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Autumn Meeting 2003 : STEM CELL BIOLOGY

Autumn Meeting 2003 : STEM CELL BIOLOGY September, 13th, 2003 Organizers: F. Lemaigre & A.M. Goffinet Faculté de Médecine UCL, 1200 Brussels Poster Award: the Poster Prize of the Autumn Meeting was awarded
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Autumn Meeting 2003 : STEM CELL BIOLOGY September, 13th, 2003 Organizers: F. Lemaigre & A.M. Goffinet Faculté de Médecine UCL, 1200 Brussels Poster Award: the Poster Prize of the Autumn Meeting was awarded to Maud JOST for her poster: Migration of stem cells from bone marrow into choroidal lesion and their implication in angiogenesis. by Jost M., Lambert V., Maillard C., Van Overstraeten-Schlögel N., Gothot A., Motte P., Humblet Ch., Defresne M.P., Rakic J.M., Foidart J.M. and Noël A. from the Laboratory of Tumor and Development Biology, Ulg, Belgium, Department of Ophthalmology, Ulg, Belgium, Laboratory of Hematology, Ulg, Belgium, Unité de Biologie Cellulaire Végétale, Ulg, Belgium, Laboratory of Cytology and Histology, Ulg, Belgium Program : 09:45 Registration and Welcome 10:00 Harry HEIMBERG (Vrije Universiteit Brussels, Brussels) The elusive pancreatic stem cell . 11:00 Peter PONSAERTS (University of Antwerp, Antwerp) Genetic loading of human adult stem cells and mouse embryonic stem cells by mrna electroporation: an efficient and safe alternative to plasmid DNA and viral vectors 11:15 Nadia SCHECROUN (Université catholique de Louvain, Brussels) In vitro growth and osteoblastic differentiation of human bone marrow stromal cells supported by autologous plasma 11:30 Catherine VERFAILLIE (University of Minnesota, Minneapolis, USA) Unexpected pluripotency of adult stem cells 12:30 Lunch and posters 14:30 Bernard ROGISTER (Université de Liège, Liège Requirements for mesenchymal stem cells to become neural stem cells 14:45 Erika BOSMAN (Katholieke Universiteit te Leuven, Leuven) Maintenance of the amnion and mesendoderm are Smad5-dependent processes 15:00 Stefan MOMMA (Institute of Neurology, Goethe University, Frankfurt) Neural stem cells for regeneration of the CNS 16:00 End of meeting THE ONECUT TRANSCRIPTION FACTOR HNF-6 CONTROLS B LYMPHOPOIESIS IN FETAL LIVER Caroline Bouzin (1), Frédéric Clotman (1), Jean-Christophe Renauld (2), Frédéric P. Lemaigre (1) and Guy G. Rousseau (1) 1: Hormone and Metabolic Research Unit, UCL-ICP, B-1200 Brussels, Belgium; 2: Experimental Medicine Unit, Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium Mouse genetic models have helped to identify transcription factors that are expressed by hematopoietic cells and control their differentiation into lymphoid cells. However, little is known on transcription factors that are involved in this process, but are expressed in nonhematopoietic cells of the microenvironment. We show here that inactivation of the gene coding for Hepatocyte Nuclear Factor-6 (HNF-6) in mice led to B lymphopenia in the bone marrow and spleen. This phenotype disappeared shortly after birth when fetal B lymphopoiesis is no longer active, pointing to a defect in fetal liver. Indeed, the number of B cells was decreased in this organ as well. An analysis of B cell differentiation markers in fetal liver cells showed that B lymphopoiesis was impaired just beyond the Pre-Pro B cell stage. Hematopoietic cells from hnf6-/- fetal liver could reconstitute the lymphoid system when injected into scid mice. Since parenchymal cells, but not hematopoietic cells, expressed hnf6 in normal liver, we concluded that HNF-6 controls B lymphopoiesis in fetal liver and that HNF-6 exerts this control indirectly by acting in parenchymal cells. The involvement, in the B cell defect of hnf6-/- fetuses, of genes known to exert such an indirect control was ruled out by expression analysis, including microarrays, and by in vivo rescue experiments. This work identifies HNF-6 as the first non cell-intrinsic transcription factor known to control B lymphopoiesis specifically in fetal liver. 1 IN VITRO STUDIES OF BONE MARROW ADIPOCYTES: FROM ADIPOCYTES TO FIBROBLAST-LIKE FAT CELLS Zakia Belaid1, Frédérique Hubin1, Chantal Humblet1, Pierre Mineur2, MyriamVerlaet3, Charles M Lapière2, Jacques Boniver1, Betty V. Nusgens2 and Marie-Paule Defresne1. 1- Dpts of Cytology & Histology and Pathological Anatomy, 2-Laboratory of Connective Tissues Biology, 3- Research Center in Cellular and Molecular Neurobiology, * Grant FNRS-Télévie, CRCE,Ulg. Bone marrow is a tissue consisting of heterogeneous populations of cells including hematopoietic stem cells, endothelial cells and other stromal cells ( adipocytes, fibroblasts, osteoclasts etc.).the bone marrow microenvironment plays a critical role in regulating the growth and differentiation of hematopoietic cells by secretion of growth factors and cytokines as well as by direct cell-to-cell contacts. Recent evidence suggests that vascular growth factor (VEGF) is involved in these interactions. We have thus compared human iliac crest bone marrow ( hematopoietic bone marrow) and femoral bone marrow (fatty bone marrow) for the expression of VEGF, its receptors (VEGFR-1, VEGFR-2/KDR) and neuropilin-1 (NRP-1), a VEGF165 specific co-receptor and have shown that adipocytes are responsible for the high expression of NRP-1 in fatty bone marrow. NRP-1 has previously been reported to be implicated in angiogenesis ; our observation suggest that it could also be implicated in the regulation of hematopoiesis. Its high expression in non hematopoietic bone marrow seems to be inconsistent with the hypothesis that NRP-1 exerts a postive effect on hematopoiesis (R.Torjman and al ;Blood 1999, 94 : ). To identify the role of NRP-1 and the role of adipocytes in the regulation of hematopoiesis, adipocytes isolated from femoral bone marrow have been studied in vitro. In culture, adipocytes ( unilocular fat cells) adopt fibroblastic morphology ( fibroblast-like fat cells), they still express NRP-1, have lost the expression of the adipocytes marker ap2 ( adipocyte lipid binding protein), they express PPAR gamma 2 (peroxisome proliferator activated receptor) and LPL (lipoprotein lipase) at low levels, and display a mitotic activity after stimulation by VEGF165. The use of adipogenic medium allows the fibrolast-like fat cells to differentiate into unilocular fat cells which reexpress adipocytes marker ap2, PPAR gamma 2 and LPL at high levels but have down regulated the expression of NRP-1. This down regulation seems to be induced by steroids in the adipogenic medium and seems to be cell type specific as it is not observed in endothelial cells. Since the fibroblast-like fat cells still express NRP-1, their influence on hematopoietic progenitors (CD34+) in vitro is under investigation ; studies are also in progress to analyse their properties in an in vivo hematopoiesis model developped in our laboratory (F.Hubin). 2 GENETIC LOADING OF HUMAN ADULT STEM CELLS AND MOUSE EMBRYONIC STEM CELLS BY MRNA ELECTROPORATION: AN EFFICIENT AND SAFE ALTERNATIVE TO PLASMID DNA AND VIRAL VECTORS. Peter Ponsaerts, Leen Van den Eeden, Dirk. R. Van Bockstaele, Viggo FI Van Tendeloo, Zwi N. Berneman Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium. BACKGROUND: Genetic manipulation of stem cells is a potentially interesting strategy for cell differentiation studies and cell replacement therapies. However, gene transfer is either done by traditional plasmid DNA transfer or by viral vectors. However, these gene transfer strategies have several disadvantages. First, gene transfer by plasmid DNA generally does not result in high numbers of transfected cells and is often associated with high cell mortality among transfected cells. The use of viral vectors for gene transfer can circumvent these problems but is associated with more complex and laborious manipulations, and with safety issues when working towards clinical applications. METHOD: We have developed a non-viral non-dna technique for efficient and rapid gene expression in several types of stem cells, based on electroporation of mrna encoding an enhanced green fluorescent protein. RESULTS: For human adult stem cells, the data show that gene transfer by mrnaelectroporation in uncultured bone marrow samples (n=3) and peripheral blood samples from G-CSF stimulated donors (n=3), results in 10-20% of gene transfer efficiency in the CD34 hematopoietic stem cell population. In contrast, gene transfer efficiency by mrna electroporation in in vitro cultured mesenchymal cells from bone marrow samples (n=3) resulted in 70-90% of efficiently transfected cells, which expressed the transgene for up to one week. Morover, the electroporation procedure did not result in significant cell mortality among transfected cells. For mouse embryonic stem cells, the data show that gene transfer efficiency by mrna electroporation in several ES cell lines (n=3) is more than 90% combined with low cell mortality (2-5%) among transfected cells. CONCLUSION: The data presented provide an easy, efficient and safe alternative to plasmid DNA and viral vectors for genetic loading of stem cells in cell differentiation studies and cell replacement therapies. 3 OC-3, A NOVEL MAMMALIAN MEMBER OF THE ONECUT CLASS OF TRANSCRIPTION FACTORS Vinciane Vanhorenbeeck, Patrick Jacquemin, Frédéric P. Lemaigre and Guy G. Rousseau Hormone and Metabolic research Unit - Universite catholique de Louvain and Institute of Cellular Pathology 75(29) Avenue Hippocrate - B-1200 Brussels, Belgium Transcription factors of the Onecut class contain a bipartite DNA-binding domain composed of a single cut domain and a divergent homeodomain. Onecut proteins have been identified in mammals (rats, mice and humans), in the nematode Caenorhabditis elegans, the ascidian Halocynthia roretzi, the fly Drosophila melanogaster, and the zebrafish Danio rerio. Our studies have shown that HNF-6 (OC-1), the founding member of the class, and OC-2 play an important role in pancreas and liver development. We have now characterized the third mammalian member of the class, which we called Onecut-3 (OC-3). The mouse Oc3 gene was cloned from a genomic DNA library and the two coding exons were sequenced. The amino acid sequence of OC-3 displays 51% of identity with HNF-6 and 50% with OC-2. The cut domains of these three Onecut factors are 97% identical and their homeodomains 84% identical. The moc-3 gene was assigned to chromosome 10. The DNA-binding properties of OC-3 were studied by electrophoretic mobility shift assays. These experiments showed that the binding specificities of OC-3 and HNF-6 are similar, but differ somewhat from those of OC-2. Cotransfection assays showed that, like HNF-6 and OC-2, OC-3 behaves as a stimulator of transcription. To determine the expression pattern of OC-3, we screened RNA from adult and embryonic (E14.5) mouse organs by RT-PCR. We found that OC-3 mrna is present only in brain, stomach and upper intestine (1). To determine the role of OC-3 during development, we inactivated the gene in the mouse. The first exon, which codes for a region of the protein required for its activity, was replaced by a sequence coding for the Green Fluorescence Protein. Oc3 -/- mice were viable. We are currently studying the consequences of inactivation the Oc3 gene on stomach and gut development. (1) Vanhorenbeeck V, Jacquemin P, Lemaigre FP, Rousseau GG (2002) Biochem. Biophys. Res. Commun. 292, PRO-ANGIOGENIC FUNCTION OF PAI-1 IN HUMAN TUMORS Maillard C (1), Jost M (1), Bajou K (1), Romer N (2), Brunner N (2), Foidart J.M. (1), Noël A (1). 1: Laboratory of Tumor and developmental Biology, B-4000 Liège; 2: Finsen Laboratory, Copenhagen, Denmark Angiogenesis and tumor expansion are associated with the remodelling of the extracellular matrix and involve various proteases such as serine proteases urokinase-type (upa), tissue-type (tpa) plasminogen activators - and their specific inhibitors plasminogen activator inhibitor type 1 or 2 (PAI-1 and PAI-2). Recent works demonstrated the key role played by PAI-1 in tumor invasion and angiogenesis in PAI-1 deficient mice in which implanted malignant murine keratinocytes were unable to induce vascularized tumors (Bajou et al, 1998; Bajou et al, 2001). This observation partially explains the paradoxical clinical data that high level of PAI-1 in the primary tumor tissue of patients with various types of solid cancer correlate with disease recurrence and reduced survival. The aim of this work was to evaluate the role of PAI-1 in the progression of various human cancers. We have generated immunodeficient mice (in Rag-1-/- and nude background) deleted for PAI-1 gene (PAI-1 -/-). Using an original in vivo transplantation model, we have studied the effect of PAI-1 deficiency on the invasive and angiogenic behaviour of different malignant human keratinocytes displaying different invasive and angiogenic phenotype in vivo. While HPV16 transformed cervical cells (CasKi) are non aggressive cells, the two cutaneous cell lines transfected with Ras oncogene (HaCaT II-4 and HaCaTA5- RT3) are characterized by an invasive and angiogenic phenotype (HaCaTA5-RT3 being the more aggressive one) (Mueller et al, 2001). Tumors formed by CasKi cells remain poorly invasive and angiogenic, independently of the presence (in Wild Type mice) or absence (in PAI-1 -/- mice) of host PAI-1. In contrast to what happens in WT mice, HaCaTII-4 cells can neither invade host tissue nor induce angiogenesis when they are transplanted into PAI-1 -/- mice, demonstrating that PAI-1 is a key element for the development of these tumors. Interestingly, the transplantation of highly invasive HaCaTA5-RT3 cells in PAI-1 KO mice is associated with a delay of vascularization rather than an angiogenesis inhibition. In parallel to these transplantation experiments, these three cell lines have been subcutaneously injected into WT and PAI-1 -/- mice. The incidence (percentage of mice exhibiting a tumor which size is at least 50mm3) and the latency period (time necessary for 100% mice to develop tumor) of the tumor induced by HaCaTII-4 cells are significantly reduced in PAI-1 -/- mice as compared to WT mice. In contrast, HaCaTA5-RT3 cells grow rapidly both in PAI-1-/- and WT mice, whereas injection of CasKi cells does not induce tumor formation, whatever the mice genotype. These data indicate that PAI-1 plays its proangiogenic role during the earlier stages of human carcinoma progression. But, once the angiogenic and invasion processes have started, the PAI-1 deficiency is not sufficient to prevent neoplastic development of aggressive tumors. In conclusion, our results demonstrate the implication o 5 IN VIVO SYNERGY OF MMP-2 AND MMP-9 FOR PATHOLOGICAL ANGIOGENESIS Vincent Lambert1, Laura Rodriguez de la Ballina1, Carine Munaut1, Véronique Masson1, Ben Wielocks3, Maud Jost1, Laetitia Devy1, Takeshi Itoh3, Zena Werb4, Claude Libert3, Jean Marie Rakic1, Jean-Michel Foidart1 and Agnès Noël1. 1 Laboratory of Tumor and Development Biology, University of Liège, Pathology Tower (B23), Sart-Tilman, B-4000 Liège, Belgium 2 Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium The matrix metallopoteinases (MMPs) are believed to play a key role in pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines and/or cytokines. MMPs may also contribute to the control of angiogenesis by generating physiological inhibitors such as angiostatin, tumstatin or endostatin. The specific function of individual MMPs as anti- or pro-angiogenic mediators remains to be elucidated. In the present study, we addressed this aspect by assessing the impact of single or combined MMP deficiencies in two in vivo models of pathological angiogenesis (malignant keratinocyte transplantation and laser-induced choroidal neoangiogenesis) and in an in vitro model of angiogenesis (the aortic ring assay). The single deficiency of MMP2, MMP3, MMP9 or MMP11 did not impair in vivo tumor invasion and vascularization. Since MMP3 can activate MMP9, double MMP9/MMP3 -/- mice were also generated. We provide evidence that although MMP9 is expressed in tumor transplants, its absence alone or in combination with MMP3 did not impair tumor growth and vascularization. However, the cooperative effect of both gelatinases is demonstrated by the inhibition of tumor vascularization and growth in double MMP2/MMP9 deficient mice. Similarly, both the incidence and severity of choroidal neoangiogenesis induced by a laser burn were strongly attenuated in these double deficient mice. In sharp contrast, single or combined lack of gelatinases did not impair the in vitro spreading of capillaries from aortic rings, suggesting the importance of a cross talk between several host cells (endothelial cells, fibroblasts and inflammatory cells) for the concerted MMP2 and MMP9- mediated formation of new blood vessels. Altogether, these data demonstrate that MMP2 and MMP9 synergize in promoting in vivo tumoral and choroidal neo angiogenesis. 6 MIGRATION OF STEM CELLS FROM BONE MARROW INTO CHOROIDAL LESION AND THEIR IMPLICATION IN ANGIOGENESIS. Jost M.1*, Lambert V.1,2*, Maillard C.1*, Van Overstraeten-Schlögel N.3, Gothot A.3, Motte P.4, Humblet Ch.5*, Defresne M.P.5*, Rakic J.M.2, Foidart J.M.1* and Noël A.1* 1 Laboratory of Tumor and Development Biology, Ulg, Belgium, 2 Department of Ophthalmology, Ulg, Belgium, 3 Laboratory of Hematology, Ulg, Belgium, 4 Unité de Biologie Cellulaire Végétale, Ulg, Belgium, 5 Laboratory of Cytology and Histology, Ulg, Belgiu Angiogenesis, the formation of new blood vessels by sprouting from pre-existing vessels, is an important feature of various diseases such as cancer and ocular pathologies (age-related macular degeneration). In addition, some angioblasts (precursor endothelial cells) from bone marrow (BM) can be activated and then can migrate to the angiogenic lesion. Angiogenesis involves different proteases including serine proteases - urokinase-type (upa), tissue-type (tpa) plasminogen activators - and their specific inhibitors (plasminogen activator type 1 and 2 : PAI-1 and PAI-2). To study pathological vascularization, we have developed an in vivo model : the choroidal neovascularization induced by impact laser. The application of this model to PAI-1 deficient mice have demonstrated the essential role of this inhibitor. Indeed, PAI-1 deficiency in host mice prevented choroidal pathological vascularization (Lambert et al., 2001 FASEB J. 15: ). Several cell types are producers of PAI-1, such as endothelial cells, inflammatory cells, mast cells, but the essential cellular type is not yet established. The aim of this work was to determine the putative role of bone marrow stem cells (hematopoietic stem cells and/or angioblasts) in the pathological neovascularization in PAI-1 KO mice. Therefore, the model was applicated in PAI-1 deficient mice sublethally irradiated and engrafted with bone marrow (BM) from Wild-Type (WT) mice. A complete restoration of the angiogenic phenotype by WT BM transplantation was observed after laserinduced choroidal neovascularization. This result suggests that vasculogenesis is associated with angiogenesis (from pre-existing vessels) in this model of pathological vascularization. To observe the localization of precursor cells in choroidal lesion, we have engrafted BM from GFP mice to WT mice, and then observe the cells GFP+ in vascularized lesion. GFP expression was often but not always co-located with blood vessels. This suggests the presence of different types of precursor cells from BM into the lesion : inflammatory cells, pericytes, endothelial cells. 7 SUPPORT OF GRANULOPOIESIS IN VIVO BY TWO DIFFERENT MURINE MARROW STROMAL CELL LINES Frédérique Hubin, Chantal Humblet, Albert Thiry, Khaled Almohamad, Zakia Belaïd, Jacques Boniver and Marie-Paule Defresne Dpts of Cytology & Histology and Pathological Anatomy, CRCE, University of Liège, B-4000 Liège, Belgium Grant FNRS, Télévie Migration, proliferation and differentiation of hematopoietic progenitors in the bone marr
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