Mature adult dystrophic mouse muscle environment does not impede efficient engrafted satellite cell regeneration and self-renewal

Abstract

Changes that occur in the skeletal muscle environment with the progress of muscular dystrophies may affect stem cell function and result in impaired muscle regeneration. It has previously been suggested that the success of stem cell transplantation could therefore be dependent both on the properties of the cell itself and on the host muscle environment. Here we engrafted young and mature adult mdx-nude mice, which are the genetic homolog of Duchenne muscular dystrophy, with a small number of satellite cells freshly isolated from young, normal donor mice. We found that the donor satellite cells contributed to muscle regeneration and self-renewal as efficiently within mature adult, as in young, dystrophic host muscle. Donor-derived satellite cells also contributed to robust regeneration after further injury, showing that they were functional despite the more advanced dystrophic muscle environment. These findings provide evidence that muscle tissue in a later stage of dystrophy may be effectively treated by stem cells.

Figure 1

Validation of host and donor mice as an appropriate experimental model. (A): Relative telomere length was significantly lower in TA of 12- and 11-month-old, respectively, mdx and mdx-nude mice compared with young/adult 2-month mdx mice; diaphragm telomere length was significantly reduced only in mature adult mdx-nude mice compared with the young/adult mdx (n = 7, three mice were used in the young/adult mdx mouse group, and two mice were used in each of the other two groups; one-way analysis of variance test, ***, p < .001). (B): The collagen VI-positive area was measured in representative sections of TA muscle from 3-week-old (n = 4) and 9-month-old (n = 3) mdx-nude mice and of 12-month-old (n = 3) and 24-month-old (n = 2) mdx mice. Scale bars: 100 μm. (C): Expression of the Myf5^nLacZ/+ locus and Pax7 expression are retained in satellite cells on myofibers from aged 1.5- to 3-year-old Myf5^nLacZ/+ mice (n = 4). Abbreviations: EDL, extensor digitorum longus; GA, gastrocnemius; TA, tibialis anterior.

Figure 2

Fresh satellite cells stripped from single fibers of young mice efficiently regenerate and self-renew when injected in irradiated mature adult mdx-nude muscles. Four hundred satellite cells physically dissociated from extensor digitorum longus myofibers of 3F-nLacZ-2E and Myf5^nLacZ/+ mice were injected into the tibialis anterior (TA) of mdx-nude recipient mice. Hindlimbs of one group of mice were exposed to 18-Gy γ-irradiation 3 days before grafting. In the irradiated TA host muscles, the amount of muscle regenerated by donor cells—quantified as dystrophin-positive fiber numbers corresponding in serial sections to the same fibers incorporating X-gal positive nucl—was generally higher (B) in comparison to the nonirradiated engrafted muscles (A). (C, D): Amount of donor muscle obtained from grafts, respectively in nonirradiated and irradiated mice (dystrophin immunostaining and serial section X-gal staining showing β-gal expressing myonuclei). However, two of eight individual engraftments in the irradiated mouse group did not show any sign of donor muscle regeneration (B), and rare β-gal-positive cells were detectable only in three of seven engraftments in nontreated mdx-nude mice, giving rise to limited groups of dystrophin-positive fibers (C). Interestingly, three of eight engraftments of 400 satellite cells mechanically isolated from Myf5^nLacZ/+ fibers adopted the satellite cell position expressing the Myf5^nLacZ/+ marker on single fibers, as seen from X-gal staining on the whole harvested muscles (F). A similar proportion (two of seven) of nonirradiated muscles grafted with the same donor cells showed the presence of myofibers carrying X-gal-positive satellite cells, as shown in E. Asterisks mark the same myofiber in each serial section. White squares show the area reported in the higher-magnification pictures. Scale bars: 100 μm.

Figure 3

Adult satellite cells regenerate and self-renew equally well in the young and mature adult muscle environment. Four hundred satellite cells isolated from 3F-nLacZ-2E single fibers were injected in young (21 day old) and mature adult (250 day old) tibialis anterior of mdx-nude mice. (A, B): Four weeks after cell injections, a comparable amount of muscle of donor origin (number of dystrophin-positive fibers colocalized with β-gal-expressing myonuclei) was found in mature adult and young host mice. A negligible amount of muscle was formed when cells were injected in nonirradiated muscles both in the young and mature adult environments. (C, D): Two comparable grafts into irradiated host muscles, in which similar amounts of muscle were formed by donor satellite cells. Injections of 400 satellite cells isolated in the same way from Myf5^nLacZ/+ donor mice gave rise to comparable numbers of self-renewed satellite cells when injected both in (E) young (seven of seven successful engraftments; magnification, ×4 and ×10) and in (F) mature adult (six of six successful engraftments; magnification, ×4 and ×10) irradiated muscles. Asterisks indicate the same myofiber in serial sections. White squares indicate the area shown in the higher-magnification pictures. Scale bars: 100 μm.

Figure 4

Donor satellite cells injected in mature adult and irradiated muscle tissue are functional. Four hundred satellite cells isolated both from 3F-nLacZ-2E and Myf5^nLacZ/+ single fibers were injected into tibialis anterior (TA) muscles of mature adult mice; recipient muscles were notexin injured 3 weeks after cell injection and removed 1 week later. (A, B): Satellite cells of donor origin were able to generate new myofibers after the notexin damage, producing groups of new myofibers coexpressing dystrophin and neonatal myosin proteins and incorporating 3F-nLacZ-2E donor myonuclei shown by X-gal staining in serial sections. (C): Myf5^nLacZ/+ donor satellite cells were detected as several β-gal-positive nuclei in X-gal-stained whole TA muscles (magnification, ×4 and ×10). (C’): Single fibers were dissected out from the X-gal-stained muscles and 4′6-diamidino-2-phenylindole stained, showing in particular the localization of the detected Myf5^nLacZ/+ satellite cells on single myofibers. Asterisks indicate the same myofiber in each serial section. White squares highlight areas shown in the pictures at higher magnification. Scale bars: 100 μm.