Mature Adult Dystrophic Mouse Muscle Environment Does Not Impede Efficient Engrafted Satellite Cell Regeneration and Self-Renewal

Muscle DNA Extraction and Real-Time Polymerase Chain Reaction Assay

Total genomic DNA used for this assay was extracted after phenol-chloroform extraction (Sigma, St. Louis, MO, http://www.sigmaaldrich.com) with isopropanol-ethanol precipitation (Sigma) from tibialis and diaphragm muscle tissues of 2-month-old mdx mice (n = 3), 12-month-old mdx mice (n = 2), and 11-month-old mdx-nude mice (n = 2). Relative quantification using real-time analysis was performed by modifying a previously published protocol [22]. Housekeeping and telomere sequences were amplified using primers previously published, and real-time polymerase chain reaction (PCR) reactions were optimized (supporting information Fig. 1) using an automated thermocycler (Abi Prism 7700 Sequence Detection System; Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) as follows: 95°C for 10 minutes, followed by 40 cycles of data collection at 95°C for 15 seconds, with 30 seconds at temperature annealing, and a final step of extension at 72°C for 30 seconds. The annealing temperature was set at 51°C for the housekeeping acidic ribosomal phosphoprotein PO (36B4) gene and at 58°C for the telomere portion. Each reaction volume contained 12.5 μl Syber Green PCR Master Mix (Applied BioSystems), 300 nM each of the forward and reverse primers, and double-distilled water to give a final volume of 25 μl. The assay was performed on three 5-ng samples of DNA from each animal sample, placed in adjacent wells in 96-well plates. For calculating the standard curve, an individual sample of DNA was serially diluted from 10 to 0.01 ng both for 36B4 and the telomere product. Real-time PCR results were exported and analyzed with Excel program (Microsoft, Redmond, WA, http://www.microsoft.com), and standard curves were generated according to the relative standard curve protocol reported in the Applied Biosystems Prism 7700 Sequence Detection System user’s manual. Input amount of amplified DNA were calculated for each reaction. The relative amount of telomere PCR was divided by the relative amount of the 36B4 obtained in each reaction (three repeated reactions for each considered sample). The average of the ratios of telomere:36B4 per sample was reported as “relative telomere length,” and the average telomere lengths in both tibialis anterior (TA) and diaphragms in young mdx mice were set as references.

Single Myofiber Isolation

Single myofibers were isolated from muscles of 3F-nLacZ-2E mice [23] and of Myf5^nLacZ/+ mice [24] as previously described [3]. For characterization of Myf5^nLacZ/+ marker expression on aged fibers, extensor digitorum longus (EDL), TA, soleus, and gastrocnemius (GA) muscles were dissected out from 1.5- to 3-year-old Myf5^nLacZ/+ mice, digested in 2% (wt/vol) collagenase type I (Sigma) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, http://www.invitrogen.com) for up to 1.5-2 hours in a 35°C water bath, and after serial washes to discard cell contaminants, they were fixed for X-gal staining and immunostaining.

For satellite cell grafting, EDL myofibers were similarly isolated from 5- to 8-week-old mice. All fibers were collected in one plate in 8 ml of plating medium (DMEM supplemented with 10% horse serum; Gibco), 005% chick embryo extract, 4 mM l-glutamine (Sigma), and 1% penicillin and streptomycin antibiotics (Sigma).

Immunohistochemistry of Single Myofibers

An aliquot of fixed single myofibers isolated from aged Myf5^nLacZ/+ mice were X-gal stained as previously described [25]. Another group of fibers from the same preparation was permeabilized with 0.5% Triton X-100 (Sigma), blocked with 10% goat and 10% swine serum, and incubated overnight with primary antibodies mouse anti-Pax7 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww.com) and rabbit anti laminin (Sigma). Secondary antibodies used were Alexa Fluor 488-conjugated goat anti-mouse Ig (Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com) and Alexa Fluor 594-conjugated goat anti-rabbit Ig (Molecular Probes).

Satellite Cell Preparation by Physical Titration for Grafting

Fibers collected in one plate in plating medium were triturated using a 1-ml syringe with a 19-gauge needle for 5 minutes to release from single fibers satellite cells. Myofibers were removed by passing the solution through a 40-μm cell sieve. Cells were centrifuged for 20 minutes, the pelleted cells were resuspended, and trypan blue-excluding cells were counted. For grafting, cells were resuspended in medium and kept on ice until injection.

Cell Grafting and Muscle Damage

Mice were bred, and experimental procedures were carried out in the Biological Services Unit, of both Imperial College Faculty of Medicine, Hammersmith Hospital, and of University College London, Institute of Child Health, in accordance with the Animals (Scientific Procedures) Act 1986. Experiments were carried out under Home Office license. Aged mice were given Septrin (Glaxo-SmithKline, UK, http://www.gsk.com) in their drinking water (sulfamethoxazole 1.2 mg/ml and trimethoprim 0.24 mg/ml) for 3 days a week, followed by no antibiotics for the remaining 4 days a week.

Three- to 4-week-old or 9-month-old mdx-nude mice were anesthetized, and both hindlimbs were irradiated with 18 Gy, as described previously [26]. Controls were age-matched mice that were nonirradiated. Three days later, both irradiated and nonirradiated host mice were anesthetized with isoflurane, and 400 satellite cells were grafted in 4 μl of medium into the TA muscles using pulled PCR pipettes under microscopic observation.

In some experiments, 3 weeks after grafting, both TA muscles of anesthetized host mice were injected with 10 μl Notechis scutatus scutatus (notexin; Latoxan Latoxan, France, http://www.latoxan.com) 10 μg/ml) using a Hamilton syringe [27].

In experiments aimed to study the regenerative capability of TA muscles in both 9-month-old and 3-week-old mdx-nude mice, 15 μl notexin (10 μg/ml) was injected per leg.

Vetergesic (Buprenorphine (Reckitt Benckiser Healthcare, UK, http://www.reckittbenckiser.com); 0.05 mg/kg) was administered for postoperative analgesia.

Muscles Harvesting and Immunohistochemistry

Engrafted muscles were removed 4 weeks after cell injection, and when notexin was used, 1 week after myotoxin injection. Muscles that had been grafted with 3F-nLacZ-2E satellite cells were frozen in isopentane cooled in liquid nitrogen. Seven-micrometer transverse cryosections were collected at 100-μm intervals from the entire muscle. Sections were X-gal stained as previously described [27] and, only if positive signal was detected (thus under-reporting the amount of donor muscle, but avoiding inclusion of host, revertant fibers in our quantification of donor-derived muscle fibers), serial sections were immunostained using primary antibodies rabbit anti-dystrophin (P7) and, in some experiments, with mouse anti-neonatal myosin (BF34; Developmental Studies Hybridoma Bank) after blocking with 10% goat serum.

Collagen VI (Abcam, Cambridge, U.K., http://www.abcam.com) and laminin (Sigma) antibodies were used in representative sections of TA muscle from 3-week-old (n = 4) and 9-month-old (n = 3) mdx-nude mice and of 12- (n = 3) and 24-month-old (n = 2) mdx mice for detection of increase in connective tissue indicative of fibrosis Secondary antibodies used were as follows: Alexa Fluor 488-conjugated goat anti-mouse Ig (Molecular Probes) and Alexa Fluor 594-conjugated goat anti-rabbit Ig (Molecular Probes).

Muscles that had been grafted with Myf5^nLacZ/+ satellite cells were fixed in paraformaldehyde and X-gal stained as described elsewhere [3, 27].

Microscopy

Fluorescence and bright-field microscopy and image were captured using a Zeiss Axiophot (Carl Zeiss, UK, http://www.zeiss.co.uk) microscope and Metamorph (Metamorph Productions, UK, http://metamorph-productions.co.uk.) software. Minor adjustments to the quality of images were done using Adobe Photoshop CS2 (Adobe Photoshop UK, http://www.Adobe.com.)

Data Analysis

Counts were made of the maximum number of donor (dystrophin positive) fibers in transverse sections of grafted muscles serial to those where donor, X-gal-positive myonuclei were detected.

INSTAT was used for statistical analysis. For quantification of fibrosis, the total area of four randomly encountered fields of one representative section of each muscle that was collagen VI positive was measured using SigmaScan software. The fibrotic index (%) was defined as the percentage of the total area that was collagen VI positive.

Dystrophin-Deficient Mdx-Nude Mouse Muscles Age Prematurely

The immunodeficient mdx-nude mouse is a good model for testing the capacity of donor-derived cells to regenerate skeletal muscle [3, 6, 21]. We wished to compare the function of grafted satellite cells in young mice at a time when the host muscles are undergoing extensive degeneration and regeneration [28] with mice whose muscles are at a later stage of the pathological process, when the dystrophic phenotype has evolved to more resemble that found in DMD [29]. However, the immunodeficiency of these mice, which has the advantage of preventing immunological rejection of donor cells, reduces their lifespan [20]. To have enough healthy old mice for our experiments, the mice were treated with septrin and were kept until they were 9 months of age, after which mice began to die despite the antibiotic regimen. We therefore performed our comparison on 3- to 4-week-old and 9-month-old mice (that we defined as “mature adult” mice), to have a statistically significant number of recipient mice able to survive the anesthesia needed for hind limb irradiation and cell injection and live long enough for analysis 4 weeks later.

Although satellite cells turnover throughout life, for homeostasis and to contribute to muscle growth or repair, this process becomes less efficient with age as cells reach the end of their proliferative lifespan and the reserve satellite cell pool therefore decreases [6, 17]. This phenomenon is particularly accentuated in aged and in dystrophic muscles, whose satellite cells have undergone the most divisions [17]. Telomere length has been suggested as a biomarker for somatic cell turnover, because telomeres shorten after cell replication [30], and it has been shown that the telomere length is significantly shorter in dystrophic [2] and in older adult muscle tissue [31].

To determine whether older mdx-nude mouse muscles exhibit telomere shortening, we compared relative telomere length in young 2-month-old mdx mice with 12-month-old mdx mice and 11-month-old mdx-nude mice. Interestingly, in TA muscles of both 12-month-old mdx mice and 11-month-old mdx-nude mice, relative telomere length was greatly and similarly reduced in comparison to the reference young mouse group (Fig. 1A; one-way analysis of variance test, ***, p < .001). Intriguingly, when we studied the diaphragm muscles of these mice, there was a significant reduction in relative telomere length in the 11-month-old mdx-nude mice (Fig. 1A; one-way analysis of variance test, ***, p < .001), in comparison with both the young 2-month-old mdx mice and the 12-month-old mdx mice. However, diaphragm telomere length was not significantly different between young 2-month-old and the mature adult 12-month-old mdx mice.

We also assessed the degree of fibrosis in the TA muscles of mature adult mdx-nude mice in comparison with mdx mice, because this is an important hallmark of the more advanced dystrophic muscle phenotype. We found no obvious difference in collagen VI content of mdx-nude (young and mature adult) compared with mdx (mature adult and aged) TA muscles (Fig. 1B and 1C), showing that the nude genotype does not significantly affect fibrosis in this particular muscle.

Furthermore, skeletal muscle regeneration persists, albeit at a lower level, with age in mdx-nude (supporting information Fig. 2), as has been shown for mdx mice [28, 29, 32]. Nevertheless, regeneration induced by an applied injury, notexin injection, elicited a similar regenerative response in TA muscles of both young and mature adult mdx-nude mice (supporting information Fig. 2).

The reduction in telomere length and features of advanced pathology—increased central nucleation and numbers of revertant fibers (supporting information Fig. 2) and fibrosis that we found in mature adult mdx-nude TA muscles is in keeping with previous literature on mdx mice [29, 32, 33]. These features of advanced muscle pathology, combined with reduced lifespan of mdx-nude mice, made us confident that our 9-month-old mdx-nude mice are a suitable model for late-stage muscular dystrophy.

Expression of Markers of Satellite Cells and Myonuclei of Donor Origin Is Retained in Mature Adult Muscle

We used myosin light chain as a marker of regenerated myofibers in mature adult host mice, because transgene 3F-nLacZ-2E expression has been shown to be retained in myofibers of older mice [6]. However, we did not know whether Myf5^nLacZ/+ expression is retained within the aged muscle environment and whether it is therefore a good marker for detecting donor satellite cells within older host muscles. To determine whether Myf5^nLacZ/+ does mark all satellite cells in aged muscles, we stained isolated fibers from different muscles (EDL, TA, soleus, GA) from 1.5- to 3-year-old Myf5^nLacZ/+ mice with either Pax7 antibody or X-gal, for detecting the two main markers of satellite cells [3]. We found comparable numbers of Pax7-positive and Myf5^nLacZ/+-expressing satellite cells, confirming that Myf5^nLacZ/+ gene expression is retained, as is Pax7, within aged muscle and that the targeted Myf5 allele of the donor mouse strain is a good model to assay satellite cells of donor origin within older host mice (Fig. 1D).

Donor Satellite Cells Regenerate and Self-Renew Within Mature Adult Dystrophic Muscle

To study the behavior of a known number of freshly isolated, pure satellite cells, we mechanically dissociated satellite cells from donor myofibers [6, 7]. To first investigate the regenerative potential of satellite cells in a mature adult dystrophic environment, satellite cells were isolated from 3F-nLacZ-2E adult myofibers to allow detection of donor-derived muscle fibers (expressing dystrophin- and containing β-gal-positive nuclei).

Irradiation is known to modify the muscle environment of young immunodeficient host mouse muscle, allowing donor-derived satellite cells to regenerate and self-renew [3, 6, 21, 34]. To test whether it also has the same effect on mature adult muscle, the right TA muscle of seven recipient mice that had not been irradiated and of eight mice that had both hind limbs irradiated with 18 Gy 3 days before grafting [19, 27] were transplanted with 400 freshly isolated 3F-nLacZ-2E satellite cells. Four weeks after cell grafting, cryosections were analyzed both for the presence of X-gal-positive nuclei and the restoration of dystrophin protein [3].

Satellite cells of donor origin contributed to regenerated muscle fibers within 9-month-old mdx-nude host muscles. However, as shown in Figure 2A and 2B, the amount of donor-derived muscle formed within irradiated host muscles was variable. It is interesting to note that, as in previous experiments in which either satellite cells [3, 6] or muscle precursor cells [21] were grafted into young mouse hosts, there is a marked variability between grafted muscles within an experimental group. Nevertheless, it is clear that large amounts of donor-derived muscle were only found within irradiated host muscles most likely close to the injection site (Fig. 2A–2D; 51 ± 26 dystrophin-positive fibers, 7 ± 4 X-gal-positive nuclei in irradiated, 7 ± 3 dystrophin-positive fibers, 1 ± 1 X-gal-positive nuclei in nonirradiated host mouse muscles).

To examine whether transplanted satellite cells also self-renewed in the mature adult dystrophic environment, donor satellite cells were prepared in the same way from Myf5^nLacZ/+ donor mice and grafted into the contralateral (left TA) of the mature adult mdx-nude host mice used above. Four weeks later, X-gal staining of whole grafted TA muscles showed cells of donor origin, presumed to be mostly satellite cells [3], expressing Myf5^nLacZ/+ in three of eight irradiated host muscles (Fig. 2F). In nonirradiated host muscles, the amount of satellite cells of donor origin (found in only two of seven grafted muscles; Fig. 2E) was appreciably reduced. It seems therefore that donor satellite cell self-renewal capability is correlated with their regenerative potential. We conclude that satellite cells can self-renew in a mature adult dystrophic environment and that preirradiation of host muscle enhances repopulation of the satellite cell pool.

Donor Satellite Cells Regenerate Skeletal Muscle and Self-Renew as Efficiently in Mature Adult as in Young Dystrophic Muscle

Four hundred freshly isolated 3F-nLacZ-2E satellite cells were injected into the right and left TA muscles, respectively, of both young (n = 10) and mature adult (n = 10) host mdx-nude mice, some of which (n = 6 in both cases) had both hindlimbs irradiated with 18 Gy 3 days before grafting. Muscles were removed for analysis 4 weeks after grafting, as described above. In irradiated host muscles, muscle of donor origin was found in five of six young and six of six mature adult host muscles, and the numbers of muscle fibers of donor origin were not significantly different (one-way analysis of variance test) between young (238 ± 90 dystrophin-positive fibers, 51 ± 21 X-gal-positive nuclei) and mature adult recipient mice (241 ± 70 dystrophin-positive fibers, 23 ± 9 X-gal-positive nuclei (Fig. 3A, 3B), as can be seen by dystrophin immunostaining and parallel X-gal staining on serial sections of grafted muscles (Fig. 3C, 3D).

The extent of donor-derived repopulation of the satellite cell pool was similar within irradiated muscles of host mice of both ages. A similar incidence and amount of donor-derived Myf5^nLacZ/+ satellite cells was obtained 4 weeks after injection of freshly isolated Myf5^nLacZ/+ satellite cells into both young (six of six positive grafts) and mature adult host mice (six of six positive grafts; Fig. 3E, 3F).

In nonirradiated host muscles, muscle fibers of donor origin were found in only two of four host muscles at both ages. The number of muscle fibers of donor origin was similarly low both in young and mature adult recipient mice (3 ± 2 dystrophin-positive fibers and 2 ± 1 X-gal-positive nuclei in young hosts and 8 ± 5 dystrophin-positive fibers, 3 ± 2 X-gal-positive nuclei in mature adults; no significant difference by one-way analysis of variance test; Fig. 3A, 3B). The incidence and number of donor-derived satellite cells was similarly low, with only sporadic X-gal-positive nuclei present in two of four host mouse muscles of both ages (data not shown).

Satellite Cells of Donor Origin Are Functional Within Mature Adult Dystrophic Host Muscle

To be true stem cells, satellite cells of donor origin must, in the presence of appropriate stimuli, be able to regenerate the muscle tissue and again repopulate the satellite cell compartment weeks after transplantation, as they are able to do in young dystrophic host muscle [3]. To study the functional ability of donor-derived satellite cells within the mature adult dystrophic host muscle environment, we injected notexin into muscles that had been grafted 3 weeks previously with either 3F-nLacZ-2E (right TA) or Myf5^nLacZ/+ (left TA) satellite cells. Notexin is a snake venom that causes myofiber destruction but spares satellite cells, leaving undamaged basal lamina and microcirculation [35]. One week later, muscles were removed for analysis.

Newly regenerated muscle fibers (expressing neonatal myosin) of donor origin (expressing dystrophin) were present within the mature adult dystrophic host muscles, showing that satellite cells of donor origin remained functional after several weeks within this environment. The mean number of newly regenerated muscle fibers of donor origin after notexin injury in our mature adult mdx-nude mice was comparable to that previously obtained in young host mice [3, 6]: (six of six successful grafts, containing a mean of 221 ± 117 dystrophin and neonatal myosin-positive fibers, 35 ± 17 X-gal-positive nuclei; Fig. 4A and 4B). In one host muscle, more than 600 newly regenerated muscle fibers of donor origin were found (Fig. 4A).

In the two grafts in nonirradiated mature adult mdx-nude mouse muscles, a negligible number of newly regenerated, donor-derived muscle fibers was obtained (4 ± 0 dystrophin- and neonatal myosin-positive fibers, 3 ± 1 X-gal-positive nuclei).

Within irradiated mature adult dystrophic host muscles that had been injected with My5^nLacZ/+ satellite cells, allowed to regenerate, and challenged with notexin, the number of X-gal-positive nuclei, found in all four grafted muscles (Fig. 4C), was appreciably greater than in nonirradiated muscles that had been grafted and injected with notexin, in which very few β-gal-expressing satellite cells were detected (data not shown). Because β-gal translocates between myonuclei of donor origin [3, 36], only cells on single fibers that expressed X-gal and were surrounded by 4′6-diamidino-2-phenylindole-positive/X-gal-negative nuclei, implying lack of cytoplasmic continuity of that cell with the myofiber [36], are confirmed as satellite cells (Fig. 4C′). Such analysis of single myofibers dissected out from the whole X-gal-stained, irradiated and notexin treated host muscles, confirmed that the X-gal activity was indeed in satellite cell nuclei.

We therefore provide clear evidence that satellite cells of donor origin had expanded and repopulated the satellite cell pool in irradiated mature adult host muscles and that they remained functional within this more advanced dystrophic environment.