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2013-12-05 - Colloque/Présentation - poster - Anglais - 1 page(s)

De La Kethulle De Ryhove Laurence , Ansseau Eugénie , Geens Mieke, Coppée Frédérique , Sermon Karen, Lagneaux Laurence, Belayew Alexandra , "DUX4 expression during Osteogenic Differentiation in MSCs" in Journée des jeunes chercheurs Télévie, Université Catholique de Louvain, Belgique, 2013

  • Codes CREF : Biologie moléculaire (DI3111), Pathologies particulières (DI3370)
  • Unités de recherche UMONS : Biologie moléculaire (M122)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) Introduction Our group has identified the Double Homeobox 4 (DUX4)1 gene within repeated DNA elements in the 4q35 chromosome region linked to the FSHD muscular dystrophy. This gene is activated in FSHD muscle cells and expresses a transcription factor that initiates a large gene deregulation cascade. In healthy individuals, Dr. S. Tapscott’s group2 (Fred Hutchinson Cancer Research Center, Seattle) has detected a full length DUX4 mRNA (fl-DUX4) in induced pluripotent stem (iPS) cells and human testis, and a longer mRNA where the gene contains 4 additional exons and a more distal polyadenylation signal. Differentiation of iPS cells to embryoid bodies decreased expression of this mRNA in control but not FSHD cells. Aims We have previously detected DUX4 at a very low level in MSC isolated from bone marrow (BM-MSC) and Wharton jelly (Wj-MSC). We wanted to investigate its expression during the osteogenic differentiation of MSCs. Methods and results Our preliminary data suggested that the DUX4 expression decreased upon differentiation of Wj-MSC at the mRNA and protein levels because we observed a decreased (-20%) number of nuclei stained by immunofluorescence after long culture times which are known to induce MSC differentiation. This observation was similar to the DUX4 inhibition shown upon differentiation of induced pluripotent stem cells (iPS) to embryoid bodies. Based on these observations we thought that DUX4 could be a “stemness” marker. We wanted to evaluate whether DUX4 expression decreased during MSC differentiation to confirm this hypothesis. We induced osteogenic differentiation with a specific medium and cells were collected after 0, 7, 14 and 21 days, nuclear protein extracts were prepared and we performed an immunodetection on western blot. To confirm the differentiation process we used at each time point the alizarin red staining which stains calcium deposits in the cell culture dish. The results we obtained were completely opposite to what we expected. We observed an increase of DUX4 expression after 14 and 21 days. These results were reproduced for three different BM-MSC samples. We hypothesize we had previously detected DUX4 protein because we had seeded more cells per well leading to a cell density that induced spontaneous differentiation, and DUX4 is known to be expressed in early differentiated cells. We then wanted to investigate whether DUX4 had a direct impact on the differentiation. In a preliminary experiment, cells were seeded and differentiated as described above. We transfected cells with antisense oligonucleotides (2’O Methyl phosphorothiate, DUX4-AO) and interfering with the protein expression. The transfection was performed one day before addition of the osteogenic differentiation medium and cells were stained with alizarin red after 0, 7 days, 14 days and 21 days of osteogenic differentiation. The cells transfected with a DUX4-AO presented weaker alizarin red staining which indicated that differentiation was delayed. We have to repeat this experiment for confirmation and follow additional differentiation markers to better understand the mechanism. Conclusion We have unexpectedly found that DUX4 expression was increased upon MSC differentiation to osteoblasts. This observation is in contrast with the data published about iPS cells differentiation to embryoid bodies in which DUX4-fl expression disappeared. An embryoid body is a 3D structure which exhibits a heterogeneous pattern of differentiated cells. Dr. M. Kyba’s group (University of Minnesota) showed DUX4 implication in neurogenesis3. They transfected murine embryonic stem cells with a DUX4 inducible vector and observed after DUX4 induction, the differentiated cells expressed neuronal expression markers. We hypothesize that DUX4 could generally be implicated in the mechanism of early differentiation. 1Dixit et al. 2007, DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. Proc Natl Acad Sci U S A 104: 18157–18162. doi:10.1073/pnas.0708659104. 2 Snider et al . 2010, Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene. PLoS Genet 6: e1001181. doi:10.1371/journal.pgen.1001181 3Dandapat et al. 2013, Expression of the Human FSHD-Linked DUX4 Gene Induces Neurogenesis During Differentiation of Murine Embryonic Stem Cells. Stem Cells Dev. doi:10.1089/scd.2012.0643.