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Etude des mécanismes impliqués dans le contrôle du destin des cellules souches intestinales et développement d'un modèle 3D d'épithélium intestinal

Justine Creff 1
1 LAAS-ELIA - Équipe Ingénierie pour les sciences du vivant
LAAS - Laboratoire d'analyse et d'architecture des systèmes
Abstract : The small intestine is a complex tissue with a crypt/villus architecture and high tissue polarity. Intestinal stem cells are located at the crypt bottom where they proliferate and differentiate while they migrate upward to the top of villi, allowing the constant renewal of the entire intestinal epithelium every 3 to 5 days. Compartmentalization in the crypt plays a key role in stem cell protection and maintenance, and this is supported by the microenvironment and tissue organization. The balance between stem cell proliferation and differentiation is necessary to maintain tissue integrity, and disruption of this balance leads to developmental anomalies and malignant transformation. Studying the mechanisms governing intestinal stem cells maintenance is therefore crucial to understand tissue homeostasis. p57Kip2 is a cyclin/CDKs inhibitor and a putative tumor suppressor. p57 is also the gene the most frequently mutated or silenced in Beckwith-Wiedemann syndrome (BWS), characterized by multiple developmental defects and tumor predisposition during childhood. Generation of knock-in mice expressing a mutant p57 (p57CK-) that cannot bind to cyclins and CDKs demonstrated that p57 exerts CDKs independent functions during development and that BWS is not entirely caused by loss of CDKs inhibition due to p57 inactivation. The first aim of this project was to investigate the role of p57 in the maintenance of intestinal stem cells. Two population of stem cells have been described in the intestine: proliferative crypt base columnar cells (CBCs), responsible of the constant renewal of the epithelium, and quiescent +4 stem cells, activated during regeneration after tissue damage. I found that p57 is involved in maintaining the quiescence of the +4 reserve stem cells in a CDK independent manner. Indeed, p57KO mice exhibit an increased proliferation in the crypt caused by amplification of +4 stem cells and of the progenitor population (transit amplifying cells), while CBCs are not affected by loss of p57. I also investigated the molecular mechanism by which p57 regulate these cells and found that p57 can bind Ascl2, a transcription factor critical for intestinal stem cell specification and maintenance. My result indicates that p57 can inhibit Ascl2 activity by participating in a transcriptional repressor complex with HDAC7 and mSin3a. This work has allowed the identification of a new function of p57 in intestinal stem cells during development and that could also be important during intestinal tumorigenesis. The second aim of this project was to develop a new culture model to study intestinal stem cells. So far, most in vitro studies have been limited to 2D surfaces or 3D organoid cultures that do not fully recapitulate the tissue’s 3D architecture, microenvironment and cell compartmentalization existing in vivo. First, I developed a new photosensitive hydrogel amenable for culture of colorectal cancer cell lines that can be processed using high-resolution 3D printing stereolithography to create scaffolds mimicking the architecture of intestinal epithelium. Finally, I showed that these 3D scaffolds support the cell growth of for 3 weeks, and that 3D culture promotes cell polarization. This model may constitute a complementary approach to organoid cultures to study intestinal homeostasis by allowing guided self-organization and controlled differentiation, as well as for in vitro drug screening and testing.
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Justine Creff. Etude des mécanismes impliqués dans le contrôle du destin des cellules souches intestinales et développement d'un modèle 3D d'épithélium intestinal. Micro et nanotechnologies/Microélectronique. Université Toulouse 3 Paul Sabatier (UT3 Paul Sabatier), 2019. Français. ⟨tel-02468021⟩

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