Establishment of Hemopoietic Stem Cell Lines By Introduction of Oncogenes and Biochemical Studies on Mechanisms of Hemopoietic Cell Differentiation Using the Established Cell Lines

1.用穿刺法将克隆基因导入人造血细胞：穿刺法已被认为是将外源基因导入成纤维细胞等黏附细胞的有效方法之一(Yamamoto F.等：Exp.Cell Res.，142：79,1982)。我们设计了一种改进的穿刺法，适用于将克隆的基因，即细胞癌基因和病毒癌基因，导入到人的无造血细胞中。简单地说，将游离的单个核细胞以低浓度悬浮在包被聚L赖氨酸的微孔板中，离心后与基质黏附并在其上延伸，就像成纤维细胞一样。将附着在底物上的细胞分别在含有质粒gAE1A的培养基中插入与Eco-gpt基因连锁的E1A基因，使其对霉酚酸(MPA)产生抗性。从单倍体…来源的单个核细胞中获得了2、3和5 Mpa抗性转化子更多的星形细胞系JOSK-I、正常脐带血和慢性粒细胞白血病。2.携带癌基因的病毒感染外源基因导入造血细胞：(1)携带IL-3的FDC-P2细胞感染携带v-abl癌基因的Abelson小鼠白血病病毒(A-MuLV)后，除获得对IL-3的依赖外，还观察到明显的生长促进作用。我们已经证明，这种明显的表型转化不是由于IL-3非依赖转化子的出现，而是由于先前存在的少量IL-3非依赖克隆的生长促进，这些克隆的生长优先被A-MuLV感染刺激。(2)用A-MuLV感染小鼠胎儿细胞，成功建立了肥大细胞系和巨核母细胞系。(3)建立了Cas-BR-M-MuLV感染的小鼠髓系白血病细胞系，并证明其具有c-myb重排和病毒掺入。我们证明，该细胞系是一种多潜能干细胞系，因为它在各种生长因子如IL-3、GM-CSF和Erypoietin的作用下表现出多向分化。G-CSF仅支持其生长，不具有诱导分化活性。(4)在缺乏已知生长因子的情况下，骨髓单个核细胞在IL-3作用下形成的正常原始细胞集落可与脂肪细胞前体MC3T3-G2/PA6细胞共培养，支持并分化为多系血细胞，所有这些细胞集落在该共培养系统中均未见表达。

1.Introduction of Cloned Genes into Human Hemopoietic Cells by the Pricking Method: The pricking procedures have been reported as one of the efficient methods for introducing exogenous genes into adhesive cells such as fibroblasts (Yamamoto F.,et al.:Exp.Cell Res.,142:79,1982). We have deviced a modification of the pricking method suitable for the introduction of cloned genes i.e., cellular and viral oncogenes, into human hemopoietic free cells. Briefly, the free mononuclear cells were suspended at a low concentration in micro-well plates which were coated with poly-L-lysine, and were centrifuged to adhere to the substrate and to extend on it just like fibroblasts. Each of the cells adherent to the substrate were pricked in the medium containing plasmid gAE1A to introduce the E1A gene linked to Eco-gpt gene, which gives resistancy to mycophenolic acid (MPA) to the introduced cells. Two, 3 and 5 MPA-resistant transformants were obtained from the mononuclear cells derived from the monobl … More astoid cell line JOSK-I, normal cord blood, and chronic myelogenous leukemia, respectively. Generally, the transforming efficiency was found to be 0.21 in this pricking method while it was 9.2x <10^(-5)> in DNA-calcium phosphate co-precipitation method.2.Introduction of Exogenous Genes into Hemopoietic Cells by Infection of Viruses Carrying Oncogenes: (1) FDC-P2 cells, of which growth is dependent upon IL-3, were infected with Abelson murine leukemia virus (A-MuLV) carrying v-abl oncogene, and then their remarkable growth enhancement was observed in addition to the acquisition of independency on IL-3. We have shown that this apparent phenotypic transformation was not due to the appearance of IL-3-independent transformants, but due to an enhancement of the growth of preexisting minor IL-3-independent clones, of which growth were preferentially stimulated by the infection of A-MuLV. (2) We infected murine fetal cells with A-MuLV, and then succeeded in establishing either mast cell or magakaryoblastic cell line. (3) NFS-60 cell line was established from murine myeloid leukemia of Cas-Br-M-MuLV infected NFS/N mice and were already demonstrated to have c-myb rearrangement and the virus incorporation. We demonstrated that this cell line was a multi-potent stem cell line because it exhibited the multi-lineage differentiation depending upon various growth factors such as IL-3, GM-CSF and Erythropoietin. G-CSF only supported its growth and showed no differentiation-inducing activity. (4) Normal blastic cell colonies, which were regularly formed from bone marrow mononuclear cells in the presence of IL-3, were shown to be supported and differentiated into multi-lineage blood cells by co-culture with preadipocytic MC3T3-G2/PA6 cells in the absence of already known growth factors, all of which mRNA expressions were never demonstrated in this co-culture sytem.Constitutive Production of Interleukin 1 Less