2000 Fiscal Year Annual Research Report

臍帯血造血幹細胞制御の分子機構の解明とその無血清体外大量培養法への応用

Research Project

Project/Area Number	10470174
Research Institution	The University of Tokyo
Principal Investigator	辻浩一郎東京大学, 医科学研究所, 助教授 (50179991)
Keywords	臍帯血造血幹細胞 / 体外増幅 / stem cell factor / Flk2 / Flt3 ligand / thrombopoietin / interleukin-6 / Soluble IL-6 receptor
Research Abstract	(目的)昨年度我々は、stem cell factor(SCF)、Flk2/Flt3 ligand(FL)、thrombopoietin(TPO)、interleukin(IL)-6/soluble IL-6 receptor複合体(IL-6/sIL-6R)を組合せにより培養することにより、臍帯血造血幹細胞は著明に増幅することを示した。本年度は、より効率的に増幅するための臍帯血造血幹細胞の純化法を検討した。そこで我々は、FLの受容体であるFlk2/Flt3に注目した。 (方法)(1)臍帯血中のCD34陽性細胞におけるFlk2/Flt3の発現をフローサイトメトリーで解析した。(2)臍帯血中のCD34+Flk2/Flt3+細胞とCD34+Flk2/Flt3-細胞を各々FACSにてソーティングした後、NOD/SCIDマウスに移植し、その長期造血再構築能を検討した。(3)臍帯血中のLineage(Lin)-CD34+CD38-Flk2/Flt3+細胞を単一細胞に分離し、SCF+FL+IL-6/sIL-6R+TPO存在下で液体培養を行った。培養細胞を培養1、2週後にNOD/SCIDマウスに移植し、ヒト造血再構築能を検討した。 (結果)(1)Flk2/Flt3は臍帯血中のCD34+細胞の約80%に発現されており、特に未分化細胞亜分画であるCD34+CD33-細胞、CD34+CD38-細胞中にもFlk2/Flk3発現細胞が認められた。(2)蛍光活性化細胞分離装置を用いて、CD34+Flk2/Flt3+細胞、CD34+Flk2/Flt3-細胞をFACSにて分画し、各々1×10^4個の細胞を4匹のNOD/SCIDマウスに移植したところ、CD34+Flk2/Flt3-細胞を移植されたレシピエントでは生着は認められなかったが、CD34+Flk2/Flt3+細胞を移植されたレシピエントでは、4匹とも生着が認められた。このことより、ヒト造血幹細胞はFlk2/Flt3を発現していると考えられた。(3)Lin-CD34+CD38-Flk2/Flt3+細胞を単細胞分離し、一個のLin-CD34+CD38-Flk2/Flt3+細胞を、FL、SCF、TPO、IL-6/sIL-6R存在下で培養したところ、61%(34/56)に増殖を認めた。培養1、2週後に増幅した細胞をNOD/SCIDマウスに移植したところ、移植後14-15週目の解析で、37.5%(6/16)でヒト造血再構築能を確認できた。以上の結果より、Flk2/Flt3を発現する造血幹細胞は単細胞レベルでも増幅が可能であることが示唆された。 (結論)結語長期造血再構築能を有するヒト造血幹細胞はFlk2/Flt3を発現しており、Lin-CD34+CD38-Flk2/Flt3+造血幹細胞はより効率的な体外増幅の標的細胞として有用である可能性が示唆された。臍帯血CD34+細胞を、種々のサイトカインの組合せで培養した後、培養中の造血前駆細胞や造血幹細胞の数を、クローナル培養法やNOD/SCIDマウス移植系を用いて検討した。

Research Products
(37 results)

All Other

All Publications (37 results)

[Publications] Ueda T.: "Expansion of human NOD/SCID-repopulating cells by a combination of stem cell factor, Flk2/Flt3 ligand,thrombopoietin and a complex of interleukin-6 and soluble interleukin-6 receptor."J.Clin.Invest.. 105. 1013-1021 (2000)
[Publications] Hayashi Y.: "SN-1, a novel leukemic cell line with t(11;16)(q23;p13): myeloid characteristics and resistance to retinoids and vitamin D3."Cancer.Res.. 60. 1139-1145 (2000)
[Publications] Ebihara Y.: "Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34^+ cells."Br.J.Haematol.. 109. 153-161 (2000)
[Publications] Ueda T.: "Hematopoietic repopulating ability of cord blood CD34^+ cells in NOD/Shi-scid mice."Stem Cells. 18. 204-213 (2000)
[Publications] Ueda T.: "Massive pericardial and pleural effusion with an asarca following allogeneic bone marrow transplantation."Int.J.Hematol.. 71. 394-397 (2000)
[Publications] Sato T.: "Erythroid progenitors differentiate and mature in response to endogenous erythropoietin."J.Clin.Invest.. 106. 263-270 (2000)
[Publications] Ishii T.: "Improvement in bronchiolitis obliterans organizing pneumonia by combination of oral predonisolone and low dose erythromycin in a child after allogegeneic bone marrow transplantation."Bone Marrow Transplant. 26. 907-910 (2000)
[Publications] Sugiyama H.: "Peroxisome proliferator-activated receptors are expressed in mouse bone marrow-derived mast cells."FEBS Lett.. 467. 259-262 (2000)
[Publications] Yoshino H.: "Natural killer cell depletion by anti-asialo GM1 antiserum treatment enhances human hematopoietic stem cell engraftment in NOD/Shi-scid mice."Bone Marrow Transplant.. 26. 1211-1216 (2000)
[Publications] Takaki S.: "Control of B cell production by the adaptor protein Lnk, a negative regulator of tyrosine kinase signaling."Immunity. 13. 599-609 (2000)
[Publications] Matsui A.: "gp130 and c-kit signals synergistically induce thrombopoietin production by hematopoietic cells."Int.J.Hematol.. 72. 455-462 (2001)
[Publications] Tsuji K.: "Expression of G-CSF receptor on myeloid progenitors."Leukemia and Lymphoma. (in press).
[Publications] Matsuoka S.: "CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages."Blood. 97. 419-425 (2001)
[Publications] Ma F.: "Cytokine requirement for the development of T-lineage potential from lymphohematopoietic progenitor cells."Br.J.Haematol.. (in press).
[Publications] Sugiyama H.: "Peroxisome proliferator-activated receptors are expressed in human cultured mast cells : a possible role of these receptors in negative regulation of mast cell activation."Eur.J.Immunol.. (in press).
[Publications] Xu M.: "Evidence for the presence of primitive megakaryopoiesis in the early yolk sac."Blood. (in press).
[Publications] Ueda T.: "Hematopoietic capability of cord blood CD34^+ cells : a comparison with that of adult bone marrow CD34^+ cells."Int.J.Hoematol.. (in press).
[Publications] Yoshimasu T.: "Prompt and durable hematopoietic reconstitution by an unrelated cord blood transplantation in a child with Fanconi anemia."Bone Marrow Transplant.. (in press).
[Publications] Miyamoto K.: "Inhibitory effect of interleukin-3 on the early development of human B-lymphopoiesis."Br.J.Haematol.. (in press).
[Publications] Matsuoka S.: "Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleura by AGM region-derived stromal cells."Blood. (in press).
[Publications] Hisakawa H.: "Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) stimulates primitive and definitive erythropoiesis in mouse embryo expressing hGM-CSF receptors but not erythropoietin receptors."Blood. (in press).
[Publications] Ma F.: "Development of human lymphohematopoietic stem/progenitor cells defined by CD34 and CD81 expressions."Blood. (in press).
[Publications] 石井武文: "急性リンパ性白血病治療後期に発症したL-アスパラギナーゼによる壊死性膵炎"日小血会誌. 14. 328-332 (2000)
[Publications] 岩本彰太郎: "大量rhG-CSF療法中にmonosomy7を伴うMDSに移行したKostmann症候群"日小血会誌. 14. 338-345 (2000)
[Publications] 三井哲夫: "造血幹細胞移植における支持療法と今後の課題"Biotherapy. 14. 292-300 (2000)
[Publications] 辻浩一郎: "増幅造血幹細胞移植ガイドライン"分子細胞療法. 1. 197-201 (2000)
[Publications] 海老原康博: "造血幹細胞の同定検査 2)コロニー法"検査と技術. 28. 971-972 (2000)
[Publications] 辻浩一郎: "造血幹細胞の起源と性状"医学のあゆみ. 194. 1021-1025 (2000)
[Publications] 杉山大介: "AGM領域"分子細胞療法. 1. 92-93 (2000)
[Publications] 辻浩一郎: "造血幹細胞の増幅"血液・免疫・腫瘍. (印刷中).
[Publications] 辻浩一郎: "試験管内造血幹細胞増幅"現代医療. (印刷中).
[Publications] 辻浩一郎: "造血幹細胞コロニー刺激因子と臍帯血造血幹細胞の体外増幅"日本産婦人科・新生児血液学会. (印刷中).
[Publications] Nakahata T.: "Cell Therapy"Springer-Verlag, Tokyo. 2 (2000)
[Publications] Nakahata T.: "Mast Cell Biology"Academic Press (in press).
[Publications] Tsuji K.: "Methods in Molecular Medicine-Cytokines and Colony Stimulating Factors"The Human Press (in press).
[Publications] 田中竜平: "造血器疾患治療マニュアル"メディカルレビュー社(大阪)(印刷中).
[Publications] 辻浩一郎: "小児の症候群"診断と治療社(東京)(印刷中).

2000 Fiscal Year Annual Research Report

臍帯血造血幹細胞制御の分子機構の解明とその無血清体外大量培養法への応用

Principal Investigator

辻 浩一郎 東京大学, 医科学研究所, 助教授 (50179991)

Research Products

[Publications] Ueda T.: "Expansion of human NOD/SCID-repopulating cells by a combination of stem cell factor, Flk2/Flt3 ligand,thrombopoietin and a complex of interleukin-6 and soluble interleukin-6 receptor."J.Clin.Invest.. 105. 1013-1021 (2000)

[Publications] Hayashi Y.: "SN-1, a novel leukemic cell line with t(11;16)(q23;p13): myeloid characteristics and resistance to retinoids and vitamin D3."Cancer.Res.. 60. 1139-1145 (2000)

[Publications] Ebihara Y.: "Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34^+ cells."Br.J.Haematol.. 109. 153-161 (2000)

[Publications] Ueda T.: "Hematopoietic repopulating ability of cord blood CD34^+ cells in NOD/Shi-scid mice."Stem Cells. 18. 204-213 (2000)

[Publications] Ueda T.: "Massive pericardial and pleural effusion with an asarca following allogeneic bone marrow transplantation."Int.J.Hematol.. 71. 394-397 (2000)

[Publications] Sato T.: "Erythroid progenitors differentiate and mature in response to endogenous erythropoietin."J.Clin.Invest.. 106. 263-270 (2000)

[Publications] Ishii T.: "Improvement in bronchiolitis obliterans organizing pneumonia by combination of oral predonisolone and low dose erythromycin in a child after allogegeneic bone marrow transplantation."Bone Marrow Transplant. 26. 907-910 (2000)

[Publications] Sugiyama H.: "Peroxisome proliferator-activated receptors are expressed in mouse bone marrow-derived mast cells."FEBS Lett.. 467. 259-262 (2000)

[Publications] Yoshino H.: "Natural killer cell depletion by anti-asialo GM1 antiserum treatment enhances human hematopoietic stem cell engraftment in NOD/Shi-scid mice."Bone Marrow Transplant.. 26. 1211-1216 (2000)

[Publications] Takaki S.: "Control of B cell production by the adaptor protein Lnk, a negative regulator of tyrosine kinase signaling."Immunity. 13. 599-609 (2000)

[Publications] Matsui A.: "gp130 and c-kit signals synergistically induce thrombopoietin production by hematopoietic cells."Int.J.Hematol.. 72. 455-462 (2001)

[Publications] Tsuji K.: "Expression of G-CSF receptor on myeloid progenitors."Leukemia and Lymphoma. (in press).

[Publications] Matsuoka S.: "CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages."Blood. 97. 419-425 (2001)

[Publications] Ma F.: "Cytokine requirement for the development of T-lineage potential from lymphohematopoietic progenitor cells."Br.J.Haematol.. (in press).

[Publications] Sugiyama H.: "Peroxisome proliferator-activated receptors are expressed in human cultured mast cells : a possible role of these receptors in negative regulation of mast cell activation."Eur.J.Immunol.. (in press).

[Publications] Xu M.: "Evidence for the presence of primitive megakaryopoiesis in the early yolk sac."Blood. (in press).

[Publications] Ueda T.: "Hematopoietic capability of cord blood CD34^+ cells : a comparison with that of adult bone marrow CD34^+ cells."Int.J.Hoematol.. (in press).

[Publications] Yoshimasu T.: "Prompt and durable hematopoietic reconstitution by an unrelated cord blood transplantation in a child with Fanconi anemia."Bone Marrow Transplant.. (in press).

[Publications] Miyamoto K.: "Inhibitory effect of interleukin-3 on the early development of human B-lymphopoiesis."Br.J.Haematol.. (in press).

[Publications] Matsuoka S.: "Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleura by AGM region-derived stromal cells."Blood. (in press).

[Publications] Hisakawa H.: "Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) stimulates primitive and definitive erythropoiesis in mouse embryo expressing hGM-CSF receptors but not erythropoietin receptors."Blood. (in press).

[Publications] Ma F.: "Development of human lymphohematopoietic stem/progenitor cells defined by CD34 and CD81 expressions."Blood. (in press).

[Publications] 石井武文: "急性リンパ性白血病治療後期に発症したL-アスパラギナーゼによる壊死性膵炎"日小血会誌. 14. 328-332 (2000)

[Publications] 岩本彰太郎: "大量rhG-CSF療法中にmonosomy7を伴うMDSに移行したKostmann症候群"日小血会誌. 14. 338-345 (2000)

[Publications] 三井哲夫: "造血幹細胞移植における支持療法と今後の課題"Biotherapy. 14. 292-300 (2000)

[Publications] 辻浩一郎: "増幅造血幹細胞移植ガイドライン"分子細胞療法. 1. 197-201 (2000)

[Publications] 海老原康博: "造血幹細胞の同定検査 2)コロニー法"検査と技術. 28. 971-972 (2000)

[Publications] 辻浩一郎: "造血幹細胞の起源と性状"医学のあゆみ. 194. 1021-1025 (2000)

[Publications] 杉山大介: "AGM領域"分子細胞療法. 1. 92-93 (2000)

[Publications] 辻浩一郎: "造血幹細胞の増幅"血液・免疫・腫瘍. (印刷中).

[Publications] 辻浩一郎: "試験管内造血幹細胞増幅"現代医療. (印刷中).

[Publications] 辻浩一郎: "造血幹細胞コロニー刺激因子と臍帯血造血幹細胞の体外増幅"日本産婦人科・新生児血液学会. (印刷中).

[Publications] Nakahata T.: "Cell Therapy"Springer-Verlag, Tokyo. 2 (2000)

[Publications] Nakahata T.: "Mast Cell Biology"Academic Press (in press).

[Publications] Tsuji K.: "Methods in Molecular Medicine-Cytokines and Colony Stimulating Factors"The Human Press (in press).

[Publications] 田中竜平: "造血器疾患治療マニュアル"メディカルレビュー社(大阪)(印刷中).

[Publications] 辻浩一郎: "小児の症候群"診断と治療社(東京)(印刷中).

辻浩一郎東京大学, 医科学研究所, 助教授 (50179991)