Lack of differential sensitivity of normal hematopoietic stem cells and murine lymphoblastic leukemic cells to a lysosomotropic agent, N-dodecyl morpholine.

The effect of N-dodecyl morpholine (NDM), a lysosomotropic compound, on the clonogenic capacity of GK15, Sp2.0, Hb131, and L1210 lymphoblastic tumor cells and CFU-GM and CFU-S progenitor cells from DBA/2 mice was measured in order to evaluate the potential use of this compound for the purging of tumor-contaminated bone marrow (BM) in autologous BM transplantation. The growth of clonogenic tumor cells from all of the tested cell lines was inhibited with doses of NDM that also killed 100% CFU-GM and CFU-S, and no optimal dose could be found in this animal model to purge marrow while sparing sufficient stem cells to ensure engraftment in syngeneic BM transplantation.

AcSDKP, an inhibitor of CFU-S proliferation, is synthesized in mice under steady-state conditions and secreted by bone marrow in long-term culture.

The peptide AcSDKP, isolated from fetal calf bone marrow, is able to prevent DNA synthesis in mouse CFU-S in vivo and in vitro. The molecule is demonstrated here to be constitutively produced in mice and synthesized by bone marrow cells in long term culture.

Membrane-bound delta-4 notch ligand reduces the proliferative activity of primitive human hematopoietic CD34+CD38low cells while maintaining their LTC-IC potential.

To examine the role of the Notch ligand Delta-4 on hematopoietic stem cells, human CD34+CD38low cord blood cells were cocultured on S17 cells transduced with transmembrane Delta-4 (mbD4/S17) or an empty vector (C/S17). By the end of a 3-week culture, mbD4/S17 induced a 25-fold reduction in nucleated cell production, as compared to C/S17, by maintaining a higher proportion of cells in G0/G1 phase. A specific retention of a high proportion of CD34+ cells throughout the culture was observed with mbD4/S17, contrary to C/S17. Although mbD4/S17 promoted expansion of cells with the phenotype of committed lymphoid precursors (CD34+CD7+CD45RA+), these cells still retained their myeloid differentiation potential. mbD4/S17 maintained a higher LTC-IC frequency in output CD34+ cells, compared to C/S17, as in the subsets of cells having completed the same number of divisions on mbD4/S17. A Delta4-Fc protein (extracellular part of human Delta4 fused to Fc human IgG1 portion), immobilized on plastic, also reduced cell production and retained the LTC-IC potential. Transplantation of cells grown on mbD4/S17 into NOD/SCID mice showed no significant enhancement of the long-term repopulating ability. Thus, Delta4 appears to inhibit hematopoietic stem cell proliferation, in association with the maintenance of short-term lymphoid and myeloid repopulation capacity.

Further studies on the biological activities of the CFU-S inhibitory tetrapeptide AcSDKP. II. Unresponsiveness of isolated adult rat hepatocytes, 3T3, FDC-P2, and K562 cell lines to AcSDKP. Possible involvement of intermediary cell(s) in the mechanism of AcSDKP action.

Because the molecular mechanisms of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP; an inhibitor of spleen colony-forming unit [CFU-S] DNA synthesis) are difficult to study on bone marrow due to the scarcity of CFU-S in this tissue, we sought a pure cell population responsive to the molecule in vitro. Although growth factor-stimulated DNA synthesis in primary culture of hepatocytes and Balb/c 3T3 cells can be inhibited by transforming growth factor beta (TGF beta) and interferon alpha/beta (IFN[alpha/beta], respectively, neither hepatocytes nor 3T3 cells were found to be sensitive to AcSDKP. DNA synthesis in stimulated murine FDC-P2 cell lines and in human K562 cell lines also remained unchanged after exposure to the tetrapeptide. The fact that hepatocytes do respond in vivo to AcSDKP implies the existence of intermediary cell(s) involved in AcSDKP action in vivo that are lacking in hepatocyte culture. Whether intermediary cell(s) are implicated in the inhibitory action of AcSDKP on CFU-S entry into DNA synthesis is now being investigated.

In vitro purging of bone marrow with mafosfamide synergizes with in vivo chemotherapy to delay the hematological recovery in a murine model of autologous bone marrow transplantation.

Very long-lasting leukopenias and thrombocytopenias have been observed in patients submitted to transplantation of autologous bone marrow incubated in vitro with cyclophosphamide derivatives. With the aim of evaluating the contribution of in vitro exposure of bone marrow to mafosfamide (Asta-Z) and of in vivo chemotherapy given before bone marrow collection in these cytopenias, we designed a murine model of syngeneic bone marrow transplantation including treatment of donor and recipient mice with high doses of cyclophosphamide and in vitro exposure of the bone marrow transplant to Asta-Z. Blood platelets and leukocytes, medullary splenic colony forming unit (CFU-S), committed megacaryocytic (CFU-Meg) and granulomacrophagic (CFU-GM) precursor cell recovery was followed up to 56 days posttransplant. The data indicate that in vitro exposure of bone marrow to Asta-Z before reinfusion increases the delay in platelet recovery already induced by the chemotherapy given to donor mice and is specifically responsible for the prolongation of leukopenia. In recipient bone marrow, a synergy between the ablative effect of the in vitro treatment of bone marrow graft and the chemotherapy given to donors and recipients on CFU-S, CFU-Meg and CFU-GM was found.

Preferential differentiation of murine CFU-S toward granulopoiesis and megakaryocytopoiesis after in vitro incubation of bone marrow with ASTA-Z 7557.

We investigated the in vitro effect of ASTA-Z 7557 on the qualitative aspects of murine CFU-S differentiation, as assessed by the histological nature of day-9 colonies generated in the spleen of irradiated mice by bone marrow exposed to the drug at concentrations ranging from 0 to 150 micrograms/ml. The proportion of erythrocytic colonies declined linearly with the logarithm of the dose (a 22% decrease per log), whereas the granulocytic and megakaryocytic colony proportions increased linearly (a 10% increase per log for both cell lineages). This suggests a preferential channeling of CFU-S differentiation toward granulopoietic and megakaryocytic cell lineages as a consequence of the in vitro chemotherapy, and supports the hypothesis that some alteration of the qualitative potential of CFU-S to differentiate after in vitro purging of bone marrow with ASTA-Z 7557 takes place prior to autologous bone marrow transplantation.

Statistical analysis of splenic colony histology: an attempt to confirm that external factors could affect the channelling of CFUS differentiation.

The aim of this work was to analyze quantitatively experimental data, which suggest that CFUS determination can be manipulated by external fibers. This hypothesis was based on the comparison of the ratio of erythroid to granulocytic spleen colonies generated by normal bone marrow exposed to factors released by either untreated or arabinoside cytosine treated mouse bone marrow. To investigate mechanisms able to produce this modification of spleen colony histology, statistical analysis was performed by testing three different schemes of evolution of the number of splenic colonies according to this histology from normal to treated groups. The total number of colonies per spleen is similar in both groups, but a significant increase of the number of erythroid colonies per spleen and a significant decrease of the number of granulocytic colonies are observed in the treated group as compared to control. Bias due to the used experimental technique are investigated but could not explain the observed differences. A unique mechanism acting on only one committed stem cell population does not fit the experimental data. Although other possible mechanisms are suggested, the experimental observations can be interpreted as the consequence of a shift of CFUS differentiation toward the erythroid pathway at the expense of at least the granulocytic lineage due to some humoral factors, secreted by treated mouse bone marrow.

The Mpl-ligand is involved in the growth-promoting activity of the murine stromal cell line MS-5 on ES cell-derived hematopoiesis.

We investigated the ability of the murine stromal cell line MS-5 to enhance the hematopoietic potential of embryonic stem (ES) cells. The presence of increasing concentrations of MS-5 cells during the differentiation of ES cells into embryoid bodies (EBs) resulted in a positive dose effect on the efficiency of EB development. Moreover, the number of myeloid progenitors derived from EBs at days 6 and 10 of differentiation significantly increased. This increase resulted from an elevation of both the proportions of positive EBs (EBs containing at least one progenitor each) and the progenitor cell content per positive EB. The stimulatory activity of MS-5 cells affected all types of myeloid progenitors except erythroid progenitors, which were depressed. However, the relative numbers of ES-derived granulocyte-macrophage progenitors (colony-forming units granulocyte/macrophage [CFU-GM], -macrophage [CFU-M], and -granulocyte [CFU-G]) and of mixed cell colonies were unchanged. In contrast, the incidence of megakaryocytic progenitors (colony-forming units-megakaryocyte [CFU-MK]) was significantly increased, that of erythroid progenitors (burst-forming units-erythroid [BFU-E]) was concomitantly decreased, and the total numbers of both progenitor types remained constant. Addition of Mpl-ligand (Mpl-L; thrombopoietin) during the growth of EBs was found to mimic the effect of the MS-5 cell line on the output of progenitor cells. No effect of Mpl-L on the efficiency of EB formation was observed. In addition, supplementation of cultures with sufficient soluble Mpl to abrogate Mpl-L activity resulted in the reversion of the quantitative and qualitative effects of MS-5 cells on progenitor cell formation but not on the efficiency of EB formation. Together, these data indicate two major effects and two levels of action of the MS-5 cell line on hematopoietic differentiation of ES cells. First, the cell line acts before hematopoietic determination, promoting the plating efficiency of ES cells via mechanisms that remain to be clarified. Second, at a later stage of differentiation, the MS-5 cells promote hematopoiesis within EBs. Mpl-L appears to be one of the components that confer this latter ability on the MS-5 cell line.

Embryonic stem cell differentiation to hematopoietic cells: A model to study the function of various regions of the intracytoplasmic domain of cytokine receptors in vitro.

To examine whether the in vitro model of embryonic stem (ES) cell hematopoietic differentiation is suitable to study the function of intracytoplasmic regions of cytokine receptors, we used the thrombopoietin receptor Mpl as a typical cytokine receptor.ES cells deficient in c-mpl (mpl(-/)-) were transfected with genes encoding the full-length or two mutated forms of the intracytoplasmic domain of Mpl using the pEF-BOS expression vector. The mutated forms lack box1 or box2.pEF-BOS was able to maintain protein production during ES cell differentiation. Reintroduction of full-length-c-mpl into mpl(-/)- ES cells restored the response of megakaryocyte progenitors to a truncated form of human Mpl-ligand conjugated to polyethylene glycol (PEG-rhuMGDF) and the formation of platelets, for which mpl(-/)- ES cells are defective. In addition, enforced expression of Mpl resulted in the development of all myeloid progenitors and mature cells in the presence of PEG-rhuMGDF. Blast colony-forming cells, the in vitro equivalent of the hemangioblast, also generated blast cell colonies with a hematopoietic potential equivalent to that of the wild type in the presence of PEG-rhuMGDF, although its growth is normally dependent on vascular endothelial cell growth factor (VEGF). Thus, Mpl acts as a substitute for other cytokine receptors and for a tyrosine kinase receptor, Flk-1, indicating that Mpl has no instructive role in hematopoietic cell commitment and differentiation. The Mpl mutant forms lacking box1 or box2 prevented response of ES cell-derived blast colony-forming cells or progenitors to PEG-rhuMGDF. Therefore, these two regions, essential for signaling by cytokine receptors, are required for the responses of ES cell-derived hematopoietic cells to PEG-rhuMGDF.These results show that the in vitro hematopoietic differentiation of ES cells is suitable for studying the role of various intracytoplasmic regions of cytokine receptors.

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.

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.

Some effects of chemotherapeutic drugs on bone marrow stem cells. I. The long-term effects of phase-specific drugs on mouse bone marrow stem cells.

Two phase-specific drugs, cytosine arabinoside and hydroxyurea, were studied with regard to their effects on various murine hematologic cell compartments of the same mouse. Effects of single and multiple injections of Ara-C were compared. Following a significant decrease in the first few days, and a subsequent overshoot of pluripotential stem cells (CFU-S), colony-forming cells (CFC), bone marrow nucleated cells, and leukocytes, the number of these cells returned to normal values with a time sequence that varied with the cell type. During the 6-month observation period the number of these cells oscillated around control values after both drugs and both types of protocols.

Some effects of chemotherapeutic drugs on bone marrow stem cells. II. Effect on non-Hodgkin lymphoma chemotherapy on various hemopoietic compartments of the mouse.

The non-Hodgkin lymphoma chemotherapy protocol used at the Gustave-Roussy Institute was adapted, in terms of drug doses and interval between doses, to normal CBA mice. The numbers of pluripotential stem cells (CFU-S), unipotential stem cells (CFC), differentiated bone marrow cells, and circulating white cells were determined. Eight hours after each drug of the first chemotherapy cycle the number of pluripotent stem cells decreased while the proportion of these cells in DNA synthesis increased. Six hours after the end of each complete cycle, the stem cell compartments were found to be considerably depleted, and they were not completely restored when the next cycle was begun, while the other hematologic compartments were completely restored at this time.

[Hematopoietic differentiation of embryonic stem cells in mice: a model to study the biology of hematopoiesis]. / La différenciation hématopoïétique des cellules souches embryonnaires de souris: modèle d'étude de la biologie de l'hématopoïèse.

The manipulation of embryonic stem (ES) cells allows to generate mice with specific alteration in any gene. This is therefore an invaluable tool for studying gene function. A number of genes involved in the regulation of hematopoiesis have been inactivated, including genes that encode transcription factors, cytokines and their receptors as well as those encoding for intracellular signalling proteins. Alternatively, ES cells are able to differentiate towards myeloid, lymphoid and endothelial lineages under specific culture conditions. The role of master genes controlling hematopoiesis can be investigated by substituting the in vitro hematopoietic differentiation model of ES cells to mice fabrication. This method can be applied for studying effects of gene inactivation or overexpression of normal or abnormal gene. Interestingly, in vitro differentiation of ES cells recapitulates some aspects of embryonic development, including the emergence of the hemangioblast, the common precursor of hematopoietic and endothelial lineages. Thus, hematopoietic differentiation of ES cells constitutes a model for studying effects of gene manipulation on both hematopoiesis and emergence and commitment of the more hematopoietic primitive cell, the hemangioblast, during embryogenesis. In our studies, we used ES cells inactivated for the c-mpl gene, the thrombopoietin receptor, for dissecting the functions of various intracytoplasmic domain of c-mpl in the response of ES cell-derived hematopoietic cells to TPO.

[Production of hematopoietic cells from ES cells]. / Production de cellules hématopoïétiques à partir des cellules ES.

Murine embryonic stem (ES) cells are cell lines established from blastocyst which can contribute to all adult tissues, including the germ-cell lineage, after reincorporation into the normal embryo. ES cell pluripotentiality is preserved in culture in the presence of LIF. LIF withdrawal induces ES cell differentiation to nervous, myocardial, endothelial and hematopoietic tissues. The model of murine ES cell hematopoietic differentiation is of major interest because ES cells are non transformed cell lines and the consequences of genomic manipulations of these cells are directly measurable on a hierarchy of synchronized in vitro ES cell-derived hematopoietic cell populations. These include the putative hemangioblast (which represents the emergence of both hematopoietic and endothelial tissues during development), myeloid progenitors and mature stages of myeloid lineages. Human ES cell lines have been recently derived from human blastocyst in the USA. Their manipulation in vitro should be authorized in France in a near future with the possibility of developing a model of human hematopoietic differentiation. This allows to envisage in the future the use of ES cells as a source of human hematopoietic cells.

Restoration of the bone marrow pluripotent stem cells in AKR mice after arabinosylcytosine treatment.

The hypothesis of repression of multipotent stem cells (CFU) by leukemic cells to explain their depletion, previously demonstrated in AKR leukemic mice, was tested. Using arabinosylcytosine to destroy leukemic cells, it was shown that the bone marrow CFU pool was acutely depressed between 2 h and 12 h after treatment. However, 5 to 7 days later, this pool was restored, surpassing the value of the bone marrow pool in normal mice. This seems to indicate that the CFU pool in leukemic mice is potentially capable of proliferating but is repressed by leukemic cells.