研究者詳細 - 山口　美樹

論文 - 山口　美樹

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A newly developed anti-Mucin 13 monoclonal antibody targets pancreatic ductal adenocarcinoma cells

Yukari Nishii, Miki Yamaguchi, Yasutoshi Kimura, Tadashi Hasegawa, Hiroyuki Aburatani, Hiroaki Uchida, Koichi Hirata, Yuji Sakuma

INTERNATIONAL JOURNAL OF ONCOLOGY ( SPANDIDOS PUBL LTD ) 46 ( 4 ) 1781 - 1787 2015年04月 [査読有り]

Pancreatic cancer is one of the most severe forms of malignancy. Patients with unresectable or metastatic pancreatic cancer usually receive chemotherapy that causes various adverse effects. Antibody-drug conjugates (ADCs), drugs developed by conjugating an anticancer agent to a monoclonal antibody (mAb), can alleviate the side effects of chemotherapy because ADCs selectively bind to cancer cells expressing a particular antigen. We recently developed the recombinant protein DT3C comprising diphtheria toxin (DT) lacking the receptor-binding domain but containing the Cl, C2, and C3 domains of Streptococcus protein G (3C). The mAb-DT3C conjugates can be used to select mAbs that are internalized by cells, because the conjugates decrease cell viability only when they are internalized by cells through Ab-antigen reactions. We developed a new mAb to be internalized by TCC-PAN2 cells, a pancreatic carcinoma cell line. The mAb, designated TCC56, recognized Mucin 13 (MUC13), while TCC56-DT3C conjugates induced cell death in TCC-PAN2 cells expressing MUC13. We found that MUC13 was expressed, at least partially, in all 40 pancreatic ductal carcinoma tissues and adjacent non-cancerous tissues analyzed. The expression levels of MUC13 in pancreatic cancer tissues were greater than those in normal tissues. Our findings suggest that MUC13 can be a target molecule for pancreatic cancer treatment. ADCs, including mAb TCC56, could be promising anticancer agents to alleviate the adverse effects of chemotherapy.

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Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope

Masahiro Ikeda, Miki Yamaguchi, Kazunori Kato, Kiminori Nakamura, Sagano Shiina, Takako Ichikawa-Ando, Hirofumi Misaka, Kensuke Myojo, Kazuyasu Nakamura, Yoshiyuki Sugimoto, Hirofumi Hamada

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 458 ( 4 ) 877 - 882 2015年03月 [査読有り]

TROP-2 is a type I transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1 E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1 E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1 E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1 E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1 E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments. (C) 2015 Elsevier Inc. All rights reserved.

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Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells

Mild Yamaguchi, Yukari Nishii, Kiminori Nakamura, Haruka Aoki, Sachie Hirai, Hiroaki Uchida, Yuji Sakuma, Hirofumi Hamada

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 454 ( 4 ) 600 - 603 2014年11月 [査読有り]

Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the Cl, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs. (C) 2014 Elsevier Inc. All rights reserved.

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Bone Marrow Stromal Cell Line Promotes the Proliferation of Mast Cell Progenitors Derived from Cord Blood CD34(+) Cells under Serum-free Conditions with a Combination of Both Cell-cell Interaction and Soluble Factors

Mitsuhiro Fujihara, Hiroshi Azuma, Hisami Ikeda, Miki Yamaguchi, Hirofumi Hamada

ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY ( INFORMA HEALTHCARE ) 39 ( 2 ) 51 - 58 2011年04月 [査読有り]

A higher production of functional mast cells (MCs) can be generated by co-culturing cord blood-derived CD34(+) cells with a human bone marrow stromal cell line under serum-free conditions supplemented with stem cell factor and IL-6. We addressed the question of whether the higher proliferation of MCs in this co-culture system might be due to the higher proliferation of MC progenitors. The stromal cell line increased the cell numbers of MC progenitors derived from cord blood-derived CD34(+) cells, in a combination of cell-cell interactions between stromal cells and CD34(+) cells, and as yet unidentified soluble factors derived from stromal cells.

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Elevated Ca²+ influx-inducing activity toward mast cells in pretransfusion sera from patients who developed transfusion-related adverse reactions.

Azuma H, Yamaguchi M, Takahashi D, Fujihara M, Sato S, Kato T, Ikeda H

Transfusion. 2009 Aug;49(8):1754-61. ( WILEY-BLACKWELL ) 49 ( 8 ) 1754 - 1761 2009年08月 [査読有り]

BACKGROUND: Type I allergic reactions such as urticaria-like manifestations constitute a large percentage of transfusion-related adverse events. Along with donor factors, patient factors might be involved in these reactions. Sera from some patients with chronic idiopathic urticaria show histamine-releasing activity (HRA). Sera from patients who develop Type I allergic reaction might possess HRA. STUDY DESIGN AND METHODS: Pretransfusion serum samples were collected. Mast cells were cultured from peripheral blood CD34+ cells and mixed with the serum samples. Cells with elevated intracytoplasmic Ca2+ concentrations were monitored using flow cytometry to evaluate Ca2+ influx-inducing activity (CaIA) in serum. The amount of histamine released into the supernatant was measured using an enzyme immunoassay kit to evaluate HRA. In some assays, cells were incubated with pertussis toxin (Ptx). RESULTS: CaIA values were higher (p < 0.05) in sera from patients with reactions (27.68 +/- 20.38 [n = 145]; range, 0.49-84.90) than in sera of patients without reactions (10.70 +/- 6.50 [n = 54]; range, 2.67-36.97) and control sera (9.67 +/- 5.88 [n = 107]; range, 1.11-25.68). No difference in CaIA was found between patients with (27.38 +/- 20.46 [n = 88]) and without (28.38 +/- 20.59 [n = 57]) skin manifestations. However, CaIA was higher (p < 0.05) in patients with pure urticaria (mean, 30.58 +/- 21.3 [n = 30]; range, 70.43-4.1) than in patients with fever alone (mean, 18.61 +/- 14.71 [n = 18]; range, 45.94-3.19). Levels of HRA were higher (p < 0.001) in CaIA-positive sera than in CaIA-negative sera. Both CaIA and HRA were blocked by Ptx. CONCLUSION: Elevated CaIA and HRA in pretransfusion sera might be attributable to adverse reactions, especially to urticaria-like manifestation. The Gi protein-coupled receptor complexes on mast cells and its ligand must be involved.

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Biocompatibility Study of Hemoglobin Vesicles, Cellular-Type Artificial Oxygen Carriers, with Human Umbilical Cord Hematopoietic Stem/Progenitor Cells Using an In Vitro Expansion System

Miki Yamaguchi, Mitsuhiro Fujihara, Shinobu Wakamoto, Hiromi Sakai, Shinji Takeoka, Eishun Tsuchida, Hirofumi Hamada, Hiroshi Azuma, Hisami Ikeda

ASAIO JOURNAL ( LIPPINCOTT WILLIAMS & WILKINS ) 55 ( 3 ) 200 - 205 2009年05月 [査読有り]

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development of a clinically useful transfusion alternative. Our previous in vivo animal studies of massive HbV dosages demonstrated the lack of any suppressive effect on hematopoiesis. Before starting clinical trials, we aimed to examine the details of the biocompatibility of HbVs with human hematopoietic stem/progenitor cells. We investigated the effect of HbVs at a concentration of up to 3 vol/vol (%) on expansion of human umbilical cord (CB) hernatopoietic stem/progenitor cells, using a coculture system of human TERT-transfected bone marrow stromal cells and CD34(+) cells in vitro. The exposure of CB CD34(+) cells to HbVs up to 14 days suppressed the expansion of total cells and the CD34(+) cells themselves, whereas the empty liposomes, that did not contain Hb, had modestly inhibitory effects on the expansion of these cells. As a result, the number of colonies obtained from the expanded CD34(+) cells was inhibited by the exposure to HbVs. In contrast, exposure to HbVs for 3 days had no effect on the expansion of CD34(+) cells and only slightly decreased the number of total cells. Our in vitro experimental condition does not fully recreate the physiological condition, and the effect of the direct contact of HbV would be magnified because of the absence of shielding by the vasculature and the lack of the reticuloendothelial system and blood stream. However, the present data raise some concern regarding hematopoiesis, and one has to pay attention to this in future human clinical trials. ASAIO Journal 2009; 55:200-205.

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Influence of hemoglobin vesicles, cellular-type artificial oxygen carriers, on human umbilical cord blood hematopoietic progenitor cells in vitro

Miki Yamaguchi, Mitsuhiro Fujihara, Shinobu Wakamoto, Hiromi Sakai, Shinji Takeoka, Eishun Tsuchida, Hiroshi Azuma, Hisami Ikeda

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A ( WILEY-LISS ) 88A ( 1 ) 34 - 42 2009年01月 [査読有り]

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development as clinically useful blood substitutes. Although HbVs are shown to distribute transiently into the bone marrow in animal models, the influence of HbVs on human hematopoietic stem/progenitor cells has not yet been studies. Therefore, we investigated the influence of HbVs at a concentration of up to 3 vol/vol % on the clonogenic activity (in semisolid culture) and proliferative activity (in liquid culture) of human hematopoietic progenitor cells derived from umbilical cord blood (CB) in vitro. Continuous exposure of CB mononuclear cells to HbVs tended to decrease the number and size of mature-committed colonies and most notably reduced the number of colonies of high-proliferative potential colony-forming cells (HPP-CFC). In contrast, exposure to HbVs for 20 h or 3 days, which is more relevant to the clinical setting, had no effect on the number of mature-committed colonies and only modestly decreased the number of HPP-CFC. Continuous exposure (10 days) to HbVs significanly suppressed the cellular proliferation and differentiation of both the erythroid and myeloid lineasges in liquid culture. Again, short exposure (20 h or 3 days) did not affect these parameters. Thus, our results show that HbVs, under conditions relevant to the clinical setting, have no adverse effect on human CB hematopoietic, progenitor activity in vitro. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 88A: 34-42, 2009

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Generation of a considerable number of functional mast cells with a high basal level of Fc epsilon RI expression from cord blood CD34(+) cells by co-culturing them with bone marrow stromal cell line under serum-free conditions

M. Yamaguchi, H. Azuma, M. Fujihara, H. Hamada, H. Ikeda

SCANDINAVIAN JOURNAL OF IMMUNOLOGY ( BLACKWELL PUBLISHING ) 65 ( 6 ) 581 - 588 2007年06月 [査読有り]

The number of mast cells (MC) that can be obtained from tissue is limited, making it difficult to study the role of MC. Cultured MC derived from cord blood (CB)-CD34(+) cells proliferate well compared with those derived from adult CD34(+) cells; however, they have been reported to be phenotypically or functionally immature regardless of culture system. For example, very few cells express Fc epsilon RI. To resolve this problem, we addressed the effect of human bone marrow stromal cell line on the development of cultured MC. CB-CD34(+) (1 x 10(4)) cells were cultured for 8 weeks in a serum-free medium containing rhIL-6 and rhSCF with or without a human bone marrow stromal cell line, namely, co-culture and liquid culture, and were compared in various regards. MC were basically determined by metachromatic staining of granules. The number of MC obtained (60.3 +/- 15.8 x 10(5) versus 2.0 +/- 1.0 x 10(5)), percentage of Fc epsilon RI+ cells (29.3 +/- 9.4% versus 1.9 +/- 0.8%), histamine content (9.7 +/- 2.8 pg/cell versus 5.8 +/- 2.3 pg/cell), and IgE-mediated histamine release (46 +/- 10% versus 17 +/- 7%) were higher (P < 0.01 and P < 0.05) in the co-culture than in the liquid culture. When CB-CD34(+) cells were developed in liquid culture with the co-culture supernatant (CM), a significant increase (P < 0.01) in the percentage of Fc epsilon RI+ cells and in cell number was observed but these values were lower than those of co-cultured MC. We concluded that this co-culture system was useful for obtaining a considerable number of mature MC with a high basal level of functional Fc epsilon RI expression from CB-CD34(+) cells. Yet unknown humoral factors in CM may partly mediate this effect.

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Generation of inflammatory cytokines and chemokines from peripheral blood mononuclear cells by HLA Class II antibody-containing plasma unit that was associated with severe nonhemolytic transfusion reactions

Hisako Sakagawa, Toru Miyazaki, Mitsuhiro Fujihara, Shinichiro Sato, Miki Yamaguchi, Kanji Fukai, Masanobu Morioka, Toshiaki Kato, Hiroshi Azuma, Hisami Ikeda

TRANSFUSION ( BLACKWELL PUBLISHING ) 47 ( 1 ) 154 - 161 2007年01月 [査読有り]

BACKGROUND: HLA Class II antibodies are thought to be involved in severe transfusion reactions including transfusion-related acute lung injury (TRALI). The activation of monocytes by HLA Class II antibody may play an important role in the etiology of TRALI. CASE REPORT: An 81-year-old man with non-Hodgkin's lymphoma (Clinical Stage IIIA) received a plateletpheresis unit containing at least 4 x 10(11) platelets because of thrombocytopenia and a bleeding tendency. Approximately 30 minutes after the start of transfusion, he developed chills, tachycardia, dyspnea, lumber, and abdominal pain and then a fever (40.3 degrees C). His SaO(2) dropped to 70 percent. The transfusion was discontinued immediately. His symptoms disappeared after treatment with oxygen and the administration of corticosteroid and aminophyrine. A chest X-ray showed no sign of pulmonary edema. RESULTS: The donor serum sample had HLA-DR antibodies against multiple DR antigens including DR13, the recipient's HLA-DR type. The cross-match between the patient's lymphocytes and the donor serum was positive. The treatment of peripheral blood mononuclear cells from healthy subjects bearing DR13 antigen with the donor plasma caused the secretion of inflammatory cytokines (i.e., interleukin [IL]-1 beta, IL-6, and tumor necrosis factor-alpha) and neutrophil-activating chemokines (i.e., IL-8 and CXCL1/GRO-alpha) in a cognate antigen-antibody relationship. In addition, the secretion of inflammatory cytokines appeared to require the involvement of CD32 and/or CD16. CONCLUSION: HLA-DR antibodies, detected in this case, had biologic functions to induce production of not only inflammatory cytokines but also neutrophil-attractant chemokines in vitro, which could contribute to the etiology of severe nonhemolytic transfusion reactions.

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Effects of hemoglobin vesicles, a liposomal artificial oxygen carrier, on hematological responses, complement and anaphylactic reactions in rats

Hideki Abe, Hiroshi Azuma, Miki Yamaguchi, Mitsuhiro Fujihara, Hisami Ikeda, Hiromi Sakai, Shinji Takeoka, Eishun Tsuchida

ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY ( TAYLOR & FRANCIS INC ) 35 ( 2 ) 157 - 172 2007年 [査読有り]

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4(+)/CD8(+) T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

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Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

S Wakamoto, M Fujihara, H Abe, M Yamaguchi, H Azuma, H Ikeda, S Takeoka, E Tsuchida

ARTIFICIAL CELLS BLOOD SUBSTITUTES AND BIOTECHNOLOGY ( TAYLOR & FRANCIS INC ) 33 ( 2 ) 101 - 111 2005年 [査読有り]

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alpha(IIb)beta(3) on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

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Cytotoxic difference of T cells expanded with anti-CD3 monoclonal antibody in the presence and absence of anti-CD28 monoclonal antibody

Y Yamada-Ohnishi, H Azuma, N Urushibara, M Yamaguchi, M Fujihara, T Kobata, H Ikeda

STEM CELLS AND DEVELOPMENT ( MARY ANN LIEBERT INC PUBL ) 13 ( 3 ) 315 - 322 2004年06月 [査読有り]

Bulk T cells can be expanded by CD3 stimulation alone (CD3-Ts) or by CD3/CD28 dual stimulation (CD3/CD28-Ts) of peripheral blood mononuclear cells (PBMC). However, few reports have described the difference of features between CD3-Ts and CD3/CD28-Ts. PBMC were stimulated with anti-CD3 monoclonal antibody (mAb) alone or co-stimulated with anti-CD3/CD28 mAbs immobilized on plastic plates, in the presence of rhIL-2 for 4 days, subsequently cultured in the presence of rhIL-2 with no antibody then analyzed. The expansion rate was significantly lower for CD3-Ts (965 +/- 510-fold, n = 5) than CD3/CD28-Ts (2263 + 856-fold, n = 5) (p < 0.05). The CD4/CD8 ratio, the percentage of CD28(+) cell, and the percentage of T cells with no ability to generate intracytoplasmic interleukin-4 (IL-4) or interferon-γ (IFN-γ) were all significantly higher, but, phenotypically, memory cells were lower in CD3/CD28-Ts than in CD3-Ts. The levels of activity of both natural killer (NK) and lymphocyte-activated killer (LAK) cells were lower in CD3/CD28-Ts than CD3-Ts. In comparison to CD3-Ts, CD3/CD28-Ts showed impaired migration toward RANTES. In conclusion, T cells expanded with anti-CD3 and anti-CD28 mAbs differ from those expanded with anti-CD3 alone with proliferation, cytotoxicity, chemotaxis, and phenotype. These differences may exert profound influences on the therapeutic potential of output cells.

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Thrombopoietin upregulates nucleolin mRNA and protein in thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.

Ito T, Fujihara M, Oda A, Wakamoto S, Yamaguchi M, Komatsu N, Miyazaki H, Azuma H, Ikeda H, Ikebuchi K

Mol Cell Biochem. 2003 May;247(1-2):75-82. 247 ( 1-2 ) 75 - 82 2003年05月 [査読有り]

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Possible contribution of apoptosis-inducing factor (AIF) and reactive oxygen species (ROS) to UVB-induced caspaseindependent cell death in the T cell line Jurkat

H Murahashi, H Azuma, N Zamzami, KJ Furuya, K Ikebuchi, M Yamaguchi, Y Yamada, N Sato, M Fujihara, G Kroemer, H Ikeda

JOURNAL OF LEUKOCYTE BIOLOGY ( FEDERATION AMER SOC EXP BIOL ) 73 ( 3 ) 399 - 406 2003年03月 [査読有り]

We analyzed the mechanism of UVB-induced cell death using the Jurkat T cell line. Apoptosis was assessed by measuring phosphatidylserine (PS) externalization, caspase activity, the decrease in mitochondrial membrane potential (DeltaPsim), nucleosomal DNA fragmentation, and morphological changes such as chromatin condensation. The mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF) was evaluated by confocal laser microscopy. The cell death pattern of UVB-irradiated cells was similar to the Fas-induced cell death pattern. However, zVAD-fmk inhibited the nucleosomal fragmentation of DNA but not the externalization of PS, decrease in DeltaPsim, or mitochondrio-nuclear translocation of AIF. N-acetyl L-cysteine significantly inhibited the translocation of AIF induced by UVB. These results suggested that caspase-dependent and -independent pathways were involved in UVB-induced cell death in Jurkat cells, and the mitochondrio-nuclear translocation of AIF was associated with the latter pathway. In addition, reactive oxygen species generated by UVB might be involved in inducing the mitochondrio-nuclear translocation of AIF. J. Leukoc. Biol. 73:399-406; 2003.

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Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion

M Yamaguchi, F Hirayama, S Wakamoto, M Fujihara, H Murahashi, N Sato, K Ikebuchi, K Sawada, T Koike, M Kuwabara, H Azuma, H Ikeda

TRANSFUSION ( AMER ASSOC BLOOD BANKS ) 42 ( 7 ) 921 - 927 2002年07月 [査読有り]

BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum-free medium. However, the stromal cells were prepared using long-term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum-based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum-based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse-reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined. RESULTS: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods. CONCLUSION: An ex vivo expansion system completely based on AB serum has been established.

Functional evaluation of ex vivo expanded cord blood lymphocytes: Possible use for adoptive cellular immunotherapy

H Azuma, Y Yamada, N Shibuya-Fujiwary, M Yamaguchi, H Murahashi, M Fujihara, N Sato, K Fukazawa, K Ikebuchi, H Ikeda

EXPERIMENTAL HEMATOLOGY ( ELSEVIER SCIENCE INC ) 30 ( 4 ) 346 - 351 2002年04月 [査読有り]

Objective. To examine the possibility of adoptive cellular immunotherapy such as donor lymphocyte infusion using ex vivo expanded cord blood (CBL) lymphocytes, the potential expansion ability of CBL lymphocytes and the function of expanded CBL lymphocytes were evaluated. Materials and Methods. Mononuclear cell fractions derived from CBL or peripheral blood (PBL) were placed in anti-CD3 monoclonal antibody-coated flasks and cultured in the presence of recombinant human interleukin-2 for 4 days. Cells then were transferred to noncoated flasks and cultured for another 2 weeks. On day 14, polyclonality, cell surface markers, killer activity, and intracelIular cytokine profiles were evaluated. Results. Cells were polyclonally expanded. The differences in cumulative fold expansion on day 14 between CBL [1174 +/- 637 (292-1939), n = 6] and PBL [ 1247 +/- 568 (517-2328), n = 9] were not significant (p = 0.95). Phenotypic patterns of both expanded CBL and PBL were similar. CD4/CD8 ratio of expanded CBL appeared to remain greater than 1 on day 8. In contrast, that of expanded PBL became less than 1. In both cases, approximately 20% of Cells had the CD3(+)CD8(+)CD56(+) phenotype. At an effector to target ratio (E/T) of 40:1, the natural killer activity of expanded CBL (64.5% +/- 10.8%, n = 9) was significantly higher than that of expanded PBL (48.3% +/- 16.8%, n = 9) (p < 0.01, Mann-Whitney U-test). However, there was no significant difference in lymphokine-activated killer activity between expanded CBL (45.3% +/- 25.2%, n = 7) and expanded PBL (67.2% +/-12.3%, n = 7). Interferon-gamma-producing cells were dominant in both cases. Conclusions. It was feasible to achieve approximately 1000-fold expansion of CBL, and the phenotype and function of expanded CBLs were essentially equivalent to those of expanded PBL. This suggested that ex vivo expanded CBL may be applied to adoptive cellular immunotherapy such as donor lymphocyte infusion. (C) 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

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Ex vivo expansion of human UC blood primitive hematopoietic progenitors and transplantable stem cells using human primary BM stromal cells and human AB serum

M Yamaguchi, F Hirayama, H Murahashi, H Azuma, N Sato, H Miyazaki, K Fukazawa, K Sawada, T Koike, M Kuwabara, H Ikeda, K Ikebuchi

CYTOTHERAPY ( TAYLOR & FRANCIS AS ) 4 ( 2 ) 109 - 118 2002年03月 [査読有り]

Background In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications, and investigators have been using xenogeneic, especially murine, stromal cells for stem-cell expansion. In addition, many such culture systems utilize FCS-containing medium or serum-free medium that contains human- or animal-derived proteins. However, the possible transmission of infectious diseases has led to a debate about the safety of the delivery of grafts expanded in culture using cells and proteins of allogeneic or xenogeneic origin. Using primary human BM stromal cells, we have established an AB serum-based co-culture system to expand human primitive progenitors and transplantable stem cells. Methods Cord blood CD34(+) cells were cultured on a monolayer of human BM-derived primary stromal cells with thrombopoietin (TPO), stem-cell factor (SCF) and flt3/flk2 ligand (FL) in the presence of either FCS or AB serum. One to three weeks later, cells were examined for total cells, CD34(+) cells, CD34(+) CD38(-) cells, and clonogenic progenitors. SCID mouse reconstituting cell (SRC) activity was also studied. Results Three weeks of culture with TPO, SCF, and FL supported more than a 250-fold expansion of CD34(+) cells, CD34(+) CD38(-) cells and CFU-C, regardless of the kind of serum used. SRC assay revealed that transplantable stem cells were moderately expanded as well. Discussion This ex vivo expansion system should prove valuable in clinical settings in which stromal cells and serum are available from recipients or stem-cell donors.

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Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU-E derived from peripheral blood CD34+cells

H Sugawara, R Motokawa, H Abe, M Yamaguchi, Y Yamada-Ohnishi, J Hirayama, H Sakata, S Sato, N Kamo, K Ikebuchi, H Ikeda

TRANSFUSION ( AMER ASSOC BLOOD BANKS ) 41 ( 4 ) 456 - 461 2001年04月 [査読有り]

BACKGROUND: Nonenveloped and thermostable viruses such as parvovirus B19 (B19) can be transmitted to patients who are receiving plasma-derived coagulation factor concentrates treated by the S/D method for inactivating enveloped viruses. Therefore, it is important to develop and validate new methods for the inactivation of nonenveloped viruses, STUDY DESIGN AND METHODS: Suspensions of B19 in coagulation factor concentrates (FVIII) were irradiated with UVC light. B19 infectivity was determined by an indirect immunofluorescence assay using CFU-E, as a host cell, derived from peripheral blood CD34+ cells. The effects of catechins on B19 infectivity and on FVIII activity after UVC illumination were also examined. RESULTS: The indirect immunofluorescence assay estimated the B19 infectivity of samples containing virus copies of 10(5) to 10(11) per 10 muL to be a median tissue culture-infectious dose of 10(0.3) to 10(5.4) per 10 muL B19 was inactivated by 3 log at 750 J per m(2) of UVC radiation and was undetectable after 1000 or 2000 J per m(2) of irradiation. However, FVIII activity decreased to 55 to 60 percent of pretreatment activity after 2000 J per m(2) of UVC radiation. This was inhibited in the presence of rutin or catechins. Epigallocatechin gallate could maintain FVIII activity at almost 100 percent of pretreatment activity after 2000 J per m(2) of UVC radiation, while B19 infectivity was decreased to undetectable levels, which resulted in >3.9 log inactivation. CONCLUSION: UVC radiation in the presence of catechins, especially epigallocatechin gallate, appears to be an effective method of increasing the viral safety of FVIII concentrates without the loss of coagulation activity.

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Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells.

Yamaguchi M, Hirayama F, Kanai M, Sato N, Fukazawa K, Yamashita K, Sawada K, Koike T, Kuwabara M, Ikeda H, Ikebuchi K

Exp Hematol. 2001 Feb;29(2):174-82. 29 ( 2 ) 174 - 182 2001年02月 [査読有り]

DOI

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucylphenylalanine-induced responses of polymorphonuclear neutrophils in vitro

T Ito, M Fujihara, H Abe, M Yamaguchi, S Wakamoto, S Takeoka, H Sakai, E Tsuchida, H Ikeda, K Ikebuchi

ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY ( MARCEL DEKKER INC ) 29 ( 6 ) 427 - 437 2001年 [査読有り]

[Poly(ethylene-lycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymorphonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-MV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus. these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.

DOI

Comparison of sensitivity to ultraviolet B irradiation between human lymphocytes and hematopoietic stem cells.

Azuma H, Ikebuchi K, Yamaguchi M, Murahashi H, Mogi Y, Sato N, Fujihara M, Hirayama F, Ikeda H

Blood. 2000 Oct 1;96(7):2632-4. 96 ( 7 ) 2632 - 2634 2000年10月 [査読有り]

Stromal cell-independent differentiation of human cord blood CD34(+)CD38(-) lymphohematopoietic progenitors toward B cell lineage

Y Yoshikawa, F Hirayama, M Kanai, S Nakajo, J Ohkawara, M Fujihara, M Yamaguchi, N Sato, M Kasai, S Sekiguchi, K Ikebuchi

LEUKEMIA ( STOCKTON PRESS ) 14 ( 4 ) 727 - 734 2000年04月 [査読有り]

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34(+)CD38(-) cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), flt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19(+)IgM(+) cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL-3 and SCF remarkably enhanced the primary colony formation and the generation of CD19(+) cells in the secondary B-lymphoid culture.

A clonal culture assay for human cord blood lymphohematopoietic progenitors

Y Yoshikawa, K Ikebuchi, J Ohkawara, F Hirayama, M Yamaguchi, N Sato, KJ Mori, M Kasai, S Sekiguchi

HUMAN IMMUNOLOGY ( ELSEVIER SCIENCE INC ) 60 ( 1 ) 75 - 82 1999年01月 [査読有り]

We describe a two-step clonal culture assay system for human lymphohematopoietic progenitors present in umbilical cord blood which are capable of differentiation along both myeloid and B-lymphoid lineages. Human cord blood CD34(+) cells were plated in methylcellulose in the presence of stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-7, and the murine stroma cell line, MS-5. The growing primary colonies were individually examined for their potentials to differentiate along both myeloid and B-lymphoid lineages by reculturing aliquots of the primary colonies in methylcellulose culture containing IL-3, G-CSF and erythropoietin (Epo), and on a monolayer of MS-5 in the presence of SCF and G-CSF. Approximately 10-15% of the primary colonies generated various combinations of myeloid cells and CD19(+) sIgM(+) cells. Subsequent studies using micromanipulated single CD34(+) cells unequivocally demonstrated the clonal origin of the lymphohematopoietic progenitors. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of human lymphohematopoiesis. (C) American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.

DOI

Role of physiologic concentrations of stem cell factor in leukemic type growth of myelodysplastic CD34+ cells.

Sawada K, Koizumi K, Tarumi T, Takano H, Ieko M, Nishio M, Fukada Y, Yasukouchi T, Yamaguchi M, Koike T

Leuk Res. 23 ( 1 ) 1 - 11 1999年01月 [査読有り]

DOI

Large scale purification of human blood CD34(+) cells using a nylon-fiber syringe system and immunomagnetic microspheres

K Koizumi, K Sawada, N Sato, M Yamaguchi, M Nishio, T Tarumi, H Takano, Y Fukada, M Ieko, T Yasukouchi, S Sekiguchi, T Koike

CYTOTHERAPY ( ISIS MEDICAL MEDIA LTD ) 1 ( 4 ) 319 - 327 1999年 [査読有り]

Background: To establish an available, economical technique for the large-scale purification of CD34(+) cells we used a nylon-fiber syringe (NF-S) for depletion of adherent cells and then selected CD34(+) cells from peripheral blood mobilized by G-CSF, using MAb and magnetic beads. Methods: PBSC were mobilized and collected from adult, healthy volunteers. With the effect of concentration of anti-CD34 MAb (9C5) on the purification of CD34(+) cells from 1 x 10(8) NF-S treated cells (NF cells), the recovery and the purity of CD34(+) cells was identical for 5, 10, 20, 40 mu g of 9C5. In this study, therefore, the concentration of 9C5 was maintained at >5 mu g/10(8) NF cells, with a cellbead ratio of 1:10. Results: When half to one-third of leukapheresis product containing 12.5 +/- 26.0 x 10(8) mononuclear cells (mean +/- SD; n = 6) was processed for CD34(+) cell purification, 5.26 +/- 3.01 x 10(7) total purified cells were obtained with a purity of CD34(+) cells at 94.9 +/- 8.5%. The numbers of CD34(+) cells and progenitor cells recovered in this process were 4.71 +/- 2.33 x 10(7) cells and 145.9 +/- 121.8 x 10(5) cells, respectively. The total recovery of CD34(+) cells was 37.0 +/- 21.0%. The depletion of monocyte by NF-S reduced the 9C5 anti-human CD34 MAb needed for purification of CD34(-) cells to one-third. Two patients were grafted with peripheral blood CD34(+) cells selected by this procedure and achieved a rapid and consistent hematopoiesis. Discussion: Our clinical data show that these cells are capable of rapid reconstitution of hematopoiesis after high-dose chemotherapy. The procedure contains an additional step; however, consistent high purity of CD34(+) cells in the purified population and cost reduction were achieved, both critical issues in the better purging of malignant cells and for a wide clinical application.

In vitro expansion of CD34(+)CD41(-) cells from human peripheral blood CD34(+)CD41(-) cells: Role of cytokines for in vitro proliferation and differentiation of megakaryocytic progenitors

K Koizumi, K Sawada, M Yamaguchi, A Notoya, T Tarumi, H Takano, Y Fukada, M Nishio, E Katagiri, T Yasukouchi, N Sato, S Sekiguchi, T Koike

EXPERIMENTAL HEMATOLOGY ( CARDEN JENNINGS PUBL CO LTD ) 26 ( 12 ) 1140 - 1147 1998年11月 [査読有り]

The aim of this study is to clarify the transitional change of the proliferation and differentiation of human peripheral blood CD34(+) cells to megakaryocytic lineage, focusing on its clinical application. We developed a rapid system to purify human peripheral blood CD34(+) cells from healthy volunteers, which produced CD34(+) cells with a 90% purity. The purified CD34(+) cells predominantly consisted of CD41(-) cells, and the rate of coexpression of CD41 was 0.6% +/- 0.5%. When the purified cells were cultured in liquid phase for 10 days in the presence of recombinant human stem cell factor (rSCF: a ligand for c-kit), interleukin-3 (rIL-3), and thrombopoietin (rTPO: a ligand for Mpl), the number of CD34(+)/CD41(+) cells increased to 19% +/- 7% of total expanded cells on day 4 (4 days of liquid culture) and then gradually decreased to 2.2% +/- 0.6% on day 10. The absolute number of CD34(+)/CD41(+) cells increased and reached a plateau on day 6, and 1.7 +/- 0.6 x 10(5) CD34(+)/CD41(+) cells were produced by 1x10(5) CD34(+)/CD41(-) day 0 cells. The CD34(-)/CD41(+) cells appeared on day 6, continuously increased in number until day 10, and constituted the main population of expanded cells on day 10, with a value of 38% +/- 18%. On day 10, 19.5 +/- 10.6 x 10(5) of CD34(-)/CD41(+) cells were produced by 1x10(5) CD34(+)/CD41(-) day 0 cells. The deletion of rTPO from this cytokine combination decreased the number of CD34(+)/CD41(+) and CD34(-)/CD41(+) cells, after days 6 and 8, respectively. Day 0 cells required rIL-3 for promoting colonies containing megakaryocytes, whereas rTPO alone promoted almost no megakaryocytic colonies from day 0 cells. Thus, a combination of IL-3 and SCF expands CD34(+)/CD41(+) cells from CD34(+)/CD41(-) cells, and TPO mainly acts to increase CD34(-)/CD41(+) cells. This study suggests that if the expansion of CD34(+)/CD41(+) is performed in vitro, the 6 days' culture of peripheral blood CD34(+)/CD41(-) cells with a combination of IL-3 and SCF with TPO provides the most rapid and stable products of CD34(+)/CD41(+) cells for the rapid recovery of platelets in patients with peripheral blood stem cell transplantation.

Effects of liposome-encapsulated hemoglobin on phorbol ester-induced superoxide production and expression of costimulatory molecules by monocytes in vitro.

Fujihara M, Ikebuchi K, Yamaguchi M, Abe H, Niwa K, Sekiguchi S

1998 Nov;26(5-6):487-95. 25 ( 5-6 ) 487 - 495 1998年11月 [査読有り]

Different adhesive characteristics and VLA-4 expression of CD34(+) progenitors in G(0)/G(1) versus S+G(2)/M phases of the cell cycle

M Yamaguchi, K Ikebuchi, F Hirayama, N Sato, Y Mogi, J Ohkawara, Y Yoshikawa, K Sawada, T Koike, S Sekiguchi

BLOOD ( W B SAUNDERS CO ) 92 ( 3 ) 842 - 848 1998年08月 [査読有り]

We identified the cell cycle status of CD34(+) cells of steady-state hone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G(2)/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G(2)/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G(0)/G(1) phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody, Taken together, these results suggest that CD34(+) progenitors in G(0)/G(1) phase of the cell cycle differ from those in S+G(2)/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment. (C) 1998 by The American Society of Hematology.

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors.

Ohkawara JI, Ikebuchi K, Fujihara M, Sato N, Hirayama F, Yamaguchi M, Mori KJ, Sekiguchi S.i

Leukemia. 1998 May;12(5):764-71. 12 ( 5 ) 764 - 771 1998年05月 [査読有り]

Inflammatory cytokine production in whole blood modified by liposome-encapsulated hemoglobin

K Niwa, K Ikebuchi, M Fujihara, H Abe, S Wakamoto, T Ito, M Yamaguchi, S Sekiguchi

ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY ( MARCEL DEKKER INC ) 26 ( 5-6 ) 559 - 570 1998年 [査読有り]

The effect of Neo Red Cells (NRC), liposome-encapsulated hemoglobin, on production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) were studied in whole blood preparations ex vivo. Venous blood was collected with heparin and incubated in a CO2 incubator. Treatment of blood samples with NRC reduced the constitutive levels of TNF-alpha and IL-6. Lipopolysaccharide (LPS) treatment for 24 h increased production of TNF-alpha and IL-6 in a dose-dependent manner. Pretreatment with NRC (5%) for 24 h markedly potentiated the I,PS-induced TNF-alpha production and, that of IL-6 to a lesser extent. Northern bloting analysis of total RNA in whole blood showed that pretreatment with NRC caused a marked increase in TNF-alpha mRNA expression in response to LPS. It is concluded that NRC potentiates LPS-induced TNF-alpha and IL-6 production in whole blood ex vivo, and that the potentiating effect of NRC on LPS-induced TNF-alpha production can be attributed, at least in part, to an increase in its mRNA expression.

Platelet-like particle production from cultured human megakaryocytic cell line

K Ikebuchi, M Yamaguchi, K Niwa, H Abe, M Fujihara, S Sekiguchi

ARTIFICIAL CELLS BLOOD SUBSTITUTES AND IMMOBILIZATION BIOTECHNOLOGY ( MARCEL DEKKER INC ) 26 ( 5-6 ) 549 - 558 1998年 [査読有り]

The human megakaryocytic cell line CMK was positive for the expression of platelet glycoprotein(GP) IIb/IIIa but few cells were positive for GPIb. To establish a GPIb-positive CMK subclone, GPIb-positive CMK cells were purified, micromanipulated and cloned, but resulting sublines were all negative for GPIb expression. The combined presence of interleukin-3 (IL-3) and the anti-virus agent ribavirin strongly induced expression of GPIb on the cells. However, the megakaryocyte growth and development factor (MGDF)/thrombopoietin (TPO) failed to enhance GPIb expression. CMK cells can produce platelet-like particles during culture, but both cells themselves and particles are negative for GPIb expression. To utilize these particles as platelet substitutes, further appropriate culture systems are necessary.

A rapid nylon-fiber syringe system to deplete CD14(+) cells for positive selection of human blood CD34(+) cells. Use of immunomagnetic microspheres

M Yamaguchi, K Sawada, N Sato, K Koizumi, S Sekiguchi, T Koike

BONE MARROW TRANSPLANTATION ( STOCKTON PRESS ) 19 ( 4 ) 373 - 379 1997年02月 [査読有り]

To achieve a rapid and an efficient purification of CD34(+) cells, we devised a nylon-fiber syringe (NF-S) and we manipulated it to deplete adherent cells from normal human blood mononuclear cells (MN cells), The cells processed by NF-S were further purified as the CD34(+) fraction, using CD34 monoclonal antibody, immuno-magnetic microspheres and chymopapain treatment to detach the microspheres, When steady-state human peripheral blood MN cells were processed by NF-S at 24 degrees C for 5 min, the frequency of monocytes (CD14(+) cells) significantly decreased from 22.0+/-5.2% to 2.5+/-0.4%, with a 73% recovery of CD34(+) cells, The subsequent immunomagnetic positive selection achieved preparations of 91+/-8% pure CD34(+) cells with a 86+/-23% yield, The overall yield of CD34(+) cells was 44+/-11%, and the time required for all procedures was 5 h. There was a tight and an inverse correlation (P<0.0001) between the frequency of CD14(+) cells in the initial cell population and the purity of CD34(+) cells in the final preparation, A recommended frequency of CD14(+) cells for achieving preparations of over 90% pure CD34(+) cells was less than 4.4%. When combining NF-S and immunomagnetic microspheres, efficient bench-top separation of CD34(+) cells in steady-state peripheral blood can be done in any laboratory, and fluorescence-activated cell-sorting is not required.

Role of cytokines in leukemic type growth of myelodysplastic CD34(+) cells

K Sawada, M Ieko, A Notoya, T Tarumi, K Koizumi, S Kitayama, H Nishio, Y Fukada, T Yasukouchi, M Yamaguchi, S Katoh, T Koike

BLOOD ( W B SAUNDERS CO ) 88 ( 1 ) 319 - 327 1996年07月 [査読有り]

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34(+) cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34(+) cells). The purified CD34(+) cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34(+) cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34(+) cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34(+) cells and that G-CSF, GM-CSF, and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34(+) cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF. (C) 1996 by The American Society of Hematology.

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors.

Sawada K, Sato N, Notoya A, Tarumi T, Yasukouchi T, Yamaguchi M, Kohno M, Mikuni C, Koike T

Eur J Haematol. 54 ( 2 ) 85 - 94 1995年02月 [査読有り]

PROLIFERATION AND DIFFERENTIATION OF MYELODYSPLASTIC CD34(+) CELLS - PHENOTYPIC SUBPOPULATIONS OF MARROW CD34(+) CELLS

K SAWADA, N SATO, A NOTOYA, T TARUMI, S HIRAYAMA, H TAKANO, K KOIZUMI, T YASUKOUCHI, M YAMAGUCHI, T KOIKE

BLOOD ( W B SAUNDERS CO ) 85 ( 1 ) 194 - 202 1995年01月 [査読有り]

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34(+) cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immuno-magnetic microspheres (MDS CD34(+) cells). Phenotypic subpopulations of these CD34(+) cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34(+) cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34(+) cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34(+) cells did not significantly differ from that of normal CD34(+) cells. The tota I number of colonies and clusters grown from 100 normal marrow CD34(+) cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34(+) cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34(+) cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34(+) cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage. (C) 1995 by The American Society of Hematology.

TRANSITIONAL CHANGES IN IMMUNOPHENOTYPIC SUBPOPULATIONS OF HUMAN PERIPHERAL-BLOOD CD34(+) CELLS EXPANDED IN-VITRO

K KOIZUMI, K SAWADA, N SATO, S HIRAYAMA, T YASUKOUCHI, M YAMAGUCHI, TA TAKAHASHI, S SEKIGUCHI, T KOIKE

EXPERIMENTAL HEMATOLOGY ( CARDEN JENNINGS PUBL CO LTD ) 22 ( 12 ) 1171 - 1178 1994年11月 [査読有り]

We determined the appropriate incubation period to expand human peripheral blood (PB) CD34(+) cells for clinical application and the role of recombinant human (rh) interleukin-3 (rhIL-3) in the expansion and differentiation of these cells. The cells were purified up to 40 +/- 16% and transitional changes in immunophenotypic subpopulations in suspension culture were examined following stimulation with three different combinations of rh colony-stimulating factors (rhCSFs): 1) rhIL-3 alone, 2) rhIL-6, rhSCF, rhG-CSF, plus rhGM-CSF, and 3) the four CSFs plus rhIL-3. With all three CSF combinations, the total cells increased continuously after day 5 until day 14, and a combination of the five CSFs always gave rise to the highest number of total cells. Immunophenotypic analysis of the expanded cells showed that with all three CSF combinations CD34(+) cells reached a maximal rate on day 5 and then decreased in an inverse correlation between the logarithm of CD34 positive rate and the duration of suspension culture. The maximum expansion of CD34(+) cells and PB progenitor cells (PBPC) with rhIL-3 alone, the four CSFs, or the five CSFs was observed on day 5, 10, or 7, respectively. The combination of the five CSFs was identi lied as the most potent stimulus for expansion of PBPC and CD34(+) cells, as it increased non-erythroid PBPC 89 +/- 69-foId, with a range of 24 to 204-fold on day 7. However, differences in the expansion rate of these cells on days 5, 7, and 10 were not statistically significant. The majority of purified CD34(+) cells coexpressed CD38 (91 +/- 3%) but were negative for CD33 (85 +/- 4%), and one-half coexpressed CD13. With all three combinations of CSFs, the mature CD34(+) cells that coexpressed CD38, CD33, or CD13 expanded in parallel with the total CD34(+) cells, while an increase in relatively immature CD34(+) cells, which do not express CD38, CD33, or CD13, was only statistically significant with the five CSFs. Thus, rhIL-3 played a critical role when combined with the four CSFs by increasing both mature and immature CD34(+) cells.

IN-VITRO EXPANSION OF HUMAN PERIPHERAL-BLOOD CD34+ CELLS

N SATO, K SAWADA, K KOIZUMI, T TARUMI, M IEKO, T YASUKOUCHI, M YAMAGUCHI, TA TAKAHASHI, S SEKIGUCHI, T KOIKE

BLOOD ( W B SAUNDERS CO ) 82 ( 12 ) 3600 - 3609 1993年12月 [査読有り]

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: response to individual colony-stimulating factors.

Sawada K, Sato N, Tarumi T, Sakai N, Koizumi K, Sakurama S, Ieko M, Yasukouchi T, Koyanagawa Y, Yamaguchi M

83 ( 3 ) 348 - 358 1993年03月 [査読有り]

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