Résumé :
|
Embryonic stem (ES) cells differentiate into multiple lineages during in vitro embryoid body (EB) formation. This makes the ES/EB system a powerful tool to study early embryonic developmental pathways and to generate specific cell populations for regenerative medicine. However, the generation of a population of myogenic progenitors from differentiating ES cells with significant regenerative potential has proven elusive. Although the process of differentiating ES cells into embryoid bodies recapitulates several early events of pre-implantation embryogenesis, structures associated with proper patterning of paraxial mesoderm, such as somites, notochord, and neural tube are missing, and thus myogenesis does not occur properly in the ES/EB system. We show that it is possible to circumvent the defective myogenic environment by over-expressing Pax3 and Pax7, master regulators of the embryonic and adult myogenic program, respectively. This strategy in concert with a cell purification method based on paraxial and lateral plate mesoderm surface markers enabled us to generate a teratoma-free population of early myogenic progenitors from ES cells, capable of promoting extensive engraftment of adult myofibers and improvement in contractile function of injured muscle. However whether these cells are able to self-renew and contribute into adult/satellite stem cell pool regeneration in vivo is still unknown. To address this question we examined the ability of Pax3, which plays a critical role in embryonic muscle formation, and Pax7, which is important for maintenance of the muscle satellite cell pool, to promote the differentiation of ES cells into myogenic progenitors that serve this dual role. Functionality of putative donor-derived satellite cells was assessed by secondary transplantation of donor-specific (GFP+) mononuclear cells isolated by FACS, from primary recipients into secondary recipients. Additionally, we took advantage of bioluminescence imaging to track the dynamic of transplanted luciferase-labeled ES-derived donor cells upon re-injury with cardiotoxin. Our results reveal that both Pax3 and Pax7 confer on ES-derived myogenic progenitors the ability to produce functional myofibers and to engraft the satellite cell compartment in vivo. To test the proof of principle that induced pluripotent stem (iPS cells) may be used in the future for the treatment of muscular dystrophies, we extended our approach to mouse and human iPS cells. The in vitro and in vivo regenerative potential of these cells will be discussed.
|