Growth of mouse megakaryocyte colonies in vitro.

Abstract

Mouse bone marrow and spleen cells formed pure or mixed colonies of up to 80 megakaryocytes in agar cultures after stimulation by medium conditioned by activated mouse lymphoid cells. Megakaryocytes were identified on the basis of their morphology, polyploid mitoses and DNA content, and high cytoplasmic content of acetylcholinesterase. Megakaryocyte colony-forming cells were relatively small with a peak sedimentation velocity of 4.2 mm/hr. Spleen, lymph node, and thymus cells produced the factor stimulating megakaryocyte proliferation after culture in medium containing 2-mercaptoethanol, with or without added mitogens or allogeneic spleen cells. Peak activity in conditioning medium was associated with the small lymphocyte fractions in mouse spleen.Culture systems are now available for the clonal growth in semisolid medium of neutrophilic granulocytes and macrophages (1, 2), eosinophil-like cells (3), and erythropoietic cells (4). Mitogen-activated mouse lymphoid cells have been shown to produce large amounts of the colony-stimulating factor that stimulates granulocytic and macrophage colony growth (5, 6) and the special type of colony-stimulating factor that stimulates eosinophil colony formation (3). The present studies have shown that lymphocyte-conditioned medium is also able to stimulate clonal growth of megakaryocytes in vitro. MATERIALS AND METHODSMarrow Culture Technique. C57BL/6 marrow cells from twomonth-old mice were cultured in 35 mm plastic petri dishes (7). The agar-medium used was a mixture of 4 parts of double strength modified Eagle's medium, 1 part of 3% trypticase soy broth, and 5 parts of 0.6% Difco bacto-agar (the last boiled for 2 min and held at 37°). The composition of the double strength medium was: Dulbecco's modified Eagle's Medium HG Instant Tissue Culture powder H-21 (13.47 g) (Grand Island Biological Co. New York); double-glass-distilled water 215 ml; 3 ml of L-asparagine (20 ug/ml); 1.5 ml of I)EAE-dextran (75 M1g/ml) (Pharmacia, Sweden, molecular weight = 2 X 106, intrinsic viscosity = 0.70); 0.575 ml of penicillin (200 units/ml); 0.375 ml of streptomycin (200 units/ ml); 175 ml of NaHCO3 (2.8% w/v); 250 of unheated fetal calf serum.In routine cultures, sufficient marrow cells were added to the agar-medium to give a concentration of 75,000 cells per ml and 1 ml volumes of the cell suspension in agar-medium were pipetted into petri dishes containing 0.2 ml of lymphocyteconditioned medium or material containing colony-stimulating factor. After mixing, culture dishes were allowed to gel and were incubated for 7 days in a fully humidified atmosphere of 10% CO2 in air.Preparation of Lymphocyte Conditioned Media. Mouse spleen cells were incubated for 7-14 days at concentrations of 5 to 20 X 106/ml in modified Dulbecco's medium (8) Scoring of Cultures. Cultures were scored for colony formation by microscopy at X40. All aggregates containing large cells were removed intact using a fine pasteur pipette, placed on microscope slides, allowed to dry, and stained with 0.6% orcein in 60...

Growth of B-lymphocyte colonies in vitro.

“…All cultures contained only 25,000 nucleated cells to permit more accurate colony counts . Use of this low number of cultured cells also prevented the formation of granulocytic and macrophage cluster and colony formation in cultures of bone marrow cells due to mercaptoethanol-stimulated production of colony-stimulating factor (CSF) by marrow lymphocytes (5) . The frequency of colony-forming cells varied between 1 in 50 and 1 in 200 cells in both spleen and lymph nodes (Table I), but the frequency of colony-forming cells in the thymus was extremely low.…”

“…It may be that mercaptoethanol substitutes for such a specific macromolecule or alternatively mercaptoethanol may actually induce the production of a specific macromolecule by other cells in the culture dish . In this context it should be noted that mercaptoethanol has been shown to induce the production by mouse lymphoid cells of large amounts of CSF and the specific factors required for growth in vitro of eosinophilic and megakaryocytic cells (5).…”

Alternative testing systems for evaluating noncarcinogenic, hematologic toxicity.

“…Results (8)(9)(10), and the burst-forming unit-erythroid (BFU-E), which produces large, multifocal colonies of hemoglobinized cells in response to erythropoietin plus cytokines with burst-promoting activity (9,(11)(12)(13)(14)(15)(16)(17)(18)(19). The megakaryocytic lineage contains the CFU-megakaryocyte (CFU-Mk) (also called CFU-Meg) (20)(21)(22), which responds to many cytokines, including the c-mpl ligand (23)(24)(25)(26)(27)(28)(29)(30). By analogy to BFU-E, several laboratories report a BFU-megakaryocyte, which forms large, multifocal colonies (31,32 recloning (43)(44)(45)(46)(47)(48)(49)(50).…”

Haemopoietic progenitor-cell responses in mice with the transplanted lymphoid leukaemia ABE-8

“…To stimulate granulocyte-macrophage colonies, 0 1 ml of mouse-lung-conditioned medium was added to the cultures (Sheridan & Metcalf, 1973). Megakaryocyte and eosinophil colonies were stimulated by adding 0-2 ml of spleen-conditioned medium to each culture (Metcalf et al, 1975b).…”