通过可调控Lhx2的表达制备小鼠造血干细胞系
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摘要
【目的】研究一种永生化小鼠造血干细胞系的制备方法。【方法】通过添加Doxycycline来诱导Lhx2基因在成年骨髓的血液祖细胞/干细胞中表达,获得造血干细胞系。而在这些永生化的造血干细胞移植前,撤销Doxycycline,终止Lhx2基因的表达。【结果】永生化的造血干细胞系和原代造血干细胞一样,能在受体小鼠骨髓中增殖,并在外周分化为T细胞、B细胞和髓系细胞。Lhx2的异位表达可能会改变这些细胞以降低其在体内的适应性。【结论】本研究通过条件性表达Lhx2获得的永生化造血干细胞系与成年小鼠骨髓中的原代造血干细胞相比,具有相似的表面标志物,从而证明Lineage~-Sca-1~+c-kit~+,永生化的造血干细胞系极易扩增,可以获得大量均一的细胞群体。
【Objective】The present paper aimed to study the preparation of an immortalized mouse hematopoietic stem cell line. 【Method】Doxycycline was added to induce the expression of Lhx2 gene in hematopoietic progenitor/stem cells of adult bone marrow to obtain hematopoietic stem cell lines. 【Result】The immortalized hematopoietic stem cell line, like the primary hematopoietic stem cell, could proliferate in the recipient mouse bone marrow and differentiate into T cells, B cells, and myeloid cells in the periphery. Ectopic expression of Lhx2 maiyht alter these cells to reduce their applicability in vivo. 【Conclusion】This study compared the immortalized hematopoietic stem cell line obtained by conditional expression of Lhx2 with that of primary hematopoietic stem cells in the bone marrow of adult mice, demonstrating that Lineage~-Sca-1~+c-kit~+, immortalized hematopoietic stem cell line could be easily expanded and a large number of uniform cell populations could be obtained.
【Objective】The present paper aimed to study the preparation of an immortalized mouse hematopoietic stem cell line. 【Method】Doxycycline was added to induce the expression of Lhx2 gene in hematopoietic progenitor/stem cells of adult bone marrow to obtain hematopoietic stem cell lines. 【Result】The immortalized hematopoietic stem cell line, like the primary hematopoietic stem cell, could proliferate in the recipient mouse bone marrow and differentiate into T cells, B cells, and myeloid cells in the periphery. Ectopic expression of Lhx2 maiyht alter these cells to reduce their applicability in vivo. 【Conclusion】This study compared the immortalized hematopoietic stem cell line obtained by conditional expression of Lhx2 with that of primary hematopoietic stem cells in the bone marrow of adult mice, demonstrating that Lineage~-Sca-1~+c-kit~+, immortalized hematopoietic stem cell line could be easily expanded and a large number of uniform cell populations could be obtained.
引文
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[12]Hole N,Graham G J,Menzel U,et al.A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro[J].Blood,1996,88(4):1266.
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[14]Sánchez M J,Holmes A,Miles C,et al.Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo[J].Immunity,1996,5(6):513-525.
[15]Müller A M,Dzierzak E A.ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients [J].Development,1993,118(4):1343.
[16]Robbins P B,Skelton D C,Yu X J,et al.Consistent,Persistent Expression from Modified Retroviral Vectors in Murine Hematopoietic Stem Cells[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(17):10182-10187.
[2]Yonemura Y,Ku H,Lyman S D,et al.In vitro expansion of hematopoietic progenitors and maintenance of stem cells:comparison between FLT3/FLK-2 ligand and KIT ligand [J].Blood,1997,89(6):1915-1921.
[3]Yadav N K,Shukla P,Omer A,et al.In-vitro,hematological toxicity prediction by colony-forming cell assays [J].Toxicology & Environmental Health Sciences,2013,5(4):169-176.
[4]Miller C L,Eaves C J.Expansion in vitro of Adult Murine Hematopoietic Stem Cells with Transplantable Lympho-Myeloid Reconstituting Ability[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(25):13648.
[5]Yagi M,Ritchie K A,Sitnicka E,et al.Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin[J].Proc Natl Acad Sci USA,1999,96(14):8126-8131.
[6]Domen J,Weissman I L.Self-renewal,differentiation or death:regulation and manipulation of hematopoietic stem cell fate [J].Molecular Medicine Today,1999,5(5):201-208.
[7]Spooncer E,Boettiger D,Dexter T M.Continuous in vitro,generation of multipotential stem cell clones from src-infected cultures [J].Nature,1984,310(5974):228-230.
[8]Tsai S,Bartelmez S,Sitnicka E,et al.Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid,myeloid,and erythroid development[J].Genes Dev,1994,8(23):2831-2841.
[9]Yanai N,Matsui N,Matsuda K I,et al.A novel stromal cell-dependent hematopoietic cell line established from temperature-sensitive SV40 T-antigen transgenic mice[J].Experimental Hematology,1999,27(6):1087-1096.
[10]Varnumfinney B,Xu L,Brashemstein C,et al.Pluripotent,cytokine-dependent,hematopoietic stem cells are immortalized by constitutive Notch1 signaling[J].Nature Medicine,2000,6(11):1278.
[11]Wong P M,Han X D,Ruscetti F W,et al.Immortalized hemopoietic cells with stem cell properties[J].Immunity,1994,1(7):571-583.
[12]Hole N,Graham G J,Menzel U,et al.A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro[J].Blood,1996,88(4):1266.
[13]Potocnik A J,Kohler H,Eichmann K.Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells [J].Proceedings of the National Academy of Sciences,1997,94(19):10295-10300.
[14]Sánchez M J,Holmes A,Miles C,et al.Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo[J].Immunity,1996,5(6):513-525.
[15]Müller A M,Dzierzak E A.ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients [J].Development,1993,118(4):1343.
[16]Robbins P B,Skelton D C,Yu X J,et al.Consistent,Persistent Expression from Modified Retroviral Vectors in Murine Hematopoietic Stem Cells[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(17):10182-10187.