Françoise Sainteny

Constitutive and specific activation of STAT3 by BCR-ABL in embryonic stem cells

BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia (CML) arises in a primitive hematopoietic stem cell with both differentiation and self-renewal ability. To study the phenotypic effects of BCR-ABL in a clonal in vitro self-renewal and differentiation model, we have introduced BCR-ABL in the ES cell line CCE. The major effect of BCR-ABL expression was the persistence of primitive morphology of ES cells despite LIF deprivation, correlated with a constitutive activation of STAT3, the major self-renewal factor of ES cells, but no evidence of activation of STAT5. The enforced expression of BCR-ABL in an ES cell line, engineered to express a tetracycline-inducible dominant-negative form of a STAT3, triggered ES cell differentiation with an increased generation of hematopoietic cells expressing erythroid and megakaryocytic phenotypes. RT–PCR analysis for Oct4, Brachyury and β-globin expression confirmed a delay of differentiation in BCR-ABL expressing clones, which could be entirely reversed upon activation of the dominant-negative form of STAT3. To study the possible relevance of STAT3 activation by BCR-ABL in human CML, Western blot analyses performed on the CD34+ cells, purified from CML patients at different stages of their disease, also demonstrated increased levels of STAT3 proteins phosphorylated both on tyrosine and serine residues. These results represent to our knowledge the first functional link between BCR-ABL oncogene and a self-renewal in the context of ES cells through constitutive activation of STAT3. Thus, the BCR-ABL embryonic stem cell model that we developed as well as the results obtained in human CML samples suggests a role for STAT3 in the pathogenesis of human CML.

Notch/Delta4 Interaction in Human Embryonic Liver CD34+ CD38− Cells: Positive Influence on BFU‐E Production and LTC‐IC Potential Maintenance

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1–4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para‐aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1–2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence‐activated cell sorter analysis showed that first‐trimester embryonic liver CD34+CD38low cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6‐fold increase in BFU‐E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long‐term culture‐initiating cells (LTC‐ICs), while control cultures quickly resulted in the extinction of this LTC‐IC potential. Furthermore, short‐term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose‐dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.

Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by the expression of a dominant-negative hTERT mutant.

As activation of telomerase represents a key step in the malignant transformation process, experimental models to develop anti-telomerase drugs provide a rational basis for anticancer strategies. We analysed the short and long-term efficacy of a stably expressed dominant-negative mutant (DN) of the telomerase catalytic unit (hTERT) in UT-7 and U937 human leukemia cell lines by using an IRES-e-GFP retrovirus. As expected, telomerase inactivation resulted in drastic telomere shortening, cytogenetic instability and cell growth inhibition in all e-GFP positive DN clones after 15–35 days of culture. However, despite this initial response, 50% of e-GFP positive DN clones with short telomeres escaped from crisis after 35 days of culture and recovered a proliferation rate similar to the control cells. This rescue was associated with a telomerase reactivation inducing telomere lengthening. We identified two pathways, one involving the loss of the DN transgene expression and the other the transcriptional up-regulation of endogenous hTERT with persistence of the DN transgene expression. Although this second mechanism appears to be a very rare event (one clone), these findings suggest that genomic instability induced by short telomeres after telomerase inhibition might enhance the probability of activation or selection of telomere maintenance mechanisms dependent on hTERT transcription.

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.

Quantitative Oct4 Overproduction in Mouse Embryonic Stem Cells Results in Prolonged Mesoderm Commitment During Hematopoietic Differentiation In Vitro

The Oct4 transcription factor is essential for the self‐renewal and pluripotency of embryonic stem cells (ESCs). Oct4 level also controls the fate of ESCs. We analyzed the effects of Oct4 overproduction on the hematopoietic differentiation of ESCs. Oct4 was introduced into ESCs via a bicistronic retroviral vector, and cells were selected on the basis of Oct4 production, with Oct4+ and Oct42+ displaying twofold and three‐ to fourfold overproduction, respectively. Oct4 overproduction inhibited hematopoietic differentiation in a dose‐dependent manner, after the induction of such differentiation by the formation of day 6 embryoid bodies (EB6). This effect resulted from defective EB6 formation rather than from defective hematopoietic differentiation. In contrast, when hematopoiesis was induced by the formation of blast colonies, the effects of Oct4 depended on the level of overproduction: twofold overproduction increased hematopoietic differentiation, whereas higher levels of overproduction markedly inhibited hematopoietic development. This increase or maintenance of Oct4 levels appears to alter the kinetics and pattern of mesoderm commitment, thereby modifying hemangioblast generation. These results demonstrate that Oct4 acts as a master regulator of ESC differentiation.

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.

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.