Multipotential hematopoietic progenitor cell lines have been established from nonadherent cell populations removed from continuous mouse bone marrow cultures. Clonal sublines of lines B6SUtA or B6JUt derived from single cells formed mixed colonies containing erythroid cells, neutrophil-granulocytes, and basophil/mast cells in semisolid medium containing erythropoietin and conditioned medium from pokeweed mitogen-stimulated spleen cells. Each of several subclones of cell line Ro cl formed colonies containing eosinophils, neutrophil-granulocytes, and basophil/mast cells in semisolid medium. Multipotentiality was maintained in vitro for over 2 1/2 years. In contrast, cell line 32D formed basophil/mast cell colonies with no detectable differentiation to other pathways. Multipotential cell lines did not produce detectable spleen colonies (CFUs) in vivo, nor did intravenous inoculation of up to 5 X 10(7) cells protect lethally irradiated mice from bone marrow failure. Newborn and adult mice inoculated with 5 X 10(7) cells showed no detectable leukemia or solid tumors after one year. Both multipotential and committed basophil/mast cell lines demonstrated absolute dependence upon a source of a growth factor(s) found in medium conditioned by WEHI-3 cells. These cell lines should be of value in studies of the regulation of hematopoietic stem cell differentiation in vitro.
Whether bone marrow stromal cells of donors contribute physiologically to hematopoietic stem cell reconstitution after marrow transplantation is unknown. To determine the transplantability of nonhematopoietic marrow stromal cells, stable clonal stromal cell line (GB1/6) expressing the a isoenzyme of glucose-6-phosphate isomerase (Glu6PI-a, D-glucose-6-phosphate ketol-isomerase; EC 5.3
The mouse T-cell clone Ly127/9, belonging to the Lyl set, displays the following functions in vitro: (i) augmentation of immunoglobulin output by B cells; (it) stimulation of bone marrow cells to produce colonies composed of granulocytes, macrophages, or both; and (iii) proliferative stimulation of T-cell clones belonging to other Ly sets. These functions are induced by Lyl'2j/9 cells themselves and by supernatants of Lyl+27/9 cultures and are not evinced by tested clones belonging to other Ly sets. The agent or agents responsible for colony formation and for B-cell stimulation had an apparent molecular weight of 45,000-50,000 and could not be physically separated. The T-cell stimulating agents(s) had an apparent molecular weight of 30,000 and could be separated from the agent(s) that acts upon colony formation and B cells. Thus, clone Lyl +2-/9 produces at least two soluble products that induce or augment activities of at least three other differently programmed cell sets.A portion of the T-lymphocyte population activates several other immunologic and nonimmunologic cells, and this T-cell set ("inducer") is identified by the surface phenotype Lyt-1+:Lyt-23-(1). Cells of the inducer set activate B cells to secrete immunoglobulin, induce hematopoietic precursor cells to make colonies, and stimulate the multiplication of T cells belonging to other sets (1-4). To further understand the multiple functions of inducer cells, we have developed a general method for producing cloned populations of this set and of other T-cell sets (5). In this report, we compare the biologic activities of a cloned line called Lyl12-/9 (Cl.Lyl12-/9) with T-cell clones that express different surface phenotypes.
MATERIALS AND METHODSAnimals. T cells for cloning were all obtained from C57BL/ 6 mice. Cells from C57BL/6 or BALB/c mice (obtained from The Jackson Laboratory, Bar Harbor, ME) were used to produce conditioned medium (see below).Antisera. Lyt-1.2 and Lyt-2.2 antisera, prepared as described (6), were kindly donated by F. W. Shen; monoclonal antibody against Thy-1.2 mc-a-Thy-1.2 was donated by Ed Clark; and mc-a-Lyt-1 and mc-a-Lyt-2 were donated by J. Ledbetter and L. Herzenberg. Expression of Lyl and Ly2 by each clone was determined with both Lyt antisera and monoclonal Lyt antibodies by immunofluorescence.Cell Culture and Cloning. Culture conditions for initiation and maintenance of cell lines have been described (5). Briefly, cells were diluted into microwells (Falcon plastic microtiter plates no. 3040) at estimated final concentrations of 100, 10, 1, and <1 cell per well. Each well contained 0.1 ml of complete conditioned medium (5) and irradiated (1500 R; 1 R = 2.58 X 10' C/kg) cell monolayers from different tissues (final concentrations 4-8 x 105 cells per well). Cultures were supplemented every 2 days with 30 ,ul of complete conditioned medium until colonies were visible (10 days-3 weeks) in wells that initially received <100 cells. Cloning efficiency was calculated by Poisson distribution (7). At least 96 wells were u...
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