hMADS cells myogenic potentiality an  in vitro  investigation Ari Massoudi UMR 6543 CNRS « Stem cells and differentiation ...
in vitro  culture  and  expansion of adherent cells adipocytes Stroma-vascular  cells Adipose tissues from human  neonate ...
Mesenchymal  Multipotence ALP Ostéonectine Ostéocalcine Ostéopontine Col I  1 … CBFA-1 Osterix SMADs VD 3 R FosB Adipocyt...
In vivo  hMADS cell myofiber contribution hMADS cells intra-muscular injection previous work of the lab  : Human dystrophi...
proliferation proliferating myoblasts cellular fusion differentiated myotubes Myogenesis quiescent  myoblasts activated my...
nuclear-LacZ hMADS cells mouse  C2C12  myoblasts + human nuclei   -gal + human- mouse hybrid myotubes hMADS cells contrib...
hMADS cells in co-culture expressed  human muscle genes D 0 D 1 D 2 D 4 hMADS cells / C2C12  co-cultures h = human specifi...
hMADS/C2C12 hybrid myotubes expressed human   -sarcoglycan protein   hMADS nuclei stained with anti- human  nuclei Ab ant...
Human Dystrophin-defective  myotubes  (from DMD myoblasts) hMADS / DMD co-culture dystrophin  staining hMADS cells complem...
3) How have hMADS cells contributed to myotube formation ? 1) hMADS cells are able to fuse with myoblasts  2) After fusion...
Hypothesis :   Do myoblasts induce myogenic  transdetermination of hMADS cells ? Strategy :   Assessing   " key &quot...
Myogenin +   myoblasts  GFP-hMADS cells in co-culture  do not express Muscle Determination Factors  hMADS cell myotube con...
Mouse encoded-myogenin  in hMADS nuclei of hybid myotubes GFP   hMADS  nucleus Hoescht  smooth ponctuate murine  nucleus m...
myoblasts  myotubes human myoblasts  GFP-hMADS + mouse C2C12  human   nestin  staining differentiation No  cross-reaction ...
Conclusion Post-fusion conversion by the myotube  differentiation factors By a classical  Determination / Differentiation ...
Perspectives Myogenic conversion  of hMADS nuclei :  sarcoglycan   + dystrophin + nestin + sarcospan + desmin + enolase3...
 
<ul><li>46 conditions de culture testées en présence ou en l’absence de sérum </li></ul><ul><li>milieux conditionnés prove...
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...
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How fat stem cells could or not form muscle cells (Eng)

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How fat stem cells could or not form muscle cells (Eng)

  1. 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. 2. 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. <ul><li>self-renewal abitily </li></ul><ul><li>high telomerase activity </li></ul><ul><li>normal karyotype </li></ul><ul><li>non-tumorigenic </li></ul><ul><li>multipotency </li></ul>hMADS cells present fundamental stem cells properties :
  3. 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. 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. 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. 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. 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. 8. hMADS/C2C12 hybrid myotubes expressed human  -sarcoglycan protein hMADS nuclei stained with anti- human nuclei Ab anti human  sarcoglycan ( confocal analysis )
  9. 9. Human Dystrophin-defective myotubes (from DMD myoblasts) hMADS / DMD co-culture dystrophin staining hMADS cells complemente dystrophin deficiency in human DMD myotubes
  10. 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. 11. Hypothesis : Do myoblasts induce myogenic transdetermination of hMADS cells ? Strategy : Assessing &quot; key &quot; 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. 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. 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. 14. myoblasts myotubes human myoblasts GFP-hMADS + mouse C2C12 human nestin staining differentiation No cross-reaction with mouse nestin Human nestin was expressed &quot; only &quot; in hMADS/C2C12 myotubes
  15. 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. 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. <ul><li>46 conditions de culture testées en présence ou en l’absence de sérum </li></ul><ul><li>milieux conditionnés provenant de myoblastes </li></ul><ul><li>membrane natives provenant de myoblastes </li></ul><ul><li>différents type de matrice extracellulaire </li></ul>Aucune des conditions testées n’a permit d’induire un programme myogénique Conditions de culture testées pour promouvoir la myogenèse des cellules hMADS
  18. 19. Expression de Nestine humaine Co-culture cellules hMADS / myoblastes C2C12 souris Myotube hybride Myotubes humain * * * *
  19. 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…)
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