血液細胞分化決定を制御する遺伝子の解析

• Project/Area Number: 07273201
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (A)
• Allocation Type: Single-year Grants
• Research Institution: Tohoku University
• Principal Investigator: 帯刀 益夫 東北大学, 加齢医学研究所, 教授 (10099971)
• Co-Investigator(Kenkyū-buntansha): 矢内 信昭 東北大学, 加齢医学研究所, 講師 (80200525) ; 井川 俊太郎 東北大学, 加齢医学研究所, 助手 (50241576) ; 東海林 互 東北大学, 加齢医学研究所, 助手 (40250831)
• Project Period (FY): 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥38,500,000 (Direct Cost: ¥38,500,000); Fiscal Year 1999: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 1998: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 1997: ¥7,500,000 (Direct Cost: ¥7,500,000); Fiscal Year 1996: ¥7,500,000 (Direct Cost: ¥7,500,000); Fiscal Year 1995: ¥7,500,000 (Direct Cost: ¥7,500,000)
• Keywords: Id / MIDA-1 / 血液細胞分化 / 造血幹細胞 / 間質細胞 / ヒト臍帯血幹細胞 / c-myc / YY-1 / 血液細胞合化 / MEL細胞 / 分化誘導 / C-Myc / 分化決定 / C-myc

Research Abstract

血液細胞分化決定をする遺伝子の解析を、間質細胞依存の血液幹細胞培養系を構築し、次のような成果を得た。
造血幹細胞の間質細胞による分化増殖の制御遺伝子を明らかにするため、SV40T-抗原遺伝子導入マウスの骨髄から我々が樹立した多数の間質細胞株をフィーダーとして、骨髄間質細胞株依存に増殖する造血幹細胞由来の細胞株の樹立を行った。その結果、胚型IgG遺伝子を持ち、条件的にIgG遺伝子の再編成を起こし、成熟することができる間質細胞依存B-リンパ球系幹細胞株(B31-1)、血球系列特異的転写因子の発現パターンから幹細胞の性状を示す細胞株であるSV40-T抗原遺伝子導入マウス骨髄幹細胞由来の細胞株(THS119)が樹立でき、これら細胞株の間質細胞依存性の増殖因子として、SCF,IL-7とともに間質細胞由来の未知分泌因子および特定の脂質が必要であることを示した。また、間質細胞依存で、ミエロイド系、リンパ球系への分化誘導が起きる骨髄幹細胞由来細胞(DFC)の培養系を構築し、その系列特異的な増殖条件を明らかにした。とくに、DFCaは顆粒球とマクロファージに分化できるミエロイド系前駆細胞であり、その維持には特定の間質細胞との共培養が必要であることがわかり、分化増殖の制御因子の探索が可能となった。さらに、マウス間質細胞株が、サイトカインとの組み合わせにより、ヒト臍帯血幹細胞の未分化な状態を維持し、増殖させるのに極めて効果的であることを示した。

Research Products

• [Publications] Okubo, T., Yanai, N. and Obinata, M: "Self-renewal and differentiation of a novel bipotent myeloid progenitor in stromadependent culture"Exp. Hematol.. ( in press ). (2000)
• [Publications] Yanai, N., Matsui, N., Furusawa, T., Okubo, T., and Obinata, M.: "Sphingosine-1-phosphate and lysophosphatidic acid trigger invasion of primitive hematopoietic cells into stromal cell layers"Blood. ( in press ). (2000)
• [Publications] Ohtaka, T., Matsui, Y. and Obinata, M.: "Hematopietic development of the primordial germ cell-derived mouse embryonic germ cells in culture"Biophys Biochem Res. Commn. 260. 475-482 (1999)
• [Publications] Inoue T., Shoji W, and Obinata, M.: "MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1 : a novel funcrion of Id protein"Biophys Biochem Res. Commn. 266. 147-151 (1999)
• [Publications] Matsuda., K., Koguma, M., Okuyama, R., Nakazawa, T., Matsuzaki, T., Nakauchi, H., Yanai, N., and Obinata, M,: "A novel stromal cell-dependent B lymphoid stem-like cell line that induces immunoglobulin gene rearrangement"J. Biochem.. 125. 602-612 (1999)
• [Publications] Yanai, N., Matsui, N., Matsuda, K-1., Furusawa, T., Ohkubo, T., Nakazawa, T., Ishibashi, K., Nawa K, and Obinata, M.: "A novel stromal cell-dependent hematopoietic cell line established from temperaturesensitive SV40 T-antigen transgenic mice"Exp. Hematol.. 27. 1087-1096 (1999)
• [Publications] Koguma,M.,Matsuda,K-I.,Okuyama,R.,Yanai,N.and Obinata,M.: "Selective Proliferation of Lymphoid Cells from Lineage c-Klt Scal Cells by a Clonal Bone Marrow Stromal Cell Line." Exp,Hematol.26. 280-287 (1998)
• [Publications] Furusawa,T.,Yanai,N.,Hara,T.,Miyajima,A.,and Obinata,M.: "Integrin-associated protein (LAP,also termed CD47) is involved in stroma-supported erythropoiesis." J.Biochem.123. 101-106 (1998)
• [Publications] Sato,Y.,Yanai,N.,and Obinata,M.: "Involvement of stromal membrane-associated protein (SMAP-1) in erythropoietic microenvironment." J.Biochem.124. 209-216 (1998)
• [Publications] Osada,M.,Ohba,M.,Kawahara,C.,Ishioka,C.,Kanamaru,R.,Katoh,I.,Ikawa,Y.,Nimura,Y.,Nakagawa,A.,Obinata,M.,and Ikawa,S.: "Cloning and functional analysis of human p51,which structurally and functionally resembles p53." Nature Medicine.4. 839-843 (1998)
• [Publications] Zhao,J-H.,Inoue,T.,Shoji,W.,Nemoto,Y.,and Obinata,M.: "Direct association of c-Myc and YY-1 in regulation of glycophorin gene expression." Oncogene.17. 1009-1017 (1998)
• [Publications] Muto,A.,Hoshino,H.,Madisen,L.,Yanai,N.,Obinata,M.,Karasuyama,H.,Hayashi,N.,Nakauchi,H.,Yamamoto,M.,Groudine,M.,and Igarashi,K.: "Identification of Bach2 as a B cell-specific partner for smal Mat proteins that negtively regulates the immunoglobulin heavy chain gene 3'enhancer." EMBO J.17. 5734-5743 (1998)
• [Publications] Obinata, M.: "The conditionally immortalized cell lines with differentiated functions established from the temperature-sonsitive T-antigen Transgenic mice." Genes to Cells. 2. 235-224 (1997)
• [Publications] Iguchi, A.et al: "Bone marrow stromal cells induce granulocytic development in the absence of G-CSF." Cell Struct.& Funct.22. 357-364 (1997)
• [Publications] Hasegawa, K.et al: "Novel smooth muscle cell lines from tramogenic mice harboring tomperature sonsitive SV40 large T-antigen gene" J.Mol.Cell.Cardiol.29. 2177-2186 (1997)
• [Publications] Yanai, N.et al.: "A new type-II membrane protein in orythropoietic organs enhances orythropoiesis." Leukemia. 11. 484-485 (1997)
• [Publications] Furusawa, T.et al: "Integrin-associated protein (IAP,also termed CD47) is involred in stroma-supported erythropoiesis." J.Biochem.123. 101-106 (1998)
• [Publications] Koguma, M.et al: "Selective proliferation of Lymphoid Cells from Lineage-C-kit_+Scal_+Cells by a Clonal Bone Marrow Stromal Cell Line." Exp.Hematol.26. (1998)
• [Publications] Nemoto, Y.: "Regulatory function of delta/YY-1 on the locus control region (LCR)-like sequence of mause glycomphorin gene in erythroleukemia cells." J. Biol. Chem.271. 13542-13548 (1996)
• [Publications] Furuchi, T.: "Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia." Development. 122. 1703-1709 (1996)
• [Publications] Yanai, N.: "v-Src interferes with the in Vitro erythropoietic stimulatory ability of the spleen stromal cells through repression of VCAM-1 and SCF." Exp. Hematol.24. 883-887 (1996)
• [Publications] Mataga, M.: "Establishment of a novel chondrocyte-like cell line derived from transgenic mice harboring temperature sensitive Simian Virus 40 large T-antigen gene." J. Bone and Mineral Res.11. 1646-1654 (1996)
• [Publications] Kodaira, K.: "Overexpression of c-Myc induces apoptosis at prophase of rat primary spermacytes." Mol. Reprod. Dev.45. 403-410 (1996)
• [Publications] Yanagisawa, M.: "Anovel serine/threonine kinase gene, Gek1, is expressed in meiotic testicular germ cells and primodial germ cells." Mol. Reprod. Dev.45. 411-420 (1996)
• [Publications] Obinata, M.: "Challenges of Modern Medicine, Vol. 10, Differentiation Therapy" ed by S. Waxman, Ares-Seveno Symposia Publications, Rome. Italy, 5 (1995)
• [Publications] Obinata, M.: "Challenge of Modern Medicine, Vol. 10, Differentiation Therapy" ed by S. Waxman, Ares-Seveno Symposia Publications, Rome, Italy, 6 (1995)
• [Publications] Shoji,W.: "MIDA1,a protein associated with Id,regulates cell growth." J.Biol.Chem.270. 24818-24825 (1995)
• [Publications] Kameoka,J.-I.: "Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors." J.Cell.Physiol.164. 55-64 (1995)
• [Publications] Okuyama,R.: "Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro." Blood. 86. 2590-2597 (1995)
• [Publications] Okuyama,R.: "Differentiation capacity towards mesenchymal cell lineages of bone marrow stromal cells established from temperature-sensitive SV40 T-antigen transgsnic mouse." Exp.Cell Res.218. 424-429 (1995)
• [Publications] Taher,A.: "Properties of incompletely immortalized cell lines generated from the line established from temperature-sensitive SV40 T-antigen transgenic mice." Exp.Cell Res.219. 332-338 (1995)
• [Publications] Nemoto,Y.: "Regulatory function of delta/YY-1 on the locus control region(LCR)-like seguence of mouse glycophorin gene in erythroleukemia cells." J.Biol.Chem.(in press). (1996)