hMADS cellsmyogenic potentiality

an in vitro investigation

Ari Massoudi

UMR 6543 CNRS« Stem cells and differentiation »

Centre de BiochimieUniversité de Nice-Sophia Antipolis

Faculte des Sciences

in vitro culture and

expansion of adherent cells

adipocytes

Stroma-vascular cells

Adipose tissues fromhuman neonate and infant donors

human Multipotent Adipose-Derived Stem cells(hMADS cells)

afterseveral months

Rodriguez et al. J Exp Med. 2005.

• self-renewal abitily

• high telomerase activity

• normal karyotype

• non-tumorigenic

• multipotency

hMADS cells present fundamental stem cells properties :

Mesenchymal Multipotence

ALPOstéonectine OstéocalcineOstéopontine

ColI1…

CBFA-1

Osterix

SMADs

VD3R

FosB

Adipocytes

CD36GAPDH

LPLaFABPLeptine

AdiponectineAdipsine

…

Pref-1

C/EBP

C/EBP

PPAR

PPAR

C/EBP

Induction adipogénique Induction ostéogénique

progéniteur

précurseur

Ostéocytes

Alizarin redORO

hMADS cells

In vivo hMADS cell myofiber contribution

hMADS cells

intra-muscularinjection

previous work of the lab :

Human dystrophin expressionhMADS 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

proliferatingmyoblasts

cellular fusion

differentiated myotubes

Myogenesis

quiescent myoblasts

activated myoblasts G0/G1transition

at confluencyG1/G0 transition

by contact inhibitioncommitment

MyoDmyf5

Pax7MyoDmyf5

myogenin

MyogeninHerculin

nuclear-LacZhMADS 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 myoblastshuman 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

hum

an m

uscl

e ce

llsm

ouse

C2C

12

RT-PCR

hMADS nucleistained with anti-human nuclei Ab

anti human sarcoglycan

( confocal analysis )

hMADS/C2C12 hybrid myotubes expressedhuman -sarcoglycan protein

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

Myoblasts : Pax7+GFP-hMADS cells : Pax7-

Myoblasts : MyoD+GFP-hMADS cells : MyoD-

Similarly :

GFP-hMADS cells

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

hu

man

mu

scle

cel

lsm

ou

se C

2C12

myoblasts myotubes

humanmyoblasts

GFP-hMADS

+mouseC2C12

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

2nd hypothesis1st 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 +

Myogenin-expressing myoblasts

hMADS cells

cellular fusion

myotube MRFs( myogenin, herculin, MEFs )

MDF involementin hMADS nuclei reprogramation

Factors involved in hMADS / myoblasts fusion

Global hMADS gene modification

• 46 conditions de culture testées en présence ou en l’absence de sérum• milieux conditionnés provenant de myoblastes

• membrane natives provenant de myoblastes• différents type de matrice extracellulaire

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…)