論文 - 山口　美樹

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]

　概要を見る

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]