This note is mainly a copy (pictures and references are skipped, part of the text is simplified or skipped) of the article:

Authors are :
Giuliana Ferrari, Egle Paolucci, Anna Stornaiuolo, Fulvio Mavilio
. Ospedale San Raffaele - Telethon Institute for Gene Theraphy (TIGET) Milan. Italy.
Gabriella Cusella-De Angelis
. Istituto di Anatomia Umana Normale. University of Pavia. Pavia. Italy.
Marcello Coletta, Giulio Cossu
. Dipartimento di Istologia ed Embriologia Medica. University of Rome La Sapienza. Rome. Italy.

The article appeared in SCIENCE - VOL. 279 - 6 MARCH 1998 - pages: 1528-1530 and can be requested for fax or mail delivery.

The study was supported by grants from the Italian Telethon Foundation and the European Union Biotechnology Program.

Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regeneration muscle, however, the number of myogenic precursors exceeds that of resident satellite cells, implying migration or recruitment of undifferentiated progenitors from other sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and partecipate in the regeneration of the damaged fibers. Genetically modified, marrow derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies.

In postnatal life, growth and repair of skeletal muscle fibers are mediated by a resident population of mononuclear myogenic precursors, the satellite cells. These cells, which are located between the sarcolemma and the basal lamina of the muscle fiber, divide at a slow rate to sustain both self-renewal and growth of differentiated tissue.

In response to muscle injury, or in individuals with chronic degenerative myopathies, satellite cells divide and fuse to repair or replace the damaged fibers. However, the self-renewal potential of adult satellite cells is limited, decreases with age, and can be exhausted by a chronic regenerative process such as that characteristic of severe muscular dystrophies, in which most muscle tissue is eventually lost and is replaced by connective tissue.

The number of resident satellite cells in adult muscle is much smaller than the number of committed myogenic precursors that populate the muscle tissue soon after an injury. Several explanations for this apparent paradox have been proposed, from migration of satellite cells from adjacent fibers, or even neighboring muscles, to recruitment to myogenesis of resident nonmyogenic cells such as fibroblast or mesenchymal progenitors.

Bone marrow (BM) stroma-derived mesenchymal cells, which serve as long-lasting precursors for bone, cartilage, and lung parenchyma in mice, can differentiate into contractile myotubes under certain conditions in vitro. However, recruitment to myogenesis of stroma-derived cells has not been observed in vivo.

To investigate wheteher BM cells can convert to myogenesis in response to physiological stimuli, muscle regeneration in the damaged tibialis anterior (TA) of 10 immunodeficient mice has been chemically induced.

Transgenic mouse line having muscle specific myosin light chain promoter modified to encode nuclear beta-galactosidase (b-gal) has been used, and BM and satellite cells have been obtained from these transgenic mice.

BM of transgenic mice was injected in TA of immunodeficient mice. As a control, satellite cells of transgenic mice was injected in TA of contralateral of all recipient animals. TA muscles were examined at various times after injection (5 days to 5 weeks) for the presence of b-gal positive nuclei.

After 2 weeks, the TA muscles injected with BM revealed fibers containing b-gal aligned nuclei similar to, although less numerous than, those observed in the contralateral leg injected with satellite cells. Transverse cryostat sections showed newly formed fibers with b-gal centrally localized nuclei in four out of six mice at 2 to 5 weeks after injection of BM cells. Conversely, centrally localized b-gal nuclei were observed in all muscles injected with satellite cells, as early as 5 days after the injection.

To test whether myogenic progenitors could be physiologically recruited from BM and access a site of muscle regeneration from the peripheral circulation, genetically marked BM cells were transplanted into 12 irradiated immunodeficient mice.

Five weeks after BM transplantation, muscle regeneration was induced in both TA muscles of nine surviving mice. Both the immune and non immune components of hematopoietic system were restored at normal level and were donor-derived. Regeneration was analyzed histochemically in the TA muscles of all transplanted mice. Transverse cryostat sections showed regenerative fibers containing b-gal nuclei in five of six reconstituted animals analyzed at 2 and 3 weeks after induction of muscle injury. b-gal nuclei were present both in immature centrally nucleated fibers and in more mature peripherally nucleated fibers.

Data indicate the existence of BM-derived myogenic progenitors that can migrate into a degenerating muscle, partecipate in the regeneration process, and give rise to fully differentiated muscle fibers. These cells appear to be recruited by long-range, possibly inflammatory, signals originating from the degenerating tissue, and they appear to access the damaged muscle from the circulation, together with granulocytes and macrophages.

The kinetics of differentiation of BM-derived progenitors differ from those for differentiation of committedc adult myogenic precursors. Injected satellite cells fused into muscle fibers within 5 days, whereas b-gal nuclei of BM origin were not detected in regenerating fibers before 2 weeks after induction of muscle damage. This observation, together with the observed clustering of b-gal nuclei in regenerating fibers, may suggest that BM-derived progenitors undergo a longer, and possibly multistep, differentiation process, which may comprise migration, cell division, commitment to the myogenic lineage, and eventual terminal maturation and fusion.

The origin of the BM-derived myogenic cells, as well as their physiological role in the homeostasis of muscle tissue, are not known. It is possible that these cells originate from multipotent, mesenchymal stem cells in the BM stroma that have been shown, by similar transplantation experiment, to give rise to bone, cartilage, and connective tissue.

Whether or not myogenic cells are derived from the same mesenchymal components, the experiments suggest that the BM could serve as a reservoir of progenitors for muscle tissue, and that, under conditions of extended damage, these progenitors might expand or maintain the pool of resident, more differentiated, muscle-forming precursors.

The existence of circulating myogenic progenitors has implications for cell or gene therapy for inherited muscle disorders. Efficient delivery to diseased muscles of genetically modified myoblasts, or even of viral vectors containing therapeutic genes, is one of the major hurdles currently limiting both ex vivo and in vitro approaches. Despite some anecdotal observations, it is generally accepted that satellite cells taken from skeletal muscle cannot colonize muscle tissue if delivered from the circulation. The availability of a cell population that could be engineered and then systematically delivered to a large number of muscles might aid in the development of a cell-mediated replacement therapy. In these experiments, BM-derived progenitors contributed only minimally to muscle regeneration. However, resident satellite cells are healthy in the used immunodeficient mice and are unaffected by the low dose radiation administered before BM transplantation.

The situation might be substantially different in a dystrophic background characterized by chronic muscle degeneration, in which genetically corrected BM-derived cells could progressively replace the exhausted pool of satellite cells. The therapeutic potential of transplanted BM cells awaits further verification in such a model.

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