Summary
We examined the effects of implantation of cultured myogenic cells from a permanent cell line into soleus muscles of histocompatible adult mice. Myogenic cells (106 or 104) were implanted into intact muscles, muscles frozen with liquid nitrogen, paralysed with botulinum toxin or reinnervated after long-term (seven months) denervation. Formation of numerous muscle fibres in myogenic cell-injected muscles raised the total number of fibres up to ten times above control by four weeks. Larger effects were found in freeze-damaged than in paralysed muscles. The new fibres had small calibers, considerable length (> 1.3mm, maximum distance over which serial sections were made), were multinucleated and were oriented parallel to the large-diameter fibres of the host muscles. In some experiments β-galactosidase, introduced into myogenic cells via retroviral transfection, was detected in small and large muscle fibres 4–20 weeks after implantation, indicating survival of the grafted cells and formation of mosaic (host-donor) and new fibres of donor origin. Muscle weight increased significantly and, rather surprisingly, a parallel increase was found in isometric tetanic tension of isolated nerve-muscle preparations; thus tension per mg muscle tissue was not different from normal. By eight weeks reduction of acetylcholine sensitivity and down-regulation of neural cell adhesion molecule to normal were observed, indicating that synaptic transmission at the new fibres was mature. After different periods of time (5–20 weeks, depending on the subclone used) tumours developed in most but not all injected limbs (37 out of 39). The tumours were destructive to the muscles and were classified asrhabdomyosarcomas. Prior to tumour formation, neural cell adhesion molecule positive cells reappeared in the muscles; since the myogenic cells initially produced differentiated muscle fibres, it appears that malignant growth is induced by factorsin vivo. Thus, at present the outcome of such implantation is unpredictable.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anzil, A. P. &Wernig, A. (1989) Muscle fibre loss and reinnervation after long-term denervation.Journal of Neurocytology 18, 833–45.
Badke, A., Irintchev, A. &Wernig, A. (1989) Maturation of transmission in reinnervated mouse soleus muscle.Muscle and Nerve 12, 580–6.
Bischoff, R. (1986) A satellite cell mitogen from crushed adult muscle.Developmental Biology 115, 140–7.
Bischoff, R. (1990) Control of satellite cell proliferation. InMyoblast Transfer Therapy (edited byGriggs, R. &Karpati, G.), pp. 147–57. New York: Plenum Press.
Bishop, J. M. (1987) The molecular genetics of cancer.Science 235, 305–11.
Blau, H. M. &Webster, C. (1981) Isolation and characterization of human muscle cells.Proceedings of the National Academy of Sciences (USA) 78, 5623–7.
Butler, J. &Cosmos, E. (1981) Enzymic markers to identify muscle-nerve formation during embryogenesis: modified myosin ATPase and silver-cholinesterase histochemical reactions.Experimental Neurology 73, 831–6.
Covault, J. &Sanes, J. R. (1985) Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles.Proceedings of the National Academy of Sciences (USA) 82, 4544–8.
Covault, J. &Sanes, J. R. (1986) Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle.Journal of Cell Biology 102, 716–30.
Gorman, C. (1985) High efficiency gene transfer into mammalian cells. InDNA Cloning. A practical Approach, vol. 2 (edited byGlover, D. M.), pp. 143–90. Oxford and Washington DC: IRL Press.
Gorman, C., Moffat, L. F. &Howard, B. H. (1982) Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells.Molecular and Cellular Biology 2, 1044–51.
Guth, L. &Samaha, F. J. (1970) Procedure for the histochemical demonstration of actomyosin ATPase.Experimental Neurology 28, 365–7.
Hughes, S. M. &Blau, H. M. (1990) Migration of myoblasts across basal lamina during skeletal muscle development.Nature 345, 350–3.
Irintchev, A., Draguhn, A. &Wernig, A. (1990) Reinnervation and recovery of mouse soleus muscle after longterm denervation.Neuroscience 39, 231–43.
Irintchev, A., Wernig, A., Härtling, A., Stephan, A., Zimmermann, K. &Starzinski-Powitz, A. (1991) Formation of muscle fibres and tumorgenesis after implantation of myogenic cells.Pflügers Archiv 418, Suppl. 1, R32.
Karnovsky, M. J. &Roots, L. (1964) A ‘direct-coloring’ thiocholine method for cholinesterases.Journal of Histochemistry and Cytochemistry 12, 219–21.
Karpati, G., Pouilot, Y., Zubrzycka-Gaarn, E., Carpenter, S., Ray, P. N., Worton, R. G. &Holland, P. (1989) Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation.American Journal of Pathology 135, 27–32.
Knebel, D., Lübbert, H. &Doerfler, W. (1985) The promoters of the late p10 gene in the insect nuclear polyhedrosis virusAutographia californica: activation by viral gene products and sensitivity to DNA methylation.EMBO Journal 4, 1301–6.
Kruczek, I. &Doerfler, W. (1983) Expression of the chloramphenicol acetyl-transferase gene in mammalian cells under the control of adenovirus type 12 promoters: effects of promoter methylation on gene expression.Proceedings of the National Academy of Sciences (USA) 80, 7586–90.
Law, P. K., Goodwin, T. G. &Li, H. -J. (1988a) Histoincompatible myoblast injection improves muscle structure and function of dystrophic mice.Transplantation Proceedings 20, Suppl. 3, 1114–19.
Law, P. K., Goodwin, T. G. &Wang, M. G. (1988b) Normal myoblast injections provide genetic treatment for murine dystrophy.Muscle and Nerve 11, 525–33.
Law, P. K., Goodwin, T. G., Li, H. -J., Ajamoughli, G. &Chen, M. (1990a) Myoblast transfer improves muscle genetics/structure/function and normalizes the behavior and life-span of dystrophic mice. InMyoblast Transfer Therapy (edited byGriggs, R. &Karpati, G.), pp. 75–84. New York: Plenum Press.
Law, P. K., Bertorini, T. E., Goodwin, T. G., Chen, M., Fang, Q., Li, H.-J., Kirby, D. S., Florendo, J. A., Herrod, H. C. &Golden, G. S. (1990b) Dystrophin production induced by myoblast transfer therapy in Duchenne muscular dystrophy.Lancet 336, 114–115.
Lojda, Z., Gossrau, R. &Schiebler, T. H. (1976)Enzymhistochemische Methoden. Berlin, Heidelberg, New York: Springer Verlag.
Morgan, J. F., Hoffman, E. P. &Partridge, T. A. (1990) Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse.Journal of Cell Biology 111, 2437–50.
Mougneau, E., Lemieux, L., Rassoulzadegan, M. &Cuzin, F. (1984) Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture.Proceedings of the National Academy of Sciences (USA) 81, 5758–62.
Ontell, M. &Feng, K. C. (1981) The three-dimensional cytoarchitecture and pattern of motor innervation of branched striated myotubes.Anatomical Record 200, 1–31.
Ontell, M., Hughes, D. &Bourke, D. (1982) Secondary myogenesis of normal muscle produces abnormal myotubes.Anatomical Record 204, 199–207.
Partridge, T. A. (1991) Myoblast transfer: a possible therapy for inherited myopathies?Muscle and Nerve 14, 197–212.
Partridge, T. A., Morgan, J. E., Coulton, G. R., Hoffman, E. P. &Kunkel, L. M. (1989) Conversion of mdx myofibres from dystrophin-negative to positive by injection of normal myoblasts.Nature 337, 176–9.
Sanes, J. R., Rubenstein, J. L. R. &Nicolas, J. -F. (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.EMBO Journal 5, 3133–42.
Schmalbruch, H. (1985)Skeletal Muscle. Berlin-Heidelberg-New York, Springer-Verlag.
Thompson, W. &Jansen, J. K. S. (1977) The extent of sprouting of remaining motor units in partly denervated immature and mature rat soleus muscles.Neuroscience 2, 253–6.
Vaughan, H. S. &Goldspink, G. (1979) Fibre number and fibre size in a surgically overloaded muscle.Journal of Anatomy 129, 293–303.
Vitadello, M., Triban, C., Fabris, M., Gorio, A. &Schiafino, S. (1986) Heterogeneity of rat neurofilament polypeptides revealed by a monoclonal antibody.Journal of Neurochemistry 46, 665–70.
Wernig, A., Irintchev, A. &Weisshaupt, P. (1990) Muscle injury, cross-sectional area and fibre type distribution in mouse soleus after intermittent wheel-running.Journal of Physiology 428, 639–52.
Wernig, A., Salvini, T. F., Langenfeld-Oster, B., Irintchev, A. &Dorlöchter, M. (1991a) Endplate and motor unit remodelling in vertebrate muscles. InPlasticity of Motoneuronal Connections (edited byWernig, A.). Amsterdam: Elsevier, in press.
Wernig, A., Salvini, T. F. &Irintchev, A. (1991b) Axonal sprouting and changes in fibre types after running-induced muscle damage.Journal of Neurocytology,20, 903–13.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wernig, A., Irintchev, A., Härtling, A. et al. Formation of new muscle fibres and tumours after injection of cultured myogenic cells. J Neurocytol 20, 982–997 (1991). https://doi.org/10.1007/BF01187916
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01187916