Effects of CFU-S inhibitors on murine bone marrow during Ara-C treatment—I. Effects on stem cells

In order to determine whether the dialysable extract of fetal calf bone marrow, which has previously been shown to inhibit CFU-S entry into DNA synthesis, was also capable of protecting bone marrow CFU-S during chemotherapy, experiments were performed in which the dialysate was injected to mice simultaneously with Ara-C. The number of modullary CFU-S was significantly higher in mice receiving the dialysate with the drug than in mice receiving the drug alone, while the numbers of nucleated cells and of GM-CFC were not different in the two groups. These results suggest a specific protective effect on bone marrow CFU-S survival. By chromatography on Sephadex G10 it was possible to isolate a low molecular weight fraction which inhibits significantly the percentage of CFU-S in DNA synthesis but does not increase CFU-S number when assayed in the same protocol. These experiments seem to imply that there is little, if any, correlation between the inhibition of CFU-S entry into cycle and an increase in survival of these cells after Ara-C treatment. These observations as well as the mechanism of the protective effect (increase of the number of CFU-S) of the dialysate will be discussed.     

Modifications of CFU-S differentiation pathways by pluripoietins: influence of treatment protocols

Similar total doses of Ara-C given as a single injection or given in a fractionated protocol have reverse effects on CFU-S differentiation pathways. Whereas a single dose of 20 mg channels CFU-S towards erythropoiesis, 5 X 5 mg given at 8 or 24 hour intervals channels CFU-S to granulopoiesis and megakaryocytopoiesis. It therefore seems possible to manipulate CFU-S differentiation not only by varying the inducing agents but also by varying the protocols using the same agent. Hypotheses to explain the mechanisms of CFU-S regulation are presented.     

Regulation of the growth fraction of CFU-S by an inhibitor produced by bone marrow

Humoral factors were extracted from cell suspensions obtained from rat bone marrow and thymus and fractionated using Amicon filters to obtain crude molecular weight bands of 50–100 K and 30–50 K. These fractions were incubated in vitro with haematopoietic stem cells (CFU-S) obtained from drug-treated mice to evaluate their effects on the proliferation of stem cells. An inhibitor was found in the 50–100 K fraction from normal rat bone marrow that would decrease the proportion of stem cells synthesising DNA in the regenerating bone marrow from ～ 50 to ～ 10%. This inhibitor was not found in the 30–50 K fraction extracted from rat bone marrow or in the 50–100 K fraction extracted from rat thymus and had no effect on the proportion of committed progenitors (CFU-C) synthesis DNA or the proliferation of mitogen-stimulated lymphocytes. The inhibitor has the same properties as that described by Lord et al. [11].     

Role of humoral factors in granulopoiesis: Colony promoting factor (CPF) and its target “pre-CFU-C’ in long-term bone marrow culture

Supernatant of long-term bone marrow cultures contains colony promoting factor (CPF). The factor has no colony stimulating activity itself but augments granulocyte-macrophage colony formation in semi-solid culture of bone marrow cells in the presence of colony stimulating factor (CSF). CPF was specifically absorbed by bone marrow cells but not by thymocytes. Incubation of bone marrow cells with CPF alone resulted in the increase of the number of granulocyte-macrophage progenitor cells (CFUc). CPF-containing supernatant did not stimulate the proliferation of pluripotent stem cells (CFUs) but inhibited CFUs proliferation. These results suggest that CPF is a separate factor from CSF or from CFUs regulators, and that CSF-non-responsive cells (pre-CFUc) in the bone marrow mature into CSF-responsive CFUc by the stimulus of CPF.     

Antigenic characteristics of circulating CFU-GM in chronic granulocytic leukaemia resemble those of CFU-GM in normal marrow and differ from those in normal blood

We studied the surface antigenic determinants of myeloid progenitor cells (Day 7 CFU-GM, Day 14 CFU-GM and BFU-E) in the peripheral blood and bone marrow of patients with chronic granulocytic leukaemia (CGL) and normal subjects by complement-mediated cytotoxicity with a panel of 8 selected murine monoclonal antibodies (McAbs) followed by culture in methyl cellulose. All classes of progenitor cell studied expressed HLA-DR antigens and also expressed other antigens recognized by two of the McAbs (S3-13 and S17-25) with myeloid specificity. Two other McAbs (R1.B19 and WGHS.29.1). Recognized antigens on Day 14 CFU-GM derived from normal marrow but not on those from normal blood. The pattern of reactivity of Day 14 CFU-GM from the blood of patients with CGL resembled to a considerable extent that of CFU-GM from normal marrow and differed from that of CFU-GM from normal blood. BFU-E from the blood of patients with CGL reacted with these McAbs in a manner very similar to that of BFU-E from normal blood; however the same two McAbs (R1.B19 and WGHS.19.1) reacted with a much higher proportion of the BFU-E from the marrows of CGL patients than of normal subjects. Our data are consistent with the hypothesis that normal blood-derived CFU-GM are more primitive than marrow-derived CFU-GM; however the CFU-GM in the circulation in CGL differ from those in normal blood, perhaps because they reflect overflow from or exchange with a hyperplastic marrow population.     

An inhibitor of murine stem cell proliferation produced by normal human bone marrow

Media conditioned by normal human bone marrow cells contain a specific inhibitor of haemopoietic stem cell proliferation. Molecular ultrafiltration and dose response studies indicate that it is similar to a previously described factor obtained from freshly isolated or long-term cultured murine bone marrow cells. It is suggested that the mechanisms involved in the control of murine and human stem cell proliferation may be essentially identical.