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

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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