¹Muscle Cell Biology Group, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London, UK

²CNRS URA 1947, Département de Biologie Moléculaire, Institut Pasteur, Paris, France

Correspondence to: P S Zammit, Muscle Cell Biology Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK

Myoblast transplantation is a potential therapeutic approach for the genetic modification of host skeletal muscle tissue. To be considered an effective, long-lived method of delivery, however, it is essential that at least a proportion of the transplanted cells also retain their proliferative potential. We sought to investigate whether transplanted neonatal myoblasts can contribute to the satellite cell compartment of adult skeletal muscle by using the Myf5^nlacZ/+ mouse. The Myf5^nlacZ/+ mouse has nlacZ targeted to the Myf5 locus resulting in -galactosidase activity in quiescent satellite cells. Following transplantation, -galactosidase-labelled nuclei were detected in host muscles, showing that donor cells had been incorporated. Significantly, -galactosidase-positive, and therefore donor-derived, satellite cells were detected. When placed in culture, -galactosidase marked myogenic cells emanated from the parent fibre. These observations demonstrate that cell transplantation not only results in the incorporation of donor nuclei into the host muscle syncytia, but also that the donor cells can become functional satellite cells. The Myf5^nlacZ/+ mouse therefore provides a novel and specific marker for determining the contribution of transplanted cells to the satellite cell pool. Gene Therapy (2001) 8, 778-783.

skeletal muscle; satellite cell; Myf5; transplantation; gene therapy

Introduction

Adult skeletal muscle is composed of myofibres that contain many postmitotic myonuclei sharing a common cytoplasm. These cells are maintained by a population of mononucleated cells - satellite cells, residing between the basal lamina and plasmalemma of the myofibres.^1,2 During postnatal development, satellite cells proliferate to supply myonuclei to the growing muscle fibres.³ In mature muscle, satellite cells become mitotically quiescent,⁴ forming a reserve myogenic cell pool. They can be activated to proliferate in response to muscle damage, subsequently fusing and differentiating to produce new myonuclei to replace those lost.^5,6

The ability of myoblasts to become incorporated into postnatal skeletal muscle during regeneration is exploited by myoblast transplantation, a potential therapeutic approach for inherited myopathies.⁷ Myoblast transplantation has been widely investigated as a possible therapy for Duchenne muscular dystrophy, a myopathy caused by the lack of dystrophin.⁸ Transplantation of muscle precursor cells (mpc) into the mdx mouse, an animal homologue of Duchenne muscular dystrophy,⁹ has shown that normal myoblasts can participate in regeneration resulting in dystrophin expression in the host muscle.⁷ However, for cell transplantation to be considered as an effective, long-lived therapy it is essential that not only is damaged tissue repaired, but that the precursor cell compartment be replenished to provide for future bouts of regeneration.

To determine whether cell transplantation can contribute to the satellite cell compartment, we used donor cells derived from the Myf5^nlacZ/+ mouse, which has a nuclear localising lacZ (nlacZ) reporter gene targeted to the Myf5 locus.¹⁰ Myf5 is a member of a family of basic helix-loop-helix transcription factors which also includes MyoD, myogenin and MRF4 (reviewed in Ref. 11). Together with MyoD, Myf5 is integral to the determination of the myogenic lineage.^10,12 Myf5^nlacZ/+ mice demonstrate -galactosidase activity in quiescent satellite cells¹³ and following muscle injury in vivo, activated satellite cells contain -galactosidase and MyoD.¹⁴

Accordingly, we have transplanted primary myoblasts, derived from neonatal Myf5^nlacZ/+ mice into pre-irradiated mdx nude (nu/nu) mice. -Galactosidase-positive nuclei could be detected in host muscle fibres 26 days after injection, indicating that donor cells had been incorporated into the muscle syncytia. Some -galactosidase-labelled nuclei were those of satellite cells which had adopted peripheral positions under the basal lamina, separated from the syncytia by the plasmalemma, the characteristic position of satellite cells. -Galactosidase-positive cells emanated from host fibres placed in culture, showing that donor-derived proliferative cells were associated with the myofibres. Together, these observations demonstrate that cell transplantation not only results in the incorporation of donor nuclei into host muscle syncytia, but importantly, also contributes functional satellite cells.

Results

-Galactosidase activity identifies quiescent satellite cells in Myf5^nlacZ/+ mouse muscle

Quiescent satellite cells of the extensor digitorum longus (EDL) muscle of Myf5^nlacZ/+ mice are -galactosidase-positive.¹³ To determine whether -galactosidase activity can be detected in quiescent satellite cells associated with both fast and slow muscle fibre types, isolated single myofibres were prepared from adult (~6 weeks postnatal) EDL, tibialis anterior (TA) and soleus muscles of Myf5^nlacZ/+ mice. These muscles were chosen as between them they contain the four major muscle fibre types; TA and EDL are predominately composed of fast IIb and IIx myosin heavy chain type, whereas the soleus muscle mainly consists of the slow type I and fast IIa phenotypes.^15,16 Isolated myofibres, fixed within 2 h of animal death, had a limited number of -galactosidase-positive nuclei that were within cells that also expressed the satellite cell marker CD34 (Figure 1a and b).¹³ These -galactosidase-labelled nuclei, therefore, could be identified as those of satellite cells. Whole mount preparations of tail and forelimb muscle also demonstrated nuclear -galactosidase activity that is also indicative of satellite cell location (data not shown).

Single myofibres prepared from Myf5^nlacZ/+ mice produce myogenic cells that are -galactosidase-positive

During muscle regeneration in the Myf5^nlacZ/+ mouse, activated satellite cells are -galactosidase- and MyoD-positive.¹⁴ Culturing single fibres in the presence of serum also activates the associated satellite cells; the cells migrate from the fibre and subsequently proliferate.¹⁷ Myofibres were prepared from EDL, soleus or TA muscles of adult Myf5^nlacZ/+ mice and placed in culture. After 24-48 h, cells had migrated from the myofibres. Incubation in X-gal or immunostaining demonstrated that these cells were -galactosidase-positive. Co-expression of desmin with -galactosidase confirmed that these cells were myogenic (Figure 1c). The majority of -galactosidase-positive cells also expressed MyoD, although the relative levels of these proteins varied between individual cells (Figure 1d-f).

Myf5^nlacZ/+-derived neonatal myoblasts are able to contribute to the satellite cell compartment

Because -galactosidase identifies quiescent satellite cells in Myf5^nlacZ/+ mice, we isolated primary muscle cultures from this mouse line to determine if transplanted cells can contribute to the satellite cell pool. The TA muscles of four pre-irradiated mdx nu/nu mice were injected with Myf5^nlacZ/+ mouse-derived neonatal myoblasts and 26 days later, single myofibres were prepared from the recipient muscles. All muscles analysed showed evidence of donor cell incorporation.

Myofibres with incorporated -galactosidase-marked nuclei fell into two categories (Figure 2). In the first, the isolated muscle fibres contained regions in which several -galactosidase-positive nuclei were congregated, some of them centrally located, a characteristic sign of recent regeneration (Figure 2a and b). This observation indicates that donor-derived myoblasts had contributed to myofibre repair. Because of translocation of -galactosidase message between myonuclei in a syncytium¹⁸ the extent of muscle repair cannot be assessed, although at least one myonucleus per myofibre must be of donor origin.

The second pattern of -galactosidase staining consisted of myofibres with occasional isolated -galactosidase-labelled nuclei, that were invariably located on the periphery of myofibres (Figure 2c-h). These -galactosidase-positive nuclei were in cells beneath the basal lamina as the process of isolating single fibres removes practically all extrafusal cells. Furthermore, -galactosidase activity was strictly localised to individual nuclei with no translocation to surrounding myonuclei, suggesting that these nuclei are those of satellite cells, separated from the syncytia by the plasmalemma (Figure 2g-h). These -galactosidase-positive nuclei were sometimes adjacent to a string of centrally placed myonuclei, which are not usually observed following irradiation of mdx nu/nu muscle at 3 weeks of age (Figure 2c-f).¹⁹

Donor Myf5^nlacZ/+-derived satellite cells are able to activate and proliferate

We then sought to establish whether a proportion of the transplanted cells were capable of activation and subsequent proliferation. The host mdx nu/nu muscles were pre-irradiated to incapacitate endogenous satellite cells and most myogenic cell proliferation should be due to mpc of donor origin.¹⁹ Single myofibres prepared from recipient TA muscles were cultured at low density and analysed for -galactosidase activity by incubation in X-gal after 24 and 48 h. -Galactosidase-positive cells were found surrounding the myofibre from which they had migrated (Figure 3a and b). The transplanted neonatal Myf5^nlacZ/+ cells, were therefore capable of activation and proliferation in a manner similar to satellite cells on myofibres isolated directly from Myf5^nlacZ/+ mice (see Figure 1).

Discussion

For cell-mediated gene therapy to be effective, the transplanted cells should contribute to the stem cell/mpc compartment to provide a long term reserve of myogenic cells that could respond to any future insult. Using the Myf5^nlacZ/+ mouse we have shown that transplanting primary myoblasts derived from neonatal muscle can not only lead to repair of damaged fibres, but can also provide a source of functional satellite cells. Whether this occurs following transplantation of human cell preparations in man remains to be seen. However, we do show that cell transplantation can result in a contribution to the satellite cell compartment in a mouse model of Duchenne muscular dystrophy and therefore illustrate the potential of myoblast transplantation as a long-term therapy.

It has been shown previously that muscle cells from neonatal mice can contribute to myofibre repair.²⁰ Other studies have also shown that some implanted myoblasts do not differentiate and remain capable of a proliferative response in vivo²¹ and in vitro.^22,23 However, the cells in question could not be identified or located in vivo. A further study did locate labelled nuclei in peripheral positions on repaired myofibres. When these fibres were placed in culture, myogenic cells were produced, although the majority of these cells were not labelled. The donor mpc used in the study were a retrovirally labelled, conditionally immortal cell line, clonally derived from the H-2K^b-tsA58 mouse.²⁴ Since the label was not a specific satellite cell marker, it did not therefore reveal the identity of donor-derived cells.

In the present study, we have used unmanipulated primary neonatal myoblasts derived from the Myf5^nlacZ/+ mouse, which has a nlacZ reporter gene targeted to the Myf5 locus. Because quiescent satellite cells continue to express Myf5, they can be readily detected in the Myf5^nlacZ/+ mouse by -galactosidase activity.¹³ Following transplantation, myofibres were observed that contained -galactosidase-labelled satellite cells. Further evidence for the contribution to the satellite cell compartment comes from in vitro studies of the recipient muscles. Irradiation of mdx muscle incapacitates endogenous satellite cells, as a result, the muscle shows few signs of spontaneous regeneration^19,25 and when cultured the vast majority of myofibres fail to produce myogenic cells.^24,26 However, a few myofibres prepared from pre-irradiated mdx nu/nu TA muscles that had been injected with Myf5^nlacZ/+-derived myoblasts, produced -galactosidase-labelled myogenic cells in culture. This demonstrates that the transplanted myoblasts give rise to a population of cells with myogenic potential that remain undifferentiated in the host muscle for at least a month, but retain the ability to activate and proliferate. Myoblast transplantation, therefore, is able to provide functional satellite cells to muscle that is otherwise largely unable to produce myogenic cells.

We also observed myofibres prepared from recipient muscles that had regions containing many -galactosidase-labelled donor-derived myonuclei. Some of these donor myonuclei were centrally positioned, a characteristic sign of muscle regeneration. In other myofibres, -galactosidase-negative, centrally placed myonuclei were observed in close proximity to a single -galactosidase-marked satellite cell. These -galactosidase-negative centrally placed myonuclei are likely to be of donor origin as endogenous satellite cells are incapacitated by the irradiation dose given at 3 weeks of age, before the characteristic cycles of muscle degeneration and regeneration begin in the mdx mouse. These centrally placed myonuclei may have down-regulated the targeted nlacZ, a situation analogous to that observed during development of Myf5^nlacZ/+ mouse muscle where perinatally, -galactosidase is detected in myonuclei and mpc,¹⁰ but as the muscle matures, expression becomes restricted to quiescent satellite cells.¹³ Therefore, it is possible that the two categories of myofibre identified in this study are examples of regeneration separated temporally. The Myf5^nlacZ/+ mouse-derived transplanted cells initially give rise to myonuclei which express -galactosidase. The expression in myonuclei is subsequently down-regulated leaving the donor-derived satellite cells as the only -galactosidase-labelled cells.

It has been shown that stem cells from other lineages, such as the haematopoietic system, can adopt a myogenic fate; bone marrow cells implanted either intramuscularly or intravenously are able to produce donor-derived muscle.²⁷ FACS sorting of both bone marrow and muscle has identified a population of cells termed side population (SP), that are able to form muscle and reconstitute the haematopoietic system. The SP is thought to consist of stem cell-like precursors although implantation studies using SP do not show whether these stem cells can renew the regenerative potential of the host tissue.^28,29 Myf5^nlacZ/+ provides an endogenous marker for following transplanted cells and determining if these cells can also become satellite cells, or whether they merely differentiate into post-mitotic muscle. In addition, primary cells from this source do not require expansion in vitro, transfection, retroviral labelling or selection. Furthermore, problems associated with the down-regulation of transgenes and retroviral labels following transplantation are avoided (Ref. 24 and references therein; J Morgan, personal communication).

In conclusion, we have shown that primary, neonatal myoblasts are able to contribute satellite cells to irradiated mdx mouse muscle, an animal model of human Duchenne muscular dystrophy. When challenged, these satellite cells are able to respond with activation and proliferation. In addition, the Myf5^nlacZ/+ mouse provides a novel and specific marker to determine if transplanted cells from various disparate origins can contribute to the satellite cell pool.

Materials and methods

Myf5^nlacZ/+ mouse

The Myf5^nlacZ/+ mouse has nlacZ-SV40poly(A)RNApolII/Neo targeted to the first exon of the Myf5 gene, thus disrupting the gene. Heterozygous Myf5^nlacZ/+ mice are viable, but homozygous Myf5^nlacZ/nlacZ mice die shortly after birth due to respiratory problems.¹⁰

mdx nu/nu mouse

The C57Bl/10 Sc/Sn mdx mouse is a genetic homologue of human Duchenne muscular dystrophy.⁹ From approximately 3 weeks of age the muscles of mdx mice undergo cycles of degeneration and regeneration. Irradiation of mdx muscle with 18 Gy incapacitates endogenous satellite cells resulting in muscle that shows few signs of regeneration.^19,25 Furthermore, myoblast transplantation is more successful following pre-irradiation of host muscle, compared with non-irradiated muscle, and we therefore adopted this strategy to maximise donor-derived cell incorporation.²⁰ Finally, using mdx on a nude (nu/nu) background prevents immunological rejection of transplanted cells.

Donor cell preparation - enzymatic disaggregation of neonatal muscle

A suspension of mononucleated primary cells was prepared according to Watt et al.³⁰ This preparation does not produce a pure myoblast culture, but does contain a high proportion of cells that will undergo myogenic differentiation in culture and produce large amounts of muscle upon transplantation.^20,30 Briefly, neonatal Myf5^nlacZ/+ mice were killed by cervical dislocation and the muscle of the limbs and back removed and digested in a solution of 0.1% pangestin and 0.6% trypsin (Sigma, Poole, UK) in Ca²⁺ and Mg²⁺ free HBSS (Gibco BRL, Paisley, UK) at 37°C for 10 min. The supernatant was then added to inhibition medium (DMEM supplemented with 20% foetal calf serum (PAA Laboratories, Somerset, UK) and filtered through a 45 m nylon filter to remove cell clumps. The suspension was centrifuged at 350 g for 10 min and a count of viable cells was made by trypan blue exclusion. Cells were briefly expanded in culture before injection.

Myoblast transplantation

Three-week-old mdx nu/nu mice were anaesthetised with 50 l of hypnorm (Janssen, Buckinghamshire, UK; fentanyl citrate, final concentration 0.79 mg/ml; fluanisone, final concentration 2.5 mg/ml) and hypnovel (Roche, East Sussex, UK; midazolam, final concentration 1.25 mg/ml), injected subcutaneously. Lower limbs were exposed to 18 Gy of gamma-irradiation.²¹ The bodies of the mice were shielded by 4 cm of lead. Mice were kept on a heated blanket until fully recovered. Three days post-irradiation mice were anaesthetised as described above and a small incision made in the skin covering the TA muscle. Immediately before implantation, donor cells were trypsinised with 0.1% trypsin and 0.04% EDTA and centrifuged at 350 g for 10 min. 5 ´ 10⁵ cells were re-suspended in 10 l of growth medium and kept on ice before implantation. The cell suspension was drawn into a 5 l Hamilton syringe and injected into the exposed TA muscle, two injections per muscle were administered. Incisions were closed with suture silk.

Single myofibre preparation

The method for single fibre preparation has been described in detail elsewhere.^17,31,32 Briefly, mice were killed by cervical dislocation and entire EDL, TA and soleus muscles, including tendons, were removed. Muscles were digested for 60-90 min in 0.2% collagenase in DMEM at 35°C, and then separated into single fibres by trituration.

Single myofibre culture

Single myofibres were placed in Primaria plates (Marathon, London, UK) which had been coated with 1 mg/ml Matrigel (Collaborative Research, London, UK) in DMEM. 0.5 ml of plating medium (DMEM supplemented with 10% horse serum (PAA Laboratories), 0.5% chick embryo extract (ICN Flow, Oxon, UK), 2% l-glutamine (Sigma), 1% penicillin and streptomycin solution (Sigma)) was added per well. Fibres were incubated at 37°C and 5% CO₂.

Histology

Myofibres and cells were fixed for 5 min in 4% paraformaldehyde/PBS at 37°C, followed by several PBS rinses. For X-gal colouration they were subsequently incubated overnight in X-gal solution (1 ´ PBS with 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl₂, 400 g/ml X-gal and 0.02% NP40) at 37°C,³³ before being rinsed in PBS and mounted in Dako Faramount aqueous mounting medium containing 100 ng/ml 4,6-diamidino-2-phenylindole (DAPI).

For immunofluorescence, isolated myofibres were treated as previously described.¹³ For cells in culture, following fixation and permeabilisation in 0.1% triton X-100 in PBS for 12 min, non-specific antibody binding was blocked using 20% goat serum in PBS for at least 30 min. Primary antibodies (anti--galactosidase (G8021 Sigma), mouse anti-human MyoD1 (clone 5.8a, Dako, Cambridge, UK) and rabbit anti-desmin (Sigma)) were added in 6% horse serum/PBS and incubated overnight at 4°C. Secondary antibodies conjugated to either biotin (Dako or Sigma) or a fluorescent chromophore (Molecular Probes, Leiden, The Netherlands; Amersham Pharmacia Biotech, Buckinghamshire, UK), were applied in 6% horse serum/PBS for 2 h at room temperature. Myofibres or cells were mounted in Dako Faramount aqueous mounting medium containing 100 ng/ml DAPI or incubated for a further 2 h in streptavidin conjugated to Texas red (Amersham) or Alexa 488 (Molecular Probes), then mounted. Images were collected on a Zeiss axiophot microscope using Metamorph software (Princeton Instruments, Surrey, UK) and assembled into montages using Adobe Photoshop 5.0.

Acknowledgements

This work was supported by EC Contract QLK3-1999-00020, EC Biotechnology Grant BIO4 CT 95-0228 and The British Council/EGIDE Alliance 2000/2001 grant PN 00.172. The Muscle Cell Biology Group was supported by The Medical Research Council, EC Biotechnology Grants BIO4 CT95-0284 and BMH4 CT97-2767. MB's laboratory was supported by grants from the Pasteur Institute, CNRS, Association Française contre les Myopathies and Ministère des Sciences et Technologies. PZ and LH were also supported by the Leopold Muller Foundation and ST by a grant from the HFSPO. We would like to thank Robert Kelly for invaluable help, and Graham Reed and Richard Newton in the MRC CSC Photography department.

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Figure 1 Satellite and myogenic cells from Myf5^nlacZ/+ mice contain -galactosidase. Freshly isolated soleus myofibres demonstrated -galactosidase (red) in satellite cells (arrowed) as identified by expression of CD34 (green) (a), a satellite cell marker.¹³ The majority of nuclei expressed neither, as shown by DAPI nuclear counter staining (b). By 48 h in culture, soleus myofibres isolated from Myf5^nlacZ/+ mice had produced many cells, the vast majority of which expressed desmin (red) with -galactosidase (green) (c). These cells also co-expressed -galactosidase (e) with MyoD (f), although the relative levels of these protein varied between individual cells (arrows in d-f). DAPI nuclear counter staining revealed all cells present (d). Bar equals 30 m.

Figure 2 Myofibres from pre-irradiated mdx nu/nu TA muscle injected with myoblasts derived from neonatal Myf5^nlacZ/+ muscle. X-gal colouration of myofibres isolated 26 days after injection demonstrated that Myf5^nlacZ/+-derived neonatal myoblasts were able to contribute to repair of TA myofibres (a). The Myf5^nlacZ/+ cells had contributed to the formation of new muscle that showed the classic central nucleation, and contained -galactosidase (a and b). Total myofibre nuclei were revealed by DAPI counter staining (b, d, f, h). In other myofibres, the -galactosidase activity was limited to a small population of peripherally located nuclei (c-f), reminiscent of satellite cells in mature Myf5^nlacZ/+ mice (see Figure 1). In addition, adjacent nuclei were not blue, indicating that translocation of -galactosidase had not occurred, again confirming that these nuclei were those of satellite cells, separated from the syncytia by the plasmalemma (g and h). Bar equals 120 m for a-d and 30 m for e-h.

Figure 3 Myogenic cells emanating from myofibres prepared from pre-irradiated mdx nu/nu TA muscles that had been injected with myoblasts derived from neonatal Myf5^nlacZ/+ muscle. Myofibres isolated 26 days after transplantation were cultured for 48 h. X-gal colouration revealed -galactosidase-positive cells had migrated from these myofibres. This indicates that cells of donor origin had remained as undifferentiated cells in the host muscle for 26 days and were subsequently activated in vitro (a and b). The myofibres recovered from irradiated and injected muscles are usually small and irregularly shaped. Bar equals 100 m.