Abstract Developmentally early murine hemopoietic progenitor cells of high proliferative potential (HPP-CFC), which are detectable in clonal agar culture in the presence of the lineage-specific hemopoietic growth factor, colony-stimulating factor-1 (CSF-1) plus hemopoietin-1 (H-1), or interleukin 3 (IL 3), express relatively high levels of the Qa-m7 antigenic determinant. This determinant is progressively lost during differentiation, and the more committed progenitors which grow in the presence of CSF-1 alone are essentially devoid of Qa-m7. Significant increases in both the proportion of Qa-m7-positive myeloid cells and the level of Qa-m7 antigen expression have been observed in bone marrow cells regenerating after the administration of the cytotoxic agent 5-fluorouracil (5-FU). By exploiting this increase in Qa-m7 antigen expression during regeneration and the HPP-CFC-sparing properties of 5-FU, we have been able to enrich HPP-CFC from marrows 8 days post-5-FU treatment (FU8d) to purities of greater than 20%. Furthermore, discontinuous gradient centrifugation and fluorescence-activated cell sorting of FU8d bone marrow cells on the basis of their light-scattering properties and Qa-m7 expression has unmasked a further subset of HPP-CFC which strictly requires the combined stimulus of three hemopoietic growth factors (H-1, IL 3, and CSF-1) for clonal growth. These highly enriched subsets of HPP-CFC are either identical to or co-fractionate with transplantable multipotential hemopoietic progenitors capable of reconstituting the hemopoietic system of lethally irradiated mice. Up to one in three cells in these highly enriched fractions is an HPP-CFC, and up to one in two cells may be CFU-S assayed 13 days post-transplantation. In addition, these fractions contain progenitors capable of reconstituting the platelet, erythroid, and myeloid compartments of the marrow.
Phenylhydrazine treatment produced an expansion of spleen CFU (10 x N) and an even greater expansion of spleen ACFU (40 x N). Blood CFU and ACFU levels also increased. While marrow CFU did not change marrow ACFU showed a pronounced drop. The ratio of ACFU/CFU varied between tissues, it was highest in marrow and lowest in peripheral blood. The changes in CFU, ACFU and blood granulocytes are discussed in relation to the radioprotective action of phenylhydrazine pretreatment of mice.
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).
Low concentrations of TGF-beta inhibited the clonal proliferation of three different preparations of mouse bone marrow progenitor cells in vitro. Normal marrow cells responsive to CSF-1; two-day post-fluorouracil cells responsive to CSF-1 plus IL-3 and IL-1 alpha; and regenerating seven-day post-fluorouracil cells responsive to CSF-1 plus IL-3, IL-1 alpha and IL-6 were all severely inhibited by TGF-beta 1; and the inhibitory effects were antagonized by increasing the concentrations of the relevant growth factors.
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