T. Morishima et al. 20haematologica | 2014; 99(1) tion system from human iPS cells 17 as well as a serum-and feeder-free monolayer hematopoietic culture system from human ES and iPS cells. 18 In this study, we generate iPS cell lines from an SCN patient with HAX1 gene deficiency and differentiate them into neutrophils in vitro. Furthermore, we corrected for the HAX1 gene deficiency in HAX1-iPS cells by lentiviral transduction with HAX1 cDNA and analyzed the neutrophil differentiation potential of these cells. Thus, this in vitro neutrophil differentiation system from patient-derived iPS cells may be a useful model for future studies in SCN patients with HAX1 gene deficiency. Methods Human iPS cell generationSkin biopsy specimens were obtained from an 11-year old male SCN patient with HAX1 gene deficiency. 19 This study was approved by the Ethics Committee of Kyoto University, and informed consent was obtained from the patient's guardians in accordance with the Declaration of Helsinki. Fibroblasts were expanded in DMEM (Nacalai Tesque, Inc., Kyoto, Japan) containing 10% FBS (vol/vol, Invitrogen, Carlsbad, CA, USA) and 0.5% penicillin and streptomycin (wt/vol, Invitrogen). Generation of iPS cells was performed as described previously. 12 In brief, we introduced OCT3/4, SOX2, KLF4, and cMYC using ecotropic retroviral transduction into patient's fibroblasts expressing mouse Slc7a1. Six days after transduction, cells were harvested and re-plated onto mitotically inactive SNL feeder cells. On the following day, DMEM was replaced with primate ES cell medium (ReproCELL, Kanagawa, Japan) supplemented with basic fibroblast growth factor (5 ng/mL, R&D Systems, Minneapolis, MN, USA). Three weeks later, individual colonies were isolated and expanded. Maintenance of cellsControl ES (KhES-1) and control iPS (253G4 and 201B6) cells were kindly provided by Drs. Norio Nakatsuji and Shinya Yamanaka (Kyoto University, Kyoto, Japan), respectively. These human ES and iPS cell lines were maintained on mitomycin-C (Kyowa Hakko Kirin, Tokyo, Japan)-treated SNL feeder cells as described previously 17 and subcultured onto new SNL feeder cells every seven days. Flow cytometric analysisCells were stained with antibodies as reported previously. 17Samples were analyzed using an LSR flow cytometer and Cell Quest software (Becton-Dickinson). Neutrophil differentiation of iPS cellsIn a previous study, we established a serum and feeder-free monolayer hematopoietic culture system from human ES and iPS cells. 18 In this study, we modified this culture system to direct neutrophil differentiation. iPS cell colonies were cultured on growth factor-reduced Matrigel (Becton-Dickinson)-coated cell culture dishes in Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich, St. Louis, MO, USA) containing the insulin-transferrin-selenium (ITS) supplement (Invitrogen) and cytokines. iPS cells were treated with cytokines as follows: bone morphogenetic protein (BMP) 4 (20 ng/mL, R&D Systems) was added for four days and then replaced with vas...

Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are T cell subpopulations that possess innate-like properties. We examined the impact of post-hematopoietic stem cell transplantation (HSCT) MAIT and iNKT cell recovery on the clinical outcomes of 69 patients who underwent allogeneic HSCT at Kyoto University Hospital. Multivariate analyses identified the absolute number of MAIT cells (< 0.48/μL on day 60 post-HSCT) as the sole independent risk factor for grade I-IV and grade II-IV acute graft-versus-host disease (aGVHD) among patients who underwent bone marrow transplantation; no correlation was observed between post-HSCT iNKT cell recovery and the development of aGVHD. Six of the 15 patients in the MAIT (≥ 0.48/μL) group developed aGVHD, five within the first 30 days post HSCT. In contrast, 13 of the 15 patients in the MAIT (< 0.48/μL) group developed aGVHD, seven after day 30 post HSCT. The overall survival of the MAIT group was slightly shorter than that of the MAIT group. Thus, the post-HSCT recovery of MAIT cells is closely related to the development of delayed onset aGVHD and the outcome of post-HSCT, suggesting its utility for identifying a subset of patients that requires more prolonged and/or intense GVHD prophylaxis.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

Nasal mucociliary clearance was measured before and after treatment in patients with chronic sinusitis. Nasal mucociliary transit time before the study was greater than 36 min in 8 out of 14 patients who were treated with S-carboxymethylcysteine, and in 9 out of 22 patients who were treated by repeated antral lavage. The nasal mucociliary clearance was significantly improved by both treatment regimens. This may indicate that the malfunction of the nasal mucociliary system is not the cause of chronic sinusitis but an effect of chronic inflammation of the respiratory mucosa.

LT has contributed to an elevation in cure rates for patients with unresectable HB; however, patients with recurrent HB after LT have poor prognosis. To analyze the prognostic and therapeutic factors that influence the clinical outcome of patients with HB receiving LT, we retrospectively analyzed 24 patients with HB who underwent LT between 1997 and 2015. The 5-year OS rate of all patients was 69.6±9.7%. The 5-year OS rate of 11 patients receiving salvage LT for recurrent tumor after a primary resection was comparable to that of 13 patients receiving primary LT. Among 12 evaluable patients receiving primary LT, six of 10 patients with a decline of serum AFP >95% at LT are currently alive and in remission, whereas two patients with a decline of AFP ≤95% experienced post-LT relapse. Among 9 evaluable patients receiving salvage LT, all three patients with any decline of AFP at LT are currently alive in remission, and three of six patients with no response to pre-LT salvage chemotherapy are also alive and in remission. Response to chemotherapy may be a reliable marker for prediction of post-LT relapse, even for patients receiving salvage LT.