Dominique Dumenil

Stimulating Factors and Cell Recruitment In Murine Bone Marrow Stem Cells and Emt6 Tumours

The role of a stimulating factor in cell recruitment and the kinetics of its secretion were investigated by in vivo and in vitro techniques. the association of these two methods made it possible to demonstrate that a non‐cycling population liberates a factor which in turn stimulates quiescent bone marrow stem cells into DNA synthesis. Moreover, it seems that undamaged cells are capable of secreting this factor. A stimulating factor responsible for cell recruitment was also demonstrated in an experimental EMT6 tumour and the kinetics of its secretion reported.

Role of pluripoietins in murine bone marrow stem cell differentiation.

Abstract The decision of the differentiation pathways taken by pluripotent stem cells seems to be under the influence, at least in part, of humoral factors acting at the CFU-S level. This differentiation is assessed by histological examinations of the recipient spleen colonies in order to determine the E/G ratios. In vitro cultures are made to determine GM-CFC and BFU E concentrations. After AraC treatment, CFU-S differentiate preferentially towards erythropoiesis. After total body irradiation, preferential differentiation is toward granulopoiesis. In both cases, there is a competitive phenomenon between the two cell lineages. This is observed in vivo and also in the in vivo-in vitro experiments where the responder cell population is in contact only with the eventual diffusible factors and not with the secreting cells or with the drug. These factors do not seem to be EPO, in the case of AraC, nor GM-CSF in the case of irradiation. We therefore suggest that humoral mediators other than those acting at the progenitor cell level can modulate CFU-S differentiation in a specific way after various types of aggression to the bone marrow.

The granulocyte colony-stimulating factor receptor supports erythroid differentiation in the absence of the erythropoietin receptor or Stat5

To evaluate the functional conservation of signal transduction mechanisms between haematopoietic receptors and to characterize the molecules activated in this phenomenon, we introduced granulocyte colony‐stimulating factor receptor (G‐CSFR) cDNA into mouse fetal liver cells using a retroviral vector. In semi‐solid medium assays, G‐CSFR‐infected cells gave rise to all types of colonies [granulocyte‐macrophage (GM), megakaryocyte (MK) and mixed lineage (GEMM) colony‐forming units (CFU) and erythroid burst‐forming units (BFU‐E)] in the presence of G‐CSF alone. The direct effect of G‐CSF on erythroid differentiation of G‐CSFR‐transduced erythroid progenitors was demonstrated by the development of erythroid colonies using G‐CSFR‐expressing Lin− cells cloned at one cell per well in liquid culture in the presence of G‐CSF. Interestingly, while Stat5, but not Stat3, was activated in erythroid cells in response to erythropoietin (EPO), both were activated in erythroid and granulocytic cells stimulated by G‐CSF. Furthermore, G‐CSF induced the growth of erythroid colonies from G‐CSFR‐expressing fetal liver cells from EPO receptor−/− (EPO‐R−/−) or Stat5a−/− Stat5b−/− mice, demonstrating that erythroid differentiation can occur in the absence of EPO‐R or Stat5. These data show that forced expression of G‐CSFR allows G‐CSF‐dependent multilineage proliferation and differentiation of haematopoietic progenitors and rescues EPO‐R−/− erythroid cells. While G‐CSF induces Stat5 activation in G‐CSFR‐expressing erythroid cells, this activation is not necessary for the terminal erythroid differentiation induced by G‐CSF.

Regulation of splenic CFU-S kinetics after cytosine arabinoside treatment in mice II. In vitro studies: Long-range modulators of the splenic CFU-S☆

A single injection of 20 mg of Ara-C to mice provokes an acceleration of splenic CFU-S differentiation, followed by their entry in DNA synthesis. In this protocol, splenic CFU-S are induced to differentiate preferentially towards erythropoiesis. The present studies show that substances secreted by spleen cells from Ara-C treated mice are responsible for the modifications in the splenic CFU-S population. This indicates that splenic CFU-S kinetics is under the control of pluripoietins as previously demonstrated for marrow CFU-S. The serum of Ara-C treated mice is shown to have stimulating effects on splenic CFU-S as well as on medullary CFU-S proliferation. It also has the capacity of channelling the differentiation of both splenic and medullar CFU-S towards erythroid lineage. These data suggest the existence of long-range humoral regulators for both populations of CFU-S.

Regulation of splenic CFU-S kinetics after cytosine-arabinoside treatment in mice—I. In vivo studies

Abstract A single dose of Ara-C injected to mice triggers differentiation and initiation of DNA synthesis in quiescent splenic CFU-S. The accelerated differentiation which precedes proliferation is preferentially oriented toward erythropoiesis at the expense of granulopoiesis. Thus, CFU-S of the spleen behave in an identical manner to bone marrow CFU-S. Therefore, splenic CFU-S do not in any way counterbalance bone marrow CFU-S perturbations and thus contribute to the efforts of hemopoietic restoration after drug treatment in mice.

MplK, a natural variant of the thrombopoietin receptor with a truncated cytoplasmic domain, binds thrombopoietin but does not interfere with thrombopoietin-mediated cell growth

OBJECTIVE Interaction of thrombopoietin (TPO) with its receptor c-Mpl is responsible for the formation of megakaryocytes and platelets. In humans, there are two major c-mpl molecules, MplP and MplK, which are generated by alternative splicing. In contrast to MplP, MplK has none of the intracellular sequences required for typical signal transduction but instead has a unique 27 amino acid sequence that is coded by intron 10. We tested to determine if MplK exerts a negative effect on TPO Mpl signal transduction by interfering with the normal homodimerization of MplP. MATERIALS AND METHODS A cassette coding for MplK cDNA was introduced into parental and MplP-expressing BaF3 cells and TPO-mediated cell growth studied. RESULTS Cells expressing MplK alone did not respond to TPO compared to cells that expressed MplP. When MplK was coexpressed with MplP on the cell surface of BaF3, no modification in cell growth was observed when compared to those expressing MplP alone. To determine if the normal homodimerization process was negatively influenced, two genetically engineered variants of c-Mpl, one lacking the box1 sequence and the other containing only the first nine amino acids of the intracellular domain, were introduced into MplP-expressing cells. In contrast to MplK, these mutants had a dominant negative effect on TPO-mediated cell growth. CONCLUSIONS MplK does not influence TPO-mediated growth of Mpl-expressing cells. Our data suggest that the absence of a dominant negative effect of MplK most probably is due to the inability of MplK to dimerize with the MplP receptor.

Modifications of pluripotent stem cell differentiation after Ara-C treatment: Clonal analyses of CFU-S progeny

The nature of the mechanisms controlling CFU-S differentiation is a crucial problem in haematology and, thus far, little is known concerning these phenomena. Work done in our laboratory has shown that the distribution of the histologic cell types represented in spleen colonies (CFU-S) differ depending on whether normal bone marrow or marrow from Ara-C treated mice is injected into the irradiated recipients. As measured by the mean of the absolute number of colonies per spleen, bone marrow from Ara-C treated mice gives more erythroid colonies and fewer granulocytic colonies than do cells from normal bone marrow. We have demonstrated that these modifications are under the control of humoral factors. Two significant questions arise from these observations. First, are the colonies after Ara-C treatment derived from a single multi-potential cell rather than from already committed progenitors and, second, is this shift in granulocytic-erythroid representation a reflection of modifications at the CFU-S level introduced by our Ara-C system? To answer these questions, we analysed the progeny of each individual spleen nodule either by reinjecting each colony unit into a secondary recipient or by cloning these cells in methyl cellulose with appropriate stimulating factors. We thus determined the number of retransplantable stem cells, as well as the number of committed precursors present in each spleen nodule. Our results demonstrate that most spleen colonies are transplantable and give rise to secondary colonies. These secondary colonies are of all haematological types, therefore proving that the nodules contain CFU-S and that these CFU-S are pluripotent. All spleen colonies contain GM-CFC, even in the nodules that were histologically erythroid. We thus conclude that modifications in the E/G ratio of spleen colonies after injection of bone marrow from Ara-C treated mice are a reflection of changes in CFU-S differentiation pathways.

CFU-S differentiation after multiple doses of cytosine arabinoside—Effect of inhibitors

Abstract After multiple doses of Ara-C, the E G ratio generated by CFU-S is dramatically decreased whereas it has previously been shown to increase after a single dose of the same drug. When inhibitors of CFU-S proliferation are given simultaneously with multiple doses of Ara-C, the E G ratio is increased to levels obtained after a single dose. Assuming that the E G ratio may reflect modifications in the determination of CFU-S differentiation, we suggest that Ara-C treatment, according to the protocol of its administration, could act on CFU-S differentiation by either changing the concentration or the nature of humoral factors, the putative pluripoietins. Although the cells secreting these factors are not yet identified, there seems to be a temporal correlation between the CFU-S pool size and the determination of their differentiation.

Further studies on the mechanism of CFU-S determination—I. Pluripoietin(s) responsible for CFU-S determination toward granulopoiesis after fractionated doses of ARA-C

Fractionated doses of Ara-C orient CFU-S differentiation towards granulopoiesis and megakaryocytopoiesis in vivo in contrast to the preferential channelling towards erythropoiesis after a similar dose of Ara-C given as a single injection. In this paper, we show that pluripoietin found in the serum of mice treated with fractionated doses of Ara-C is responsible for the choice of CFU-S differentiation towards granulopoiesis. Therefore, we confirm our previous results involving single doses of Ara-C, that humoral factors can commit CFU-S preferentially towards one of the cell lineages. It is not as yet known whether there is a specific pluripoietin for each cell lineage or whether the same pluripoietin has a different effect according to its concentration in the organism. The difference in response of CFU-S commitment to single and fractionated doses of Ara-C is not due to the difference in the kinetic status of CFU-S:CFU-S are cycling after both treatments.

Further studies on the mechanism of CFU-S determination—II. Feedback regulation

In previous papers, we have demonstrated that the determination of CFU-S differentiation is under the control of humoral factors, which we have called pluripoietins. The activity of these regulators varies according to the experimental protocol. The aim of this work was to determine the mechanisms involved in the regulation of pluripoietin secretion after each treatment. The results suggest that the production of pluripoietin(s) is under the control of a feedback mechanism, originating, at least in part, in the progenitor compartments. After one dose of 20 mg of Ara-C, CFU-S determination is channelled towards erythropoiesis when the BFU-E compartment is more depleted than the GM-CFC compartment. During fractionated doses of Ara-C, at the time of the third injection, GM-CFC survival is lower than BFU-E survival and CFU-S orient their differentiation towards granulopoiesis. After bleeding, there is no difference of survival between the two compartments and CFU-S determination is not changed. These results seem to indicate that CFU-S determination is modified in order to restore normal homeostasis of the hemopoietic tissues by preferentially replenishing the more depleted compartment. This is most likely achieved by the secretion of pluripoietin(s) that vary in either their nature or their concentration according to the events occurring in the more mature compartments.