FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...
How fat stem cells could or not form muscle cells (Eng)
1. hMADS cells myogenic potentiality an in vitro investigation Ari Massoudi UMR 6543 CNRS « Stem cells and differentiation » Centre de Biochimie Université de Nice-Sophia Antipolis Faculte des Sciences
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3. Mesenchymal Multipotence ALP Ostéonectine Ostéocalcine Ostéopontine Col I 1 … CBFA-1 Osterix SMADs VD 3 R FosB Adipocytes CD36 GAPDH LPL aFABP Leptine Adiponectine Adipsine … Pref-1 C/EBP C/EBP PPAR PPAR C/EBP Induction adipogénique Induction ostéogénique progéniteur précurseur Ostéocytes Alizarin red ORO hMADS cells
4. In vivo hMADS cell myofiber contribution hMADS cells intra-muscular injection previous work of the lab : Human dystrophin expression hMADS nuclei detected in muscles Rodriguez et al. J Exp Med . 2005. several months later mdx mouse Aims : to understand how hMADS cells contibute to skeletal muscle
6. nuclear-LacZ hMADS cells mouse C2C12 myoblasts + human nuclei -gal + human- mouse hybrid myotubes hMADS cells contribute to myotube formation Similar results with : primary wt or mdx mouse myoblasts primary wt human myoblasts human myoblasts from Duchenne Muscular Dystrophy
7. hMADS cells in co-culture expressed human muscle genes D 0 D 1 D 2 D 4 hMADS cells / C2C12 co-cultures h = human specific primers m = mouse specific primers h sarcospan h dystrophin h muscle creatine kinase h enolase 3 h desmin h actin m hprt muscle markers human muscle cells mouse C2C12 RT-PCR
8. hMADS/C2C12 hybrid myotubes expressed human -sarcoglycan protein hMADS nuclei stained with anti- human nuclei Ab anti human sarcoglycan ( confocal analysis )
9. Human Dystrophin-defective myotubes (from DMD myoblasts) hMADS / DMD co-culture dystrophin staining hMADS cells complemente dystrophin deficiency in human DMD myotubes
10. 3) How have hMADS cells contributed to myotube formation ? 1) hMADS cells are able to fuse with myoblasts 2) After fusion, hMADS cells express muscle genes
11. Hypothesis : Do myoblasts induce myogenic transdetermination of hMADS cells ? Strategy : Assessing " key " Muscle Determination Factors (MDF) expression by hMADS cells in co-cultures How have hMADS cells contributed to myotube formation ? Pax7 ? MyoD ? Myogenin ? hMADS cells Myoblasts inducing factors ? transdetermination « myo »-hMADS cells
12. Myogenin + myoblasts GFP-hMADS cells in co-culture do not express Muscle Determination Factors hMADS cell myotube contribution was not due to transdetermination Similarly : GFP-hMADS cells Myoblasts : Pax7+ GFP-hMADS cells : Pax7- Myoblasts : MyoD+ GFP-hMADS cells : MyoD-
14. myoblasts myotubes human myoblasts GFP-hMADS + mouse C2C12 human nestin staining differentiation No cross-reaction with mouse nestin Human nestin was expressed " only " in hMADS/C2C12 myotubes
15. Conclusion Post-fusion conversion by the myotube differentiation factors By a classical Determination / Differentiation process 2 nd hypothesis 1 st hypothesis Myogenic conversion of hMADS nuclei : sarcoglycan + dystrophin + nestin + sarcospan + desmin + enolase3 + muscle creatin kinase + Myogenin-expressing myoblasts hMADS cells cellular fusion myotube MDFs ( myogenin, herculin, MEFs ) Model of hMADS myogenic conversion
20. Cellules souches (CS) Progéniteurs (Pro) Auto-renouvellement Condition obligatoire, mais… L’auto-renouvellement peut aussi concerner les Pro Multipotence Certaines CS sont « unipotentes » (spermatogonies) Certains Pro sont « multipotents » Clonogénicité Certaines cellules souches ne poussent pas ou mal en dilution clonal Clonogénicté est aussi vrai pour les Pro Phénotype « side population » Controversé ! Ne concernent que quelques rares CS - Activité télomérase Ne concernent que quelques rares CS - Activité phagocytaire ++++ + Non-immunogénicité Vrai pour quelques rares cellules souches (MSC) +/- Marqueurs spécifiques Ne concernent que quelques rares CS (cellules ES, HSC, cellules satellites…)