Abstract The properties of the mouse embryo cell conditioned medium (ECM) colony stimulating factor(s) from six day mouse embryo cultures have been examined. The general properties were similar to those described previously for the human urine colony stimulating factor. The ECM colony stimulating activity (CSA) was not lost following treatment with nucleases, glycosidases, phospholipases and proteolytic enzymes with the exception of α‐chymotrypsin. ECM CSA was lost following mild periodate treatment. Fractionation of ECM CSA revealed a slight size heterogeneity on gel‐filtration and on zone sedimentation in sucrose gradients. There was a discrepancy between the apparent molecular weights determined by gel‐filtration (70,000–150,000) and by zone sedimentation (64,000) as has been reported previously for other colony stimulating factors. A gross charge heterogeneity of ECM CSA was apparent on electrophoresis and DEAE‐cellulose chromatography, and a heterogeneity of the elution profile on stepwise elution from calcium phosphate gel was observed. This heterogeneity was still apparent in the presence of 6M urea and appeared to be unchanged following re‐chromatography on DEAE‐cellulose under the usual fractionation conditions. These studies suggested that the heterogeneity was not due to easily reversible combinations of active subunits. The electrophoretic heterogeneity of six day ECM CSA was found to develop gradually from an electrophoretically monodisperse band at day 2 of culture. Experiments in which preparations containing concentrated monodisperse ECM CSA were added back to culture dishes during and after ECM production suggested that the development of heterogeneity was related to the production or release of factor(s) from the cells rather than the action on the colony stimulating factor(s) of an extracellular enzyme in the medium. Alteration of the electrophoretic mobility of six day ECM CSA by incubation with purified sialidase suggested the presence of sialic acid on the active molecules. Purification procedures for the ECM factor(s) were not developed to any large extent primarily in view of the charge heterogeneity. The results of this study suggest that the ECM colony stimulating factor(s) is a glycoprotein(s).
Summary Incubation of culture medium with cells from 8‐day‐old mouse kidney or cells from 16‐ to 18‐day mouse embryos resulted in production of conditioned medium which was capable of stimulating colony growth in vitro of mouse bone marrow cells. A linear relationship was demonstrated between the dose of conditioned medium and the number of colonies developing. A similar relationship was also found between conditioned medium dosage and the size of the colonies grown. The activity of conditioned medium was not lost on extensive dialysis; it showed a high degree of heat stability and was precipitated by 50‐100 saturation with ammonium sulphate. The morphology of cells in colonies grown with conditioned medium was typical of mouse metamyelocytes early in the incubation period but later there was a transition to mononuclear cell types.
Abstract Two doses of 1 mg/g of hydroxyurea (HU), injected 7 hr apart into irradiated mice in which CFU‐S were proliferating during marrow regeneration, killed about 90% of CFU‐S. This same dose regime injected into normal female mice, with non‐proliferating CFU‐S killed 92 % of CFU‐C, 99 % of ESC and only 30 % of CFU‐S. One day after the treatment CFU‐S had decreased to 50 % and remained at about this level for a further day then returned to normal values. In spleen the increase in CFU‐S was delayed by a day and showed a marked overshoot. During the period that CFU‐S were decreased in number they were actively proliferating. Marrow CFU‐C recovered in an exponential manner with a doubling time of 16 hr. Spleen CFU‐C recovered 1 day later than marrow and showed a pronounced overshoot. ESC recovered very rapidly with doubling time of 5 hr. The changes in 59 Fe incorporation into RBC, and the peripheral blood picture, were a delayed reflection of the changes in ESC and CFU‐C.
S ummary . Liquid suspension cultures of mouse bone marrow cells at high and low density were prepared in supplemented Eagle's medium containing 10% of a partially purified extract of mouse embryos and pregnant mouse uterus (PMU). In the low cell density cultures the number of cells decreased for 2 days; by 4 days the agar colony‐forming cells (agar CFC) had risen ten‐fold and the spleen colony‐forming units (spleen CFU) had fallen to one tenth; between 4 and 8 days the total cell count showed a four‐fold increase and the final cell number exceeded the number of the original culture. The cells produced were mainly macrophages. If PMU was not included in the culture the agar CFC disappeared after 4 days and there was no cell multiplication. In the high cell density cultures a similar pattern was observed; in the presence of PMU the agar CFC showed an increase in number, the spleen CFU decreased and an increase in cell number occurred between 4 and 8 days. However, the cells produced were predominantly granulocytes. In the absence of PMU from this cell culture, agar CFC were maintained for 6 days and the cell population remained predominantly granulocytic. These methods of growing cell enable cell recovery from the cultures to be made at any stage and provide an opportunity to study the kinetics and functional capacity of the cells produced.
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