Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.

Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majority.

Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

The ontogeny of soleus muscles in mdx and wild type mice.

The satellite cell, the organotypic muscle stem cell, is the key element in ontogenetic and load induced muscle fibre growth and repair. It is therefore possible that the satellite pool becomes exhausted with age, especially in mdx mice where dystrophin deficiency results in skeletal muscle degeneration. We compared structural criteria and satellite cell frequencies in soleus muscles of 26 mdx and 23 wild type mice aged between 26 and 720 days. The total number of muscle fibres was similar in both groups and remained stable throughout life, except for an early increase in wild type mice. However, in mdx muscles there was always a proportion of small-diameter fibres which resulted in a reduction in the effective myogenic area on cross-section, whereas total cross-sectional area and muscle weights were increased relative to controls throughout life. In adult animals, the frequency and numbers of satellite cells remained stable with age and were similar in both animal groups. Satellite cell numbers showed some considerable variation between individual animals, although with a markedly smaller variability between results of the same animal, pointing to the satellite cell pool being an individual variant.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

TNF inhibits insulin induced STAT5 activation in differentiated mouse muscle cells pmi28.

Tumor necrosis factor (TNF) plays a central role in the state of insulin resistance leading to type II diabetes. We here describe the crosstalk of TNF with insulin signaling cascades in the mouse muscle cell line pmi28. TNF downregulated insulin induced insulin receptor kinase activity and insulin induced activation of the transcription factor STAT5. Our results provide evidence that the inhibitory crosstalk between TNF and insulin in skeletal muscle cells comprises an interference with the expression of STAT5 regulated genes which may play an important role in the manifestation and/or progression of insulin resistance in muscle cells.

Regenerative capacity and the number of satellite cells in soleus muscles of normal and mdx mice.

Satellite cells are potential myogenic cells that participate in repair and growth of muscle fibres. In this investigation, the change in the number of satellite cells following severe muscle damage was monitored in soleus muscle of age-matched mdx and C57Bl/10 mice. Satellite cells were identified immunohistochemically in the light microscope by their association with a recently described marker protein, M-cadherin, and their location between the muscle fibre's sarcolemma and the surrounding basal lamina. In cross-sections of untreated soleus muscle of C57Bl/10 mice at 11-14. 5 months of age, nuclei of M-cadherin positive satellite cells on average amounted to 3.4% of the total number of myonuclei. Surprisingly, significantly higher numbers of satellite cell nuclei, both in absolute numbers (mean 24+/-11 versus 40+/-11 satellite cells per section) and relative to the total number of myonuclei (5. 3%), were found in similarly aged animals in which severe muscle damage had been inflicted 3-6 months before. Cross-sectional area, muscle tissue area and myonuclei counts had recovered to control values. In untreated muscles of age-matched mdx mice satellite cell counts were not different (2.7% of myonuclei) from C57Bl/10 mice. However, regeneration showed marked deficits, as there was a loss of about 36% total cross-sectional area, about 48% total muscle fibre area and about 43% myonuclei per section compared to the untreated mdx muscles. Furthermore, the absolute number of satellite cells decreased from 20+/-11 to 12+/-8 per section. The relative number of satellite cell nuclei remained comparable to, but did not exceed, the undamaged muscles. The poor recovery of muscle and the missing post-regeneration rise in satellite cell numbers may indicate the reproductive limits of the satellite pool.

Impaired functional and structural recovery after muscle injury in dystrophic mdx mice.

We compared functional and structural recovery from imposed muscle injury in mdx and wild type mice to test their regenerative capacity. Soleus muscle, known to be particularly affected by the disease process, was subjected to most severe damage caused by freeze injury plus 'bystander damage'; the latter causes destruction of host muscle cells in the course of immune rejection of implanted non-histocompatible myogenic cells. Freezing/implantation was performed in mdx and control mice at two ages (4-6 months, "young' and 10-12 months, 'old' age). While recovery of muscle force in the control groups reached 77 and 88% of contralateral by 3 and 6 months, it was 60% and only 43% in mdx mice damaged at young and old age, respectively. Larger force deficits in mdx mice were due to loss of muscle tissue as measured from desmin-positive areas. Worse recovery of dystrophic muscles in general, and old muscles in particular, is interpreted to indicate pronounced exhaustion of the regenerative capacity, possibly caused by previous cycles of degeneration and regeneration.

Acetylcholine receptor bars and transmitter release in frog neuromuscular junctions.

After labelling with rhodaminated alpha-bungarotoxin, acetylcholine receptors in cutaneous pectoris muscles of normal adult frogs (Rana temporaria) appear as brightly fluorescent straight bars, usually extending over the whole gutter. Here we investigated first whether receptor bars can undergo changes and secondly whether they would provide a structural correlate for the strength of a junction. Bars of low fluorescence intensity, as well as short or discontinuous receptor bars consisting of two or three segments, suggest plasticity at the receptor/active zone level. In order to elucidate this notion, receptor bars were studied at different seasons which have previously been shown to be associated with structural changes. In two groups of frogs kept under laboratory conditions simulating wintertime and summertime, respectively, the length and number of receptor bars and the amount of discontinuous bars were investigated. Synaptic contact length, which is the summed length of labelled synaptic branches, and the number and total length of receptor bars did not differ significantly. A clear difference between Group I ("winter" frogs) and Group II ("summer" frogs) was found in the number of discontinuous bars, which was almost twice as high in Group I compared with Group II (6.4 +/- 3.3% S.D. vs 3.4 +/- 1.3% S.D., n = 8 and 7 muscles, respectively, P < 0.05). In addition, the average length of individual bars was slightly longer in Group I frogs (2.16 +/- 0.7 micron S.D. vs 2.07 +/- 0.12 microns S.D., 0.1 < P < 0.05). Transmitter release has been shown to be different in these two groups--as determined from endplate potential measurements in tubocurarine-containing bathing solutions--although it was equal when measured in low Ca2+/high Mg2+ [Dorlöchter M. et al. (1991) Pflügers Arch. 418, Suppl. 1, R31]. We also investigated whether receptor bars would be a reasonable structural correlate of synaptic function by comparing different measures of transmitters release with different structural parameters in 19 identified junctions. The mean quantal content (m) of a junction was positively correlated with the number and total length of receptor bars, but not with synaptic contact area or length. Amplitudes of the first, maximum, and plateau endplate potentials (corrected for a common resting potential and apparent input resistance) at tetanic nerve stimulation (40 Hz for 2 s) in tubocurarine block were strongly correlated with both synaptic contact length and total receptor bar length (r = 0.90 for maximum endplate potential); correlations between m and any structural measure were significantly worse.(ABSTRACT TRUNCATED AT 400 WORDS)

Reinnervation and recovery of mouse soleus muscle after long-term denervation.

Reinnervation and recovery of the mouse soleus muscle were studied 2-10 months after denervation periods of about 7 months. To maintain denervation the right sciatic nerve was frozen 14 times at 2-week intervals. Though initially intermittent muscle reinnervation occurred, contractile force of denervated muscles was reduced to less than 10% of the contralateral muscles by the fifth nerve freezing and further declined thereafter. Following reinnervation, recovery of soleus muscle force proceeded slowly to reach plateau values after 5-6 months. Tetanic muscle force reached on average 72% (range 58-86%, n = 12) of contralateral muscles after 5-10 months, (P less than 0.01, t-test for absolute values) and 87% of unoperated animals after 10 months (P less than 0.05, n = 5). Muscle fibre diameters were significantly reduced in reinnervated muscles, but frequency distributions were normal and similarly shaped in reinnervated and control muscles, suggesting complete muscle reinnervation and the absence of denervated fibres even at 2 months of reinnervation. Total numbers of muscle fibres were similar in reinnervated (842 +/- 73 S.D., n = 15), contralateral (854 +/- 104 S.D., n = 15) and control soleus muscles (853 +/- 77 S.D., n = 5). The number of myelinated axons in regenerating soleus nerves reached control values by 3 months after the last freezing, continued to increase till 6 months (150% of control), and declined thereafter (125% at 9-10 months). In the contralateral soleus nerves the number of myelinated axons remained constant during this period. Nerve fibre diameters remained abnormally small; even after 10 months of reinnervation fibre diameters were unimodally distributed with a mean diameter of 3.3 microns in contrast to the bimodal distribution in intact nerves (mean values 3.9 and 9.0 microns, respectively). Total fibre cross-section area per nerve increased with time but reached only 54% +/- 6 S.D., (n = 3) of contralateral nerves by 10 months. The relative thickness of the myelin sheath (g-ratio) returned to normal after 9-10 months. Anatomically, muscle reinnervation appeared to be complete by 7-8 weeks since unusually small muscle fibre profiles were absent.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects on recovery of soleus and extensor digitorum longus muscles of prolonged wheel running during a period of repeated nerve damage.

The right sciatic nerve in NMRI mice was frozen under anaesthesia 13 times at three-week intervals for a total period of 8.5 months. During this period, but not afterwards, one sub-group of these mice had access to running wheels in which the animals ran several kilometres per night, thereby actively or passively training reinnervated or denervated leg muscles, as well as the intact contralateral muscles. A number of distinct effects persisted for as long as 14-18 weeks after the termination of this "endurance training". In reinnervated soleus muscle, tetanic force was significantly higher (37%) in the trained muscles as was muscle weight (36%); in general, negative effects of the nerve damage persisted. In the reinnervated extensor digitorum longus, tetanic force and muscle weight were significantly smaller in the trained animals (by 11 and 16%, respectively) which are considered typical effects of endurance training. The resistance of the soleus neuromuscular junction to block by both curare and Mg2+ was depressed on the damaged side but this property was not influenced by the training; in extensor digitorum longus the pattern was similar. It is concluded that training during the period of repeated cycles of denervation-reinnervation produced significant effects which impressively outlasted the training period. The possible nature of these effects is discussed.

Sprouting and nerve retraction in frog neuromuscular junction during ontogenesis and environmental changes.

Based on recent evidence for a physiological remodeling of neuromuscular contacts (Wernig et al.), a morphometric study was performed on axon- and cholinesterase-stained cutaneous pectoris muscle of frog. The aim of this investigation was to separate changes due to aging, growth, and environmental conditions. Within a single muscle, fiber diameters, synaptic lengths, number of intraterminal branches, and lengths of abandoned gutters differ considerably (with coefficients of variation from 40 to 56%). On the other hand, these parameters are correlated and correlations hold when muscle fibers grow during ontogenesis: large muscle fibers bear larger and more complex junctions than small fibers. Obviously there exist growth regulating interactions between muscle fiber and the presynaptic nerve. To dissociate between age- and growth-related changes muscle fibers of equal diameters in frogs of different age are compared. With increase in age there is an additional increase in abandoned gutters, synaptic length, and complexity independent of muscle fiber growth. Possibly, abandoned gutters accumulate with time and synaptic length increases with age as the net outcome of continual synapse remodeling. When freshly caught frogs (October) were compared with frogs kept under laboratory conditions for a period of 16 weeks (which in addition included a change in season) the number of sprouts in a junction increased by about 2, the average length of presynaptic nerve terminals with small circumscribed contacts increased by 30-150 microns, and abandoned gutters tended to be shorter on fibers with large junctions. The hypothesis is discussed that remodeling is "inherent" to nerve terminals whereby sprouting is counterbalanced and reversed by nerve activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistence of nerve sprouting with features of synapse remodelling in soleus muscles of adult mice.

An increase in the number of nerve branches of the unmyelinated axon terminals with increasing age was observed in normal adult mouse motor endplates. In addition, ultrastructural investigation revealed signs of nerve retraction. A combined light and electron microscopic investigation was performed on zinc-iodine-osmium stained endplates in soleus muscles. The number of branch points in a synapse, endplate length and muscle fiber diameter were evaluated in "young adult" (3 months) and adult (6 and 11 months) mice. For all 3 parameters, 3-month-old animals had the lowest values. Eleven-month-old animals had more branch points and larger endplate lengths than 6-month-old animals while there was no significant difference in fiber diameters. Branch point numbers and endplate length were correlated in each muscle while fiber diameters did not correlate with any of the other parameters. The ultrastructure of 15 thin nerve branches--likely candidates for new branches--was investigated in serial section and in 14 of them synaptic contacts were found. Near such contacts, empty gutters, possibly abandoned former synaptic sites, were present in several cases. It is concluded that there is continual nerve sprouting in synapses of adult mice and that sprouts form synaptic contacts. The possible signs of nerve retraction observed indicate that, as in the frog, synaptic contacts in mouse muscles undergo some continual remodeling.

Laufband (treadmill) therapy in incomplete paraplegia and tetraplegia.

Recent reports indicate that intensive training of upright walking on a treadmill (German: Laufband, LB), significantly improves walking capability in spinal cord-damaged persons. The aids provided initially are body weight support by a harness and passive setting of one or both limbs by therapists. To facilitate stepping and evoke motor automatisms, "rules of spinal locomotion" need to be applied during training. The effects of this novel locomotion therapy on patients with chronic and acute incomplete paralysis are summarized and discussed here. Many patients with chronic paralysis, still wheelchair-bound and not capable of walking without help from others, became independent and learned to walk for some distance without help. Assessment of voluntary muscle activity in resting position before and after the period of therapy often showed only small increases, rendering the involvement of complex motor reflexes (motor programs) and better utilization of remaining muscle function during walking as main sources for the improvements in locomotion. This idea is supported by electromyographic recordings. Follow-up assessments performed 0.5 to 6.5 years after discharge from the hospital show that the significant improvements achieved by LB-therapy in patients with initially chronic paralysis can be maintained under domestic surrounding. Patients with initially acute paralysis improved their walking capabilities even further. It is suggested that LB therapy may be generally applied in the motor rehabilitation of persons with acute and chronic incomplete paraplegia and tetraplegia. Its use in other diseases is discussed.

Recruitment of spinal motor pools during voluntary movements versus stepping after human spinal cord injury.

We investigated the activation of lower limb motor pools by supraspinal and spinal networks after human spinal cord injury (SCI). We compared electromyographic (EMG) activity from six muscles during voluntarily attempted non-weight-bearing single-joint movements, multijoint movements approximating stepping in a supine position, and weight-bearing stepping on a treadmill with body weight support (BWST) in seven clinically incomplete and three clinically complete SCI subjects. Seven SCI subjects had previously completed Laufband therapy (a specific step training using variable levels of body weight support and manual assistance). Significant coactivation of agonists and antagonists and multijoint flexion or extension movements of the entire limb occurred during attempts at isolated knee or ankle single-joint movements in clinically incomplete SCI subjects. Further, some muscles that were not recruited during voluntary attempts at single-joint movements were activated during voluntary step-like multijoint movements (5/16 comparisons). This suggests that the residual voluntary motor control in incomplete SCI subjects evokes more generalized motor patterns (limb flexion or extension) rather than selective activation of individual muscles. Clinically incomplete and clinically complete SCI subjects could achieve greater activation of motor pools and more reciprocal patterns of activity between agonists and antagonists during weight bearing stepping than during non-weight-bearing voluntary movements. The EMG mean amplitudes were higher during stepping than during voluntary movements in 50/60 muscles studied (p < 0.05). These results suggest that stepping with knee and hip extension and flexion and alternating lower limb loading and unloading provides proprioceptive inputs to the spinal cord that increases motor recruitment and improves reciprocity between agonists and antagonists compared to voluntary efforts.

Localization of active sites in the neuromuscular junction of the frog.

Spontaneous quantal discharges at the frog neuromuscular junction were simultaneously monitored with three glass microelectrodes. Two extracellular electrodes were located at the synaptic cleft a few microns apart from each other so that each quantal discharge occurring nearby evoked signals in both electrodes. From the observed ratios of the signal amplitudes it is inferred that quantal discharges exclusively occur at discrete sites. The distance between active sites is in the same range as that between "active zones" seen from electron micrographs.

Prolonged running does not improve muscle coordination after cross-union of tibial and peroneal nerves in mice.

Several months after cross-union of the tibial and peroneal nerves and full muscle reinnervation motor behavior was monitored and was tested by electromyographic recordings from both musculus tibialis anterior (TA) and musculus gastrocnemius medialis (MG). In addition spinal motoneuron pools were labelled by injecting wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) into experimental and control TA muscles. Though the location of TA motoneurons was similar in all 9 mice tested and suggested successful cross-reinnervation, running behavior and EMG patterns varied remarkably. In some animals the alternating activities of TA and MG muscles remained more or less clearly separated from each other but were out of phase with the running cycle. A somewhat better motor behavior was accompanied by simultaneous activity in both muscles. Voluntary running in wheels for several kilometers per day did not visibly improve motor coordination indicating the absence of activity-related synaptic readjustments.

Differential sensitivity to Mg(2+)-and tubocurarine-block of frog neuromuscular junctions in summer and winter.

Several parameters of transmitter release in neuromuscular junctions were compared in "winter' and "summer' frogs. In low Ca2+/high Mg(2+)-block, the quantal content m of endplate potentials was similar for both groups. In curarized junctions, however, endplate potentials were about twice as large in winter as in summer, and facilitation was lower and depression higher. This indicates that transmitter release is higher in winter junctions. This is not reflected in Mg(2+)-block, suggesting that strong calcium deprivation may suppress release in winter more strongly than in summer junctions.