amino acids in myeloma cells for producing abundant immunoglobulin [1]. In the present case, MET-PET/CT was very useful for determining the precise localization of the myelomatous lesions and evaluating therapeutic effects. Although FDGPET/CT is reported to have higher sensitivity for localized myelomatous lesions than other imaging modalities, our case suggests the possibility that MET-PET/CT detects myelomatous lesions more clearly than FDG-PET/CT. Considering that in myeloma patients higher FDG uptake or a larger number of FDG-avid lesions is reported to be associated with inferior overall survival and event-free survival, the lower FDG uptake in this patient is possibly related to the slowly progressive nature of her myeloma. In addition, in 30% of myeloma patients, FDG-PET/CT reportedly failed to show the abnormal findings in the spine and pelvis; this may account for the lower FDG uptake in these lesions. Although many imaging modalities, including FDGPET/CT are now widely available, the findings of the present case suggest that MET-PET/CT can provide valuable information for patients with myeloma and has a potential to become an important imaging test. However, further investigation to compare the MET-PET/CT with FDG-PET/CT or other imaging modalities are needed to confirm the diagnostic efficiency and the clinical feasibility of MET-PET/CT for multiple myeloma.

A male with primary breast lymphoma

A 64-year-old man was referred to our hospital with a history of painless, gradually growing lump in his left breast. On physical examination, nontender, irregular surface, demarcated elastic firm tumor (6 cm 3 7 cm in size) was palpable in the medial area of his left breast (Image 1A). Fine needle aspiration cytology of the tumor cells showed that lymphocytic cells with prominent nucleoli were dispersed as single cells (Image 1B). Microscopic examination of the tumor with core needle biopsy (CNB) confirmed diffuse proliferation of CD20-positive large atypical lymphocytes (Image 1C,D). A magnetic resonance imaging (MRI) examination showed a lobulated, well-demarcated breast tumor of high-intensity in T2-weighted fat-saturated image (T2WI F-SAT, Image 1E) and of isointensity in T1-weighted image (T1WI) to the adjacent muscles (Image 1F). The breast tumor was diagnosed with non-Hodgkin lymphoma, classified as diffuse large B-cell lymphoma. Blood examination was normal including serum lactate dehydrogenase and soluble interleukin-2 receptor. There was no evidence of lymphoma involvement in bone marrow and cerebrospinal fluid. A [F]-fluorodeoxyglusoce (FDG) positron emission tomography/computed tomography (PET/ Image 1. (A) Appearance of breast lump (blue arrows). Histological findings of the breast tumors, (B) Papanicolou stain shows dispersed lymphocytic cells with prominent nucleoli, (C) HE stain shows diffuse proliferation of large atypical lymphocytes, and (D) the atypical lymphocytes are positive for CD20, a B-cell marker, confirmed by the immunohistochemical analysis. (E–J) Radiological findings of the breast tumors. A lobulated, well-demarcated, high-intensity tumor in T2-weighted fat-saturated image (T2WI F-SAT) of MRI before the treatment (E, blue arrow) is disappeared after six cycles of chemotherapy (J). (F) The breast tumor with iso-intensity to the adjacent muscle in T1-weighted image (T1WI) of MRI before the treatment (blue arrow). A PET/CT demonstrates increased focal FDG uptake in the breast tumor before treatment (G and H, blue arrow) whereas no FDG uptake is observed after the chemotherapy (I).

Neurolymphomatosis as a manifestation of relapsed primary cardiac lymphoma

A 68-year-old Japanese woman was admitted to our hospital because of congestive heart failure and sinus node dysfunction. F-Fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) revealed a cardiac tumor involving right atrium of the heart (Fig. 1a, b), and the tumor was diagnosed as primary cardiac diffuse large B cell lymphoma by open chest biopsy. The immunophenotype of the lymphoma cells was CD3, CD5, CD10, CD19, CD20, CD22, smIgM, and smIg-j. The

Adult-onset autoimmune neutropenia with antineutrophil antibodies to an unknown neutrophil-specific antigen analyzed by using five cell-lineage immunofluorescence test and reactivity against cell lines expressing human neutrophil antigens

Autoimmune neutropenia (AIN) is a rare autoimmune disorder characterized by the presence of antineutrophil antibodies in the patient’s blood, resulting in increased neutrophil destruction [1–5]. Herein, we report the case of a patient with adult-onset AIN harboring antineutrophil antibodies to an unknown neutrophil-specific antigen (or antigens). These antibodies were analyzed by using two newly developed assay methods: five cell-lineage immunofluorescence test (IFT) [6] and reactivity against a panel of cell lines expressing particular human neutrophil antigens (HNAs), named KY-cell lines [7, 8]. An 80-year-old Japanese man with a 4-year history of prostate cancer was referred to our hospital because of neutropenia. Three-and-a-half years earlier, his white blood cell and neutrophil counts were within the normal range; thereafter, the white blood cell count gradually decreased. Although the gonadotropin-releasing hormone analog he had been receiving for limiting the prostate cancer was discontinued, his neutropenia did not resolve. He was asymptomatic, in good general health, had no history of infection and autoimmune disorder, and had never received cytotoxic drugs. Blood tests revealed severe neutropenia (white blood count of 1880/lL, with an absolute neutrophil count of 230/lL), a normal hemoglobin level, and a normal platelet count. Tests for autoantibodies, including anti-Ro/ SSA antibody, antinuclear antibody, proteinase-3-antineutrophil cytoplasmic antibody (ANCA), and myeloperoxidase-ANCA, were negative, indicating the absence of Sjögren’s syndrome, systemic lupus erythematosus, or an ANCA-associated disorder. A bone marrow smear showed normocellular marrow without abnormal or dysplastic cells, but a markedly decreased number of myelocytes and more mature myeloid cells than myelocytes, suggesting maturation arrest at the promyelocyte stage. Myeloblasts and promyelocytes, megakaryocytes, lymphocytes, and erythroid cells were normal. We analyzed the patient’s serum for the presence of antineutrophil antibodies by using the five cell-lineage IFT [6], a modified granulocyte IFT (G-IFT) in which antibodies against neutrophils, monocytes, T cells, B cells, and platelets from healthy volunteers can be detected simultaneously with low background interference (Fig. 1). The HNA-1 allotypes of the volunteers were previously determined. We found immunoglobulin (Ig)G and IgM class antineutrophil antibodies in his serum. The antibodies reacted in a similar manner with HNA-1a-homozygous neutrophils, HNA-1a/HNA-1b-heterozygous neutrophils, and HNA-1b-homozygous neutrophils and did not react with the other cell lineages. Consequently, the diagnosis of AIN was confirmed. To elucidate the specificity of the antibodies, we examined their reactivity against gene-transfected panel cell lines (termed KY-cell lines) selectively expressing the molecules belonging to the HNA system (HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, and -5b) except HNA-3a, the gene of which was not identified until very recently, by flow cytometry [7, 8]. These antibodies reacted with none of the cell lines (data not shown), and their specificity was not identifiable. M. Nishizawa N. Sugino M. Tsudo Department of Hematology, Osaka Red Cross Hospital, Osaka, Japan