ALBERT

Description: Hemogenic stem cells (HSC) emerge at distinct allocation territories during ontogenesis. They irrigate freely the whole body by the blood stream, but the adult HSC expand and differentiate exclusively in the bone marrow (BM). This implies that stem cells are not autonomous units of development; rather, tissue specific niches control their destiny. We investigated the spatio-temporal emergence of stem cells and nest-forming cells, and basic mechanisms by which they shape together the functional-proliferative units in the liver and BM during ontogenesis. Video microscopy in gut/liver primordium cultures provided direct evidence for intrinsic emergence of a primitive vasculo-hemogenic meshwork guided by pulsative, cardiomyogenic cells around nascent hepatic cords in E8.75-E10 mouse embryos. Gain-of-competence and development of hepatic units required firm aggregation of cholangio-hepatic (CK19+/CK18+), hemogenic (CAFC) and vasculogenic stem/progenitor cells and nest-forming cells into compact 3-D structures, similarly to BM hematons. Colonization and expansion of HSC in the mouse liver at E12, then in the BM at postnatal d2, numerically correlated with the increase in isolatable niches/hematons. Time-lapse video microscopy, quantitative colony assays, FACS and immunocytochemistry analyses of the adult human BM showed that the hematon is enriched in mesenchymal stem cells, preadipocytes/adipocytes, contractile myogenic cells, a vascular meshwork, guardian macrophages and hemopoietic stem/progenitor cells (Sca-1/c-kit/CD34/Thy-1/CD31+). A hematon module plays the role of an organizing center that emits growth signalling factors and accumulates morphogen/differenciator factors, retinoids and 1,25-di(OH)-vitaminD3. The physical attachment of many hematons to spongy bone suggests that they form the basic structural-functional units in the ultimate, fully competent BM. We suggest designating these units osteo-hematons. A comparative analysis of BM aspirates from healthy donors (n=75) and patients (n=210) revealed the disorganization of osteo-hematon modules in AML prior to therapy and in CML during all stages of the disease.

Description: Administration of 5-fluorouracil (5-FU) to mice results in a marked increase in the level of circulating platelets in 10 days. Mice lacking Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To gain insight into the mechanism by which 5-FU produces such a substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/kg) produced thrombocytosis in c-mpl−/− mice, thus establishing whether TPO was required for this response. A 5- to 6-fold increase in platelet levels in c-mpl−/− mice (to approximately 1000 × 109/L) was observed on days 20 and 25 after 5-FU injection. Thus, at the peak of the response, c-mpl−/− mice had platelet levels comparable to those in normal mice. Administration of 5-FU also produced thrombocytosis in previously splenectomized c-mpl−/− mice. Comparison of the platelet response to 5-FU in young (6-12 weeks) and old (33-46 weeks) c-mpl−/− mice found that older mice produced a much more marked response than younger mice, with a mean maximum platelet level of approximately 1700 × 109/L. To determine whether this increase in circulating platelets was preceded by an increase in hematopoietic progenitors, serial cultures of bone marrow and spleen were evaluated. A considerable increase in all colony types studied was observed on days 15 and 20 in spleens of c-mpl−/− mice, but no similar elevations were detected in bone marrow. These results indicate that c-mpl−/− mice can achieve a normal level of platelets after 5-FU injection, by means of a TPO-independent mechanism, and that they respond to 5-FU myelosuppression by producing large numbers of megakaryocytic, myeloid, and erythroid progenitors.

Description: The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

Description: The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.