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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
in vitro  culture  and  expansion of adherent cells adipocytes Stroma-vascular  cells Adipose tissues from human  neonate  and  infant donors human Multipotent Adipose-Derived Stem cells (hMADS cells) after several months Rodriguez et al.  J Exp Med . 2005. ,[object Object],[object Object],[object Object],[object Object],[object Object],hMADS cells present fundamental stem cells properties  :
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
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
proliferation proliferating myoblasts cellular fusion differentiated myotubes Myogenesis quiescent  myoblasts activated myoblasts G0/G1transition  at confluency G1/G0 transition  by contact inhibition commitment MyoD myf5 Pax7 MyoD myf5 myogenin Myogenin Herculin
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
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
hMADS/C2C12 hybrid myotubes expressed human   -sarcoglycan protein   hMADS nuclei stained with anti- human  nuclei Ab anti  human    sarcoglycan  ( confocal analysis )
Human Dystrophin-defective  myotubes  (from DMD myoblasts) hMADS / DMD co-culture dystrophin  staining hMADS cells complemente dystrophin deficiency  in human DMD myotubes
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
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
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-
Mouse encoded-myogenin  in hMADS nuclei of hybid myotubes GFP   hMADS  nucleus Hoescht  smooth ponctuate murine  nucleus merge  myoG   hum myogenin hum actin mouse hprt hMADS/C2C12  co-culture myotubes RT-PCR human muscle cells mouse C2C12
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
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
Perspectives Myogenic conversion  of hMADS nuclei :  sarcoglycan   + dystrophin + nestin + sarcospan + desmin + enolase3 + muscle creatin kinase + myotube MRFs ( myogenin, herculin, MEFs ) MDF involement in hMADS nuclei reprogramation Factors involved in hMADS / myoblasts fusion Global hMADS  gene modification Myogenin-expressing  myoblasts hMADS cells cellular fusion
 
[object Object],[object Object],[object Object],[object Object],Aucune des conditions testées n’a permit d’induire un programme myogénique Conditions de culture testées pour promouvoir la myogenèse des cellules hMADS
Expression de Nestine humaine Co-culture cellules hMADS / myoblastes C2C12 souris Myotube hybride Myotubes   humain * * * *
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…)

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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
  • 2.
  • 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
  • 5. proliferation proliferating myoblasts cellular fusion differentiated myotubes Myogenesis quiescent myoblasts activated myoblasts G0/G1transition at confluency G1/G0 transition by contact inhibition commitment MyoD myf5 Pax7 MyoD myf5 myogenin Myogenin Herculin
  • 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-
  • 13. Mouse encoded-myogenin in hMADS nuclei of hybid myotubes GFP hMADS nucleus Hoescht smooth ponctuate murine nucleus merge myoG hum myogenin hum actin mouse hprt hMADS/C2C12 co-culture myotubes RT-PCR human muscle cells mouse C2C12
  • 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
  • 16. Perspectives Myogenic conversion of hMADS nuclei :  sarcoglycan + dystrophin + nestin + sarcospan + desmin + enolase3 + muscle creatin kinase + myotube MRFs ( myogenin, herculin, MEFs ) MDF involement in hMADS nuclei reprogramation Factors involved in hMADS / myoblasts fusion Global hMADS gene modification Myogenin-expressing myoblasts hMADS cells cellular fusion
  • 17.  
  • 18.
  • 19. Expression de Nestine humaine Co-culture cellules hMADS / myoblastes C2C12 souris Myotube hybride Myotubes humain * * * *
  • 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…)