- Email: [email protected]
Myogenicity in vitro and in vivo of mouse muscle cells separated on discontinuous Percoll gradients
Journal of the Neurological Sciences, 1988, 85:197-207 Elsevier
197
JNS 03004
Myogenicity in vitro and in vivo of mouse muscle cells separated on discontinuous Percoll gradients Jennifer Elizabeth Morgan Department of Histopathology, Chafing Cross and Westminster Medical School, London W6 8RF (U.K.) (Received 4 November, 1987) (Revised, received 1 February, 1988) (Accepted 1 February, 1988)
SUMMARY
Mouse muscle ceils, obtained by enzymatically disaggregating newborn mouse muscle, were separated on a discontinuous Percoll gradient. The myogenicity in vitro of the resultant cell fractions was examined by counting the percentage of nuclei in myotubes. Myogenicity in vivo was assessed by implanting a cell suspension of one of the allotypes of glucose-6-phosphate isomerase (GPI) into a regenerating skeletal muscle graft of a second GPI allotype: the finding of hybrid GPI indicated that the implanted cells were myogenic. Separation of mouse muscle cells on a discontinuous Percoll gradient gave rise to two myogenic fractions, one of which was more myogenic in vitro than were the unseparated cells and one of which was less myogenic. Both of these fractions were myogenic in vivo. A cell fraction was also produced which was non-myogenic in vitro as well as in vivo. In vitro and in vivo measurements of myogenicity were therefore in broad agreement.
Key words: Mouse muscle cells; Cell separation; Myogenicity
INTRODUCTION
The long-term aim of our work is to implant normal muscle precursor ceils into growing or regenerating myopathic muscle, in an attempt to alleviate the myopathy. In Correspondence to: Dr. J.E. Morgan, Dept. of Histopathoiogy, Chafing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF U.K. 0022-510X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
198 previous work (Watt et al. 1982, 1984), we have implanted muscle cells obtained by enzymic disaggregation of newborn mouse muscle into growing or regenerating mouse muscle and have shown that the implanted cells had been incorporated into host muscle fibres. In order to apply this technique to the treatment of myopathies, the implanted cell suspension should contain as high a proportion as possible of myogenic cells capable of fusion with the host muscle fibres, so as to maximise the numbers of myonuclei of donor origin expressing their normal genes within the resultant mosaic muscle fibres. Enzymic dissaggregation of skeletal muscle gives rise to a single-cell preparation containing both myogenic cells and non-myogenic cells, such as fibroblasts (Konigsberg 1979). Several methods have been used in attempts to separate myoblasts from fibroblasts in such suspensions (Yaffe 1968; Puri and Turner 1978; Turner 1978; Konigsberg 1979). Clonal analysis studies have shown that in the chick at least, myogenic cells are not a homogeneous population (Hauschka and White 1972; White et al. 1975; Seed and Hauschka 1984). The myogenicity of the resultant cell populations is invariably assessed in vitro (Morris and Cole 1972; Shainberg et al. 1971). This however leaves open the question of the extent to which in vitro myogenesis reflects the in vivo potential of a cell preparation. Here, we have separated newborn mouse muscle cells on discontinuous Percoll gradients to produce a fraction containing high proportions of myogenic, and another fraction consisting largely of non-myogenic cells. We present an assessment in vivo as well as in vitro of the myogenicity of the highly myogenic and the non-myogenic fraction of separated cells. The two methods of assessment were found to give broadly comparable results. MATERIALS AND METHODS Mouse strains
Various strains of mice were used as sources of muscle precursor cells, depending on availability. Thus, muscle cells were prepared from newborn 129/ReJ, CBA or C57B1/6J mice. CBA and C57B1/6J muscle cells (both of Gpi-ls b allotype) were implanted into minced tibialis anterior (TA) autografts in nu/nu mice (Gpi-ls a allotype). In some instances, 129/ReJ muscle cells (Gpi-ls a allotype) were implanted into C57B 1/6J minced TA isografts (Gpi-ls b allotype). Because it was found to be difficult to make the ICR/IAn strain of mouse (which was of particular interest because its skeletal muscles lack the enzyme phosphorylase kinase), tolerant to any of our usual strain of donor cells, 129/ReJ muscle cells were also implanted into ICR/IAn whole extensor digitorum longus (EDL) muscles (Gpi-ls b allotype) which were themselves grafted into nu/nu mice of Gpi-ls a allotype, thus avoiding the problem of immune rejection. Tolerance induction
Where 129/ReJ muscle cells were implanted into muscle grafts in C57B 1/6J hosts, the latter were made tolerant to the donor strain by neonatal intravenous injection of
199 5 X 10 6 129/ReJ spleen cells (Billingham et al. 1956). Tolerance induction was not necessary where grafts were made in nu/nu athymic mice. Preparation of mouse muscle cell suspensions Suspensions of muscle cells were obtained by enzymic dissaggregation of newborn mouse muscle, as described previously (Watt et al. 1982; Morgan et al. 1988). After resuspension of the cells in the growth medium used in the particular experiment, a count of viable, trypan blue-excluding, cells was made. Separation of muscle cell suspensions on discontinuous Percoll gradients An aliquot of the single cell suspension was kept on ice in growth medium (unseparated cells). The remaining mouse muscle cells were centrifuged at 350 x g and resuspended in 5 ml of calcium and magnesium-free Hanks balanced salt solution (HBSS; Gibco), buffered with 25 mM Hepes (Gibco). Under this were layered 5 ml aliquots of 26% and 34% Percoll, both solutions being made up in calcium and magnesium-free HBSS, buffered with 25 mM Hepes. The gradient was then centrifuged at 350 x g for 15 rain at 20 °C. Cells from each interface and from under the 34% Percoll were collected with a Pasteur pipette and added to an equal volume of HBSS containing calcium and magnesium, penicillin, streptomycin (P 5000 IU/ml; S 5000 #g/ml; Flow) and 20% FCS. Fraction 1 refers to cells from the 0-26% Percoll interface, fraction 2 to cells from the 26-34% Percoll interface and fraction 3 to cells from under the 34% Percoll. The unseparated cells and the three cell fractions were again centrifuged at 350 × g for 10 rnin, resuspended in a known volume of the particular growth medium to be used in that experiment and a count of viable cells was performed. The cells were then adjusted to the required cell density. Where the cells were to be implanted into muscle grafts, centrifuged pellets containing known numbers of unseparated cells and cells from each of the Percoll fractions were prepared. The unseparated cells and cells from each of the Percoll fractions were also grown in tissue culture. Culture of separated muscle cells and assessment of their myogenicity in vitro Separated and control cells were plated directly into 35-mm petri dishes (Nunc) containing a glass coverslip coated with rat tail collagen (Paul 1972). Plating densities varied from 2 × 104 to 1 x 105 viable cells per 35-ram petri dish. The culture medium was based on Medium 199 containing Earle's salts and 25mM Hepes buffer (Gibco), penicillin and streptomycin (P 5000 IU/ml; S 5000 #g/ml; Flow) (M199). Initially, different proportions of fetal calf serum (FCS; Sera Lab) and chick embryo extract (CEE; Flow) were used, but the particular batches of FCS and CEE used in these experiments were found to support the greatest extent of myoblast fusion when 20 parts FCS and 10 parts CEE were used. Cultures were only grown until fusion had occurred in the cultures of unseparated cells (i.e., approx. 5-7 days) and the culture medium was not changed during this time. When fusion had occurred in the control cultures, the coverslips bearing the monolayer of cells were removed from the petri dishes, washed twice in PB S and fixed
200 for 15 min in methanol. The cells were then stained with haematoxylin and eosin. Stained cultures were then examined microscopically in order to determine the proportion of nuclei present in multinucleated myotubes and those present in mononucleated cells. The number of nuclei in each culture present in one-hundred 80 x 80 #m squares of a Gallenkampf M N H 420A graticule at x 100 magnification were counted in 25 randomly encountered fields. Multinucleated myotubes were defined as those structures which contained 3 or more nuclei. The percentage of nuclei in myotubes was calculated for each of the 100 squares and these figures were averaged to give a measure of the percentage of nuclei in myotubes for the entire culture.
Grafting techniques Minced and whole muscle grafts were prepared as described previously (Watt et al. 1982, 1987). Muscle cells were added to and mixed into, minced muscle grafts. In the case of whole muscle grafts, the pellet of donor muscle precursor cells was inserted by means of a fine Pasteur pipette into a slit made in the anterior aspect of the graft.
Implantation of cells separated on discontinuous PercoUgradients into minced muscle grafts From 2 to 8 x 105 donor muscle cells were implanted into minced TA muscle grafts in nu/nu (9 mice) or neonatally tolerant C57BL/6J mice (12 mice). The grafts were removed for analysis 20-30 days later.
Implantation of cells separated on discontinuous PercoU gradients into whole muscle grafts In these experiments, 3 x 105-106 129/ReJ muscle cells of Gpi-1 s ~ allotype were separated on discontinuous Percoll gradients and implanted into whole ICR/IAn muscle grafts (Gpi-ls b allotype) in nu/nu hosts of Gpi-ls ~ allotype. Grafts were removed for analysis from 20 to 60 days after grafting.
Analysis of GPI isoenzymes Mice were killed by cervical dislocation at intervals after grafting. The muscle graft was removed, mounted in Tissue-tec OTC compound and frozen in isopentane cooled to - 165 °C in liquid nitrogen. Five #m thick cryostat sections were cut at three levels through the graft and these sections were taken up onto microscope slides. One section on each slide was overlaid for a few seconds with a small rectangle of filter paper, approx. 6 x 2 mm, dampened with distilled water. Within 30 sec, sufficient GPI for analysis was absorbed into the filter paper for isoelectric focusing (IEF) analysis. The sections on the slides were then stained with haematoxylin and eosin. The filter paper rectangles containing GPI were placed on the surface of an 0.8 mm thick polyacrylamide gel containing a mixture of cartier ampholytes (LKB) pH range 9-11 and 8-9.5 in a ratio of 2 : 1. They were subject to isoelectric focusing for 2.5 h at 150 V/cm. Two standards were run on each gel. One was a cryostat section cut from the muscle of a C57BL/6J x 129/ReJ F1 hybrid mouse; this sample contains the AA, AB and BB GPI isoenzymes in a ratio of 1 : 2 : 1. The other was a cryostat section cut from
201 a block containing pieces of C57BL/6J muscle and 129/ReJ muscle; this was to show that the AA and BB GPI isoenzymes did not dissociate and reassociate on focusing to give rise to the AB isoenzyme. After focusing, the gel was stained to indicate the presence of the isoenzymes of GPI by means of an agar overlay mixture (Partridge et al. 1978). The BB GPI isoenzyme, characteristic of the ICR/IAn, CBA and C57B1/6J strains of mouse, is cathodal and focuses at a pH of 8.6, while the AA isoenzyme, characteristic of the 129/ReJ strain and the nu/nu mice used here, is anodal, focusing at a pH of 8.2. The hybrid AB GPI isoenzyme is a heterodimer containing one A and one B subunit and focuses between the anodal and cathodal forms. The finding of donor GPI isoenzyme in the grafts indicates the presence of the implanted muscle-derived cells, but the identity of the tissue to which these cells had contributed cannot be ascertained. The finding of the hybrid AB isoenzyme means that nuclei of Gpi-ls a and Gpi-ls h allotype are present within a common cytoplasm. In our muscle grafts, this indicates that muscle precursor cells from host and donor origin have fused to form a mosaic muscle fibre, within which the donor as well as the host myonuclei are expressing their genes. This shows that some at least of the implanted cells must have been myogenic.
Histological examination of grafts Cryostat sections stained with haematoxylin and eosin were examined microscopically. The proportions of constituent tissues were estimated using a 25 random point graticule, according to the method of Curtis (1960) and the percentage of the graft consisting of new muscle was recorded. RESULTS
Assessment of myogenicity of mouse muscle cell preparations in vitro In initial experiments (results not shown) the discontinuous Percoll gradients consisted of layers corresponding closely in specific gravities to those in the Ficoll gradient used by Turner (1978). It was found that mouse muscle cells which had densities in the range of 34~ Percoll were less myogenic and those which were denser than 34~ Percoll were in general more myogenic in culture than were unseparated cells. A stepped gradient was finally arrived at which gave a non-myogenic fraction as well as one which was rich in myogenic cells. Fraction 1 cells, from the 0 - 2 6 ~ Percoll interface, were only cultured at one seeding density (5 × 104 cells/dish) and were less than a quarter as myogenic as the unseparated cells grown at the same initial seeding density (6°/o nuclei in myotubes compared with 27~ ; Fig. la). At all of the initial seeding densities, the fraction 2 cells were virtually non-myogenic, one culture containing only a few myotubes (0.4~o nuclei in myotubes; Fig. lb), the rest containing none. At all of the initial seeding densities, fraction 3 cells (Fig. lc) were more myogenic (36-45 ~o nuclei in myotubes) than the unseparated cells (23-32°/o nuclei in myotubes;
C,
203
m o
0
r-
Od O
O O
D
'rp
Oo
°0 •
~i~/¸¸¸!
o
,)1o 4
Q
O
Fig. 1. Cultures of newborn 129/ReJ mouse muscle cells separated on a discontinuous Percoll gradient and cultured for 7 days in M199, 20 parts FCS and 10 parts CEE, at an original cell seeding density of 5 x 104 cells per 35-mm petri dish.. (a) Fraction I, from the 0-26% Percoll interface. (b) Fraction 2, from the 26-34% Percoll interface. (c) Fraction 3, from under the 34% Percoll interface. (d) Unseparated cells. H&E, × 44.
204 Fig. ld). Both the unseparated and fraction 3 cells underwent fusion into myotubes earlier (day 5) when plated at a higher initial cell density (105 cells/dish) than at a lower cell density (2-5 × 104 cells/dish), when they underwent fusion on day 7. Direct comparison of cultures plated at different cell densities is of little value, as the cells plated at the higher densities had been fixed at an earlier time than those plated at the lower densities.
Assessment of myogenicity of mouse muscle cell preparations in vivo (Fig. 2) Implantation of cells separated on discontinuous Percoll gradients into minced muscle grafts Four out of 5 grafts to which unseparated muscle cells had been added contained host, donor and hybrid GPI isoenzymes (Fig. 2). The remaining graft contained only host isoenzyme; this graft was made in a C57B 1/6J mouse in which tolerance induction had been attempted and was the only graft to contain no new muscle. It seems possible therefore that the implanted cells had been immunologically rejected. All four of the grafts to which fraction 1 cells from the Percoll gradient had been added contained host, donor and hybrid GPI isoenzymes. One of the grafts to which fraction 2 cells had been added contained host, donor and a very small amount of hybrid GPI isoenzyme. Two of the grafts contained only host and donor GPI isoenzymes and the fourth contained the host GPI isoenzyme alone.
GPI i8oenzymes g
Fig. 2. GPI isoenzymes, separated by IEF, from cryostat sections of the following: (a) 30 day old minced TA isograR, made in a neonatally tolerant C57B1/6J host, to which 6 x 105 unseparated 129/ReJ muscle cells had been added. This graft contained host and a trace of donor and hybrid GPI isoenzymes. (b) 25-day-old minced TA autograR, made in a nu/nu mouse host, to which 8 x 105 fraction 1 (from the 0-26 % Percoll interface) C57B 1/6J muscle cells had been added. This graft contained host and a trace of donor and hybrid GPI isoenzymes. (c) 30-day-old minced TA isograR, made in a neonatally tolerant C57B1/6J host, to which 6 x 105 fraction 1 (from the 0 - 2 6 ~ Percoll interface) 129/ReJ muscle cells had been added. This grail contained host, donor and hybrid GPI isoenzymes. (d)30-day-old minced TA isograR, made in a neonatally tolerant C57B1/6J host, to which 6 x 105 fraction 2 (from 26-34% Percoll interface) 129/ReJ muscle cells had been added. This graft contained only host GPI isoenzyme. (e) 25-day-old minced TA autograft, made in a nu/nu mouse host, to which 8 × 105 fraction 3 (from under the 34% Percoll) C57B1/6J muscle cells had been added. This graft contained host, donor and hybrid GPI isoenzymes. (f) 30-day-old minced TA isograft made in a neonatally tolerant C57B1/6J host, to which 6 x 105 fraction 3 (from under the 3 4 ~ Percoll) 129/ReJ muscle cells had been added. This graft contained host, donor and hybrid GPI isoenzymes. (g) Control: a mixture of 129/ReJ and C57B1/6J skeletal muscles, to show that the AA and BB GPI isoeazymes do not re-associate under the conditions o f l E F to form the 'hybrid' AB GPI isoenzyme. (h) Standard: skeletal muscle from a C57B1/6J × 129/ReJ F] mouse, to show the localisation of the AA, BB and AB GPI isoenzymes.
205 Six out of 8 grafts to which fraction 3 cells had been added contained host, donor and hybrid GPI isoenzymes; the remaining two grafts (one in a nu/nu host and the other in a C57B 1/6J host) contained only host isoenzyme. Ceils which were virtually non-myogenic in vitro were therefore, with one exception, non-myogenic in minced muscle grafts in vivo. The addition of a cell pellet which was more myogenic (fraction 3) or less myogenic (fraction 1) in vitro than the unseparated ceils did not give rise to a noticably different incidence of hybrid isoenzyme in minced muscle grafts.
Implantation of cells separated on discontinuous Percoll gradients into whole muscle grafts In these experiments, muscle cells of Gpi-ls a allotype were implanted into whole ICR/IAn muscle grafts (Gpi-ls a allotype) in nu/nu hosts of Gpi-ls a allotype. Under these circumstances, any AA GPI isoenzyme found in the graft could be derived from the host or from the implanted donor muscle cells. Control grafts, to which no cell were added, were performed to test the possibility that hybrid AB GPI isoenzyme in the grafts was derived from muscle ceils of host origin rather than from the implanted muscle cells. Three out of 5 such grafts contained only the ICR/IAn-type BB GPI isoenzyme; the remaining two grafts contained the AA, BB but not the hybrid AB GPI isoenzyme. This implies that cells of host origin did enter the graft, but that these cells showed no evidence of myogenicity. One out of 4 grafts to which unseparated cells had been added contained AA, BB and hybrid AB GPI isoenzymes; 2 contained only BB GPI and the remaining 1 contained AA and BB GPI isoenzymes. Three out of 8 grafts to which fraction 1 muscle cells had been added contained AA, BB and hybrid AB GPI isoenzymes; 4 contained AA and BB GPI isoenzymes and the remaining 1 contained only BB GPI. None of the grafts to which fraction 2 cells had been added contained hybrid AB GPI isoenzyme; all 7 grafts contained only the AA and BB GPI isoenzymes. Three out of 7 grafts to which fraction 3 cells had been added contained AA, BB and hybrid AB GPI isoenzymes; 3 contained only AA and BB GPI isoenzymes and the remaining 1 contained only BB GPI. In whole muscle grafts, therefore, muscle ceils which were non-myogenic in vitro were also non-myogenic in vivo. Cells from fraction 3, which were more myogenic than fraction 1 in vitro, however, gave rise to no higher incidence of hybrid GPI in whole muscle grafts in vivo.
DISCUSSION
We have shown that it is possible to separate suspensions of newborn mouse muscle cells by centrifugation on discontinuous Percoll gradients into populations which, in vitro, are more myogenic and populations which are less myogenic than the original cell preparation. The in vitro myogenicity of our mouse muscle cells separated on discontinuous Percoll gradients shows some similarities to chick muscle cells sepa-
206 rated on discontinuous Ficoll (Turner 1978) or Percoll (Yablonka-Reuveni and N ameroff 1986)gradients. The gradients used by these workers separated non-myogenic cells as the least dense fraction, whereas we found that myogenic cells were present in both the least dense fraction, and in a high proportion in the densest fraction, but were absent from the intermediate fraction. It is possible that the myogenic cells in fraction 1 were in a different stage of the cell cycle from those found in fraction 3; the denser myogenic cells may, for example, have been in the S and G2 phases of the cell cycle. Proliferative myogenic cells may thus have been separated from quiescent stem cells, or from non-dividing, fusion-capable myogenic cells present in the newborn mouse muscle, but this was not further investigated. By implanting the separated muscle cell suspensions into regenerating mouse muscle grafts, we were also able to assess their myogenicity in vivo. This is the first attempt to correlate the in vivo and in vitro myogenicity of muscle cell populations. The finding of hybrid GPI was the only certain indication that the implanted cells had actually taken part in muscle fibre formation: it can only have been derived from mosaic muscle fibres, formed by the fusion of host and donor myogenic cells. Donor-type GPI could have been derived from muscle but could also have come from other cells present in the graft site. In the absence of hybrid GPI therefore, we can neither exclude nor confirm the possibility that the cell preparation in question was myogenic. As a general rule, mouse muscle cells which were myogenic in vitro were also myogenic in vivo when implanted into regenerating muscle grafts. Likewise, cells which showed no evidence of myogenicity in vitro also showed none in vivo. Implantation of a mouse muscle cell suspension which was more myogenic in vitro than unseparated cells did not give rise to a higher percentage of grafts containing hybrid isoenzyme. However, the presence or absence of hybrid GPI is a crude indicator of whether or not myogenic cells were present in a particular cell suspension. It should be possible to obtain a more precise measure of the myogenicity of an implanted muscle cell preparation by making a quantitative study of the proportions of the GPI isoenzymes present in the graft as has been attempted by Friar and Peterson (1983) and Jockusch et al. (personal communication): the proportion of hybrid GPI should be in correlation with the myogenicity of the cell preparation added to them. Such fully quantitative methods however, do require that the proportions of the isoenzymes present are not too disparate so that the reaction for each of them can be measured while it is still in the linear phase. An alternative method of assessing myogenicity is by a dilution assay: the more myogenic a cell preparation is, the fewer cells would need to be implanted into grafts to produce a detectable amount of hybrid GPI. Approximately half of the nuclei in skeletal muscle belong to non-muscle cells and tissue culture studies (Sanderson et al. 1986) have shown that muscle fibroblasts are necessary for the formation of basal lamina. We are now using a model system whereby we can make a new muscle in vivo by grafting a scaffolding of freeze-killed muscle and repopulating it with muscle precursor cells (Morgan et al. 1987), to investigate the interaction of non-myogenic cells and myogenic cells, separated on discontinuous Percoll gradients, to the formation of new muscle in vivo. Our preliminary findings suggest that implanted muscle fibroblasts play a helper role in the formation of new muscle in freeze-killed grafts.
207 ACKNOWLEDGEMENTS
I wish to thank Dr. T. A. Partridge for his advice and criticism and Mr. R. Barnett and the Department of Medical Illustration for photography. This work was supported by the Muscular Dystrophy Group of Great Britain.
REFERENCES Billingham, R.E., L. Brant, and P.B. Medawar (1956) Quantitative studies on tissue transplantation immunity III: Actively acquired tolerance. Phil. R. Soc. Lond. Series B Biol. Series, 239: 357-424. Curtis, A. S. G. (1960) Area and volume measurements by random sampling methods. Med. Biol. Illustr., 10: 261-266. Frai L P. M. and A. C. Peterson (1983) The nuclear-cytoplasmic relationship in 'mosaic' skeletal muscle fibres from mouse chimaeras. Exp. Cell Res., 145: 167-178. Hauschka, S.D. and N.K. White (1972) Studies ofmyogenesis in vitro. In: B.Q. Banker, R.J. Pryzybylski, J.P. Van der Meulen and M. Victor (Eds.), Research in Muscle Development and the Muscle Spindle, Excerpta Medica, Amsterdam, pp. 53-71. Konigsberg, I. R. (1979) Skeletal myoblasts in culture. Methods Enzymol., 58:511-527. Morgan, J.E., G.R. Coulton and T.A. Partridge (1987) Muscle precursor cells invade and repopulate freeze-killed muscles. J. Muscle Res. Cell Motil., 8: 386-396. Morris, G.E. and R.J. Cole (1972) Cell fusion and differentiation in cultured chick muscle cells. Exp. Cell Res., 75: 191-199. Paul, J. (1972). In: Cell and Tissue Culture, Churchill Livingstone, Edinburgh, pp. 73-74. Partridge, T. A., M. Grounds and J.C. Sloper (1978) Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature (Lond.), 273: 306-308. Puri, E.C. and D.C. Turner (1978) Serum-free medium allows chick myogenic cells to be cultivated in suspension and separated from attached fibroblasts. Exp. Cell. Res., 115: 159-173. Sanderson, R.D., J.M. Fitch, T.R. Lisenmayer and R. Mayne (1986) Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro. J. Cell Biol., 102: 740-747. Seed, J. and S.D. Hauschka (1984) Temporal separation of the migration of distinct myogenic precursor populations into the developing chick wing bud. Dev. Biol., 106: 389-393. Shainberg, A., G. Yagil and D. Yaffe (1971) Alterations of enzymatic activities during muscle differentiation in vitro Dev. Biol., 25: 1-29. Turner, D.C. (1978) Differentiation in cultures derived from embryonic chicken muscle. The postmitotic, fusion-capable myoblast as a distinct cell type. Differentiation, 10: 81-93. Watt, D.J., K. Lambert, J. E. Morgan, T.A. Partridge and J. C. Sloper (1982) Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse. J. Neurol. Sci., 57: 319-331. Watt, D.J., J.E. Morgan and T.A. Partridge (1984) Use of mononuclear muscle precursor cells to insert allogeneic genes into growing mouse muscles. Muscle Nerve, 7: 741-750. Watt, D.J., J. E. Morgan, M. A. Clifford and T.A. Partridge (1987) The movement of muscle precursor cells between adjacent regenerating muscles in the mouse. Anat. Embryol., 175: 527-536. White, N.K., P.H. Bonnet, D.R. Nelson and S.D. Hauschka (1975) Clonal analysis of vertebrate myogenesis. Dev. Biol., 44: 346-361. Yablonka-Reuveni, Z. and M. Nameroff (1986) Analysis of skeletal muscle cell populations using density centrifugation J. Cell Biol., 103: 120a. Yaffe, D. (1968) Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. U.S.A., 61: 477-483.
197
JNS 03004
Myogenicity in vitro and in vivo of mouse muscle cells separated on discontinuous Percoll gradients Jennifer Elizabeth Morgan Department of Histopathology, Chafing Cross and Westminster Medical School, London W6 8RF (U.K.) (Received 4 November, 1987) (Revised, received 1 February, 1988) (Accepted 1 February, 1988)
SUMMARY
Mouse muscle ceils, obtained by enzymatically disaggregating newborn mouse muscle, were separated on a discontinuous Percoll gradient. The myogenicity in vitro of the resultant cell fractions was examined by counting the percentage of nuclei in myotubes. Myogenicity in vivo was assessed by implanting a cell suspension of one of the allotypes of glucose-6-phosphate isomerase (GPI) into a regenerating skeletal muscle graft of a second GPI allotype: the finding of hybrid GPI indicated that the implanted cells were myogenic. Separation of mouse muscle cells on a discontinuous Percoll gradient gave rise to two myogenic fractions, one of which was more myogenic in vitro than were the unseparated cells and one of which was less myogenic. Both of these fractions were myogenic in vivo. A cell fraction was also produced which was non-myogenic in vitro as well as in vivo. In vitro and in vivo measurements of myogenicity were therefore in broad agreement.
Key words: Mouse muscle cells; Cell separation; Myogenicity
INTRODUCTION
The long-term aim of our work is to implant normal muscle precursor ceils into growing or regenerating myopathic muscle, in an attempt to alleviate the myopathy. In Correspondence to: Dr. J.E. Morgan, Dept. of Histopathoiogy, Chafing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF U.K. 0022-510X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
198 previous work (Watt et al. 1982, 1984), we have implanted muscle cells obtained by enzymic disaggregation of newborn mouse muscle into growing or regenerating mouse muscle and have shown that the implanted cells had been incorporated into host muscle fibres. In order to apply this technique to the treatment of myopathies, the implanted cell suspension should contain as high a proportion as possible of myogenic cells capable of fusion with the host muscle fibres, so as to maximise the numbers of myonuclei of donor origin expressing their normal genes within the resultant mosaic muscle fibres. Enzymic dissaggregation of skeletal muscle gives rise to a single-cell preparation containing both myogenic cells and non-myogenic cells, such as fibroblasts (Konigsberg 1979). Several methods have been used in attempts to separate myoblasts from fibroblasts in such suspensions (Yaffe 1968; Puri and Turner 1978; Turner 1978; Konigsberg 1979). Clonal analysis studies have shown that in the chick at least, myogenic cells are not a homogeneous population (Hauschka and White 1972; White et al. 1975; Seed and Hauschka 1984). The myogenicity of the resultant cell populations is invariably assessed in vitro (Morris and Cole 1972; Shainberg et al. 1971). This however leaves open the question of the extent to which in vitro myogenesis reflects the in vivo potential of a cell preparation. Here, we have separated newborn mouse muscle cells on discontinuous Percoll gradients to produce a fraction containing high proportions of myogenic, and another fraction consisting largely of non-myogenic cells. We present an assessment in vivo as well as in vitro of the myogenicity of the highly myogenic and the non-myogenic fraction of separated cells. The two methods of assessment were found to give broadly comparable results. MATERIALS AND METHODS Mouse strains
Various strains of mice were used as sources of muscle precursor cells, depending on availability. Thus, muscle cells were prepared from newborn 129/ReJ, CBA or C57B1/6J mice. CBA and C57B1/6J muscle cells (both of Gpi-ls b allotype) were implanted into minced tibialis anterior (TA) autografts in nu/nu mice (Gpi-ls a allotype). In some instances, 129/ReJ muscle cells (Gpi-ls a allotype) were implanted into C57B 1/6J minced TA isografts (Gpi-ls b allotype). Because it was found to be difficult to make the ICR/IAn strain of mouse (which was of particular interest because its skeletal muscles lack the enzyme phosphorylase kinase), tolerant to any of our usual strain of donor cells, 129/ReJ muscle cells were also implanted into ICR/IAn whole extensor digitorum longus (EDL) muscles (Gpi-ls b allotype) which were themselves grafted into nu/nu mice of Gpi-ls a allotype, thus avoiding the problem of immune rejection. Tolerance induction
Where 129/ReJ muscle cells were implanted into muscle grafts in C57B 1/6J hosts, the latter were made tolerant to the donor strain by neonatal intravenous injection of
199 5 X 10 6 129/ReJ spleen cells (Billingham et al. 1956). Tolerance induction was not necessary where grafts were made in nu/nu athymic mice. Preparation of mouse muscle cell suspensions Suspensions of muscle cells were obtained by enzymic dissaggregation of newborn mouse muscle, as described previously (Watt et al. 1982; Morgan et al. 1988). After resuspension of the cells in the growth medium used in the particular experiment, a count of viable, trypan blue-excluding, cells was made. Separation of muscle cell suspensions on discontinuous Percoll gradients An aliquot of the single cell suspension was kept on ice in growth medium (unseparated cells). The remaining mouse muscle cells were centrifuged at 350 x g and resuspended in 5 ml of calcium and magnesium-free Hanks balanced salt solution (HBSS; Gibco), buffered with 25 mM Hepes (Gibco). Under this were layered 5 ml aliquots of 26% and 34% Percoll, both solutions being made up in calcium and magnesium-free HBSS, buffered with 25 mM Hepes. The gradient was then centrifuged at 350 x g for 15 rain at 20 °C. Cells from each interface and from under the 34% Percoll were collected with a Pasteur pipette and added to an equal volume of HBSS containing calcium and magnesium, penicillin, streptomycin (P 5000 IU/ml; S 5000 #g/ml; Flow) and 20% FCS. Fraction 1 refers to cells from the 0-26% Percoll interface, fraction 2 to cells from the 26-34% Percoll interface and fraction 3 to cells from under the 34% Percoll. The unseparated cells and the three cell fractions were again centrifuged at 350 × g for 10 rnin, resuspended in a known volume of the particular growth medium to be used in that experiment and a count of viable cells was performed. The cells were then adjusted to the required cell density. Where the cells were to be implanted into muscle grafts, centrifuged pellets containing known numbers of unseparated cells and cells from each of the Percoll fractions were prepared. The unseparated cells and cells from each of the Percoll fractions were also grown in tissue culture. Culture of separated muscle cells and assessment of their myogenicity in vitro Separated and control cells were plated directly into 35-mm petri dishes (Nunc) containing a glass coverslip coated with rat tail collagen (Paul 1972). Plating densities varied from 2 × 104 to 1 x 105 viable cells per 35-ram petri dish. The culture medium was based on Medium 199 containing Earle's salts and 25mM Hepes buffer (Gibco), penicillin and streptomycin (P 5000 IU/ml; S 5000 #g/ml; Flow) (M199). Initially, different proportions of fetal calf serum (FCS; Sera Lab) and chick embryo extract (CEE; Flow) were used, but the particular batches of FCS and CEE used in these experiments were found to support the greatest extent of myoblast fusion when 20 parts FCS and 10 parts CEE were used. Cultures were only grown until fusion had occurred in the cultures of unseparated cells (i.e., approx. 5-7 days) and the culture medium was not changed during this time. When fusion had occurred in the control cultures, the coverslips bearing the monolayer of cells were removed from the petri dishes, washed twice in PB S and fixed
200 for 15 min in methanol. The cells were then stained with haematoxylin and eosin. Stained cultures were then examined microscopically in order to determine the proportion of nuclei present in multinucleated myotubes and those present in mononucleated cells. The number of nuclei in each culture present in one-hundred 80 x 80 #m squares of a Gallenkampf M N H 420A graticule at x 100 magnification were counted in 25 randomly encountered fields. Multinucleated myotubes were defined as those structures which contained 3 or more nuclei. The percentage of nuclei in myotubes was calculated for each of the 100 squares and these figures were averaged to give a measure of the percentage of nuclei in myotubes for the entire culture.
Grafting techniques Minced and whole muscle grafts were prepared as described previously (Watt et al. 1982, 1987). Muscle cells were added to and mixed into, minced muscle grafts. In the case of whole muscle grafts, the pellet of donor muscle precursor cells was inserted by means of a fine Pasteur pipette into a slit made in the anterior aspect of the graft.
Implantation of cells separated on discontinuous PercoUgradients into minced muscle grafts From 2 to 8 x 105 donor muscle cells were implanted into minced TA muscle grafts in nu/nu (9 mice) or neonatally tolerant C57BL/6J mice (12 mice). The grafts were removed for analysis 20-30 days later.
Implantation of cells separated on discontinuous PercoU gradients into whole muscle grafts In these experiments, 3 x 105-106 129/ReJ muscle cells of Gpi-1 s ~ allotype were separated on discontinuous Percoll gradients and implanted into whole ICR/IAn muscle grafts (Gpi-ls b allotype) in nu/nu hosts of Gpi-ls ~ allotype. Grafts were removed for analysis from 20 to 60 days after grafting.
Analysis of GPI isoenzymes Mice were killed by cervical dislocation at intervals after grafting. The muscle graft was removed, mounted in Tissue-tec OTC compound and frozen in isopentane cooled to - 165 °C in liquid nitrogen. Five #m thick cryostat sections were cut at three levels through the graft and these sections were taken up onto microscope slides. One section on each slide was overlaid for a few seconds with a small rectangle of filter paper, approx. 6 x 2 mm, dampened with distilled water. Within 30 sec, sufficient GPI for analysis was absorbed into the filter paper for isoelectric focusing (IEF) analysis. The sections on the slides were then stained with haematoxylin and eosin. The filter paper rectangles containing GPI were placed on the surface of an 0.8 mm thick polyacrylamide gel containing a mixture of cartier ampholytes (LKB) pH range 9-11 and 8-9.5 in a ratio of 2 : 1. They were subject to isoelectric focusing for 2.5 h at 150 V/cm. Two standards were run on each gel. One was a cryostat section cut from the muscle of a C57BL/6J x 129/ReJ F1 hybrid mouse; this sample contains the AA, AB and BB GPI isoenzymes in a ratio of 1 : 2 : 1. The other was a cryostat section cut from
201 a block containing pieces of C57BL/6J muscle and 129/ReJ muscle; this was to show that the AA and BB GPI isoenzymes did not dissociate and reassociate on focusing to give rise to the AB isoenzyme. After focusing, the gel was stained to indicate the presence of the isoenzymes of GPI by means of an agar overlay mixture (Partridge et al. 1978). The BB GPI isoenzyme, characteristic of the ICR/IAn, CBA and C57B1/6J strains of mouse, is cathodal and focuses at a pH of 8.6, while the AA isoenzyme, characteristic of the 129/ReJ strain and the nu/nu mice used here, is anodal, focusing at a pH of 8.2. The hybrid AB GPI isoenzyme is a heterodimer containing one A and one B subunit and focuses between the anodal and cathodal forms. The finding of donor GPI isoenzyme in the grafts indicates the presence of the implanted muscle-derived cells, but the identity of the tissue to which these cells had contributed cannot be ascertained. The finding of the hybrid AB isoenzyme means that nuclei of Gpi-ls a and Gpi-ls h allotype are present within a common cytoplasm. In our muscle grafts, this indicates that muscle precursor cells from host and donor origin have fused to form a mosaic muscle fibre, within which the donor as well as the host myonuclei are expressing their genes. This shows that some at least of the implanted cells must have been myogenic.
Histological examination of grafts Cryostat sections stained with haematoxylin and eosin were examined microscopically. The proportions of constituent tissues were estimated using a 25 random point graticule, according to the method of Curtis (1960) and the percentage of the graft consisting of new muscle was recorded. RESULTS
Assessment of myogenicity of mouse muscle cell preparations in vitro In initial experiments (results not shown) the discontinuous Percoll gradients consisted of layers corresponding closely in specific gravities to those in the Ficoll gradient used by Turner (1978). It was found that mouse muscle cells which had densities in the range of 34~ Percoll were less myogenic and those which were denser than 34~ Percoll were in general more myogenic in culture than were unseparated cells. A stepped gradient was finally arrived at which gave a non-myogenic fraction as well as one which was rich in myogenic cells. Fraction 1 cells, from the 0 - 2 6 ~ Percoll interface, were only cultured at one seeding density (5 × 104 cells/dish) and were less than a quarter as myogenic as the unseparated cells grown at the same initial seeding density (6°/o nuclei in myotubes compared with 27~ ; Fig. la). At all of the initial seeding densities, the fraction 2 cells were virtually non-myogenic, one culture containing only a few myotubes (0.4~o nuclei in myotubes; Fig. lb), the rest containing none. At all of the initial seeding densities, fraction 3 cells (Fig. lc) were more myogenic (36-45 ~o nuclei in myotubes) than the unseparated cells (23-32°/o nuclei in myotubes;
C,
203
m o
0
r-
Od O
O O
D
'rp
Oo
°0 •
~i~/¸¸¸!
o
,)1o 4
Q
O
Fig. 1. Cultures of newborn 129/ReJ mouse muscle cells separated on a discontinuous Percoll gradient and cultured for 7 days in M199, 20 parts FCS and 10 parts CEE, at an original cell seeding density of 5 x 104 cells per 35-mm petri dish.. (a) Fraction I, from the 0-26% Percoll interface. (b) Fraction 2, from the 26-34% Percoll interface. (c) Fraction 3, from under the 34% Percoll interface. (d) Unseparated cells. H&E, × 44.
204 Fig. ld). Both the unseparated and fraction 3 cells underwent fusion into myotubes earlier (day 5) when plated at a higher initial cell density (105 cells/dish) than at a lower cell density (2-5 × 104 cells/dish), when they underwent fusion on day 7. Direct comparison of cultures plated at different cell densities is of little value, as the cells plated at the higher densities had been fixed at an earlier time than those plated at the lower densities.
Assessment of myogenicity of mouse muscle cell preparations in vivo (Fig. 2) Implantation of cells separated on discontinuous Percoll gradients into minced muscle grafts Four out of 5 grafts to which unseparated muscle cells had been added contained host, donor and hybrid GPI isoenzymes (Fig. 2). The remaining graft contained only host isoenzyme; this graft was made in a C57B 1/6J mouse in which tolerance induction had been attempted and was the only graft to contain no new muscle. It seems possible therefore that the implanted cells had been immunologically rejected. All four of the grafts to which fraction 1 cells from the Percoll gradient had been added contained host, donor and hybrid GPI isoenzymes. One of the grafts to which fraction 2 cells had been added contained host, donor and a very small amount of hybrid GPI isoenzyme. Two of the grafts contained only host and donor GPI isoenzymes and the fourth contained the host GPI isoenzyme alone.
GPI i8oenzymes g
Fig. 2. GPI isoenzymes, separated by IEF, from cryostat sections of the following: (a) 30 day old minced TA isograR, made in a neonatally tolerant C57B1/6J host, to which 6 x 105 unseparated 129/ReJ muscle cells had been added. This graft contained host and a trace of donor and hybrid GPI isoenzymes. (b) 25-day-old minced TA autograR, made in a nu/nu mouse host, to which 8 x 105 fraction 1 (from the 0-26 % Percoll interface) C57B 1/6J muscle cells had been added. This graft contained host and a trace of donor and hybrid GPI isoenzymes. (c) 30-day-old minced TA isograR, made in a neonatally tolerant C57B1/6J host, to which 6 x 105 fraction 1 (from the 0 - 2 6 ~ Percoll interface) 129/ReJ muscle cells had been added. This grail contained host, donor and hybrid GPI isoenzymes. (d)30-day-old minced TA isograR, made in a neonatally tolerant C57B1/6J host, to which 6 x 105 fraction 2 (from 26-34% Percoll interface) 129/ReJ muscle cells had been added. This graft contained only host GPI isoenzyme. (e) 25-day-old minced TA autograft, made in a nu/nu mouse host, to which 8 × 105 fraction 3 (from under the 34% Percoll) C57B1/6J muscle cells had been added. This graft contained host, donor and hybrid GPI isoenzymes. (f) 30-day-old minced TA isograft made in a neonatally tolerant C57B1/6J host, to which 6 x 105 fraction 3 (from under the 3 4 ~ Percoll) 129/ReJ muscle cells had been added. This graft contained host, donor and hybrid GPI isoenzymes. (g) Control: a mixture of 129/ReJ and C57B1/6J skeletal muscles, to show that the AA and BB GPI isoeazymes do not re-associate under the conditions o f l E F to form the 'hybrid' AB GPI isoenzyme. (h) Standard: skeletal muscle from a C57B1/6J × 129/ReJ F] mouse, to show the localisation of the AA, BB and AB GPI isoenzymes.
205 Six out of 8 grafts to which fraction 3 cells had been added contained host, donor and hybrid GPI isoenzymes; the remaining two grafts (one in a nu/nu host and the other in a C57B 1/6J host) contained only host isoenzyme. Ceils which were virtually non-myogenic in vitro were therefore, with one exception, non-myogenic in minced muscle grafts in vivo. The addition of a cell pellet which was more myogenic (fraction 3) or less myogenic (fraction 1) in vitro than the unseparated ceils did not give rise to a noticably different incidence of hybrid isoenzyme in minced muscle grafts.
Implantation of cells separated on discontinuous Percoll gradients into whole muscle grafts In these experiments, muscle cells of Gpi-ls a allotype were implanted into whole ICR/IAn muscle grafts (Gpi-ls a allotype) in nu/nu hosts of Gpi-ls a allotype. Under these circumstances, any AA GPI isoenzyme found in the graft could be derived from the host or from the implanted donor muscle cells. Control grafts, to which no cell were added, were performed to test the possibility that hybrid AB GPI isoenzyme in the grafts was derived from muscle ceils of host origin rather than from the implanted muscle cells. Three out of 5 such grafts contained only the ICR/IAn-type BB GPI isoenzyme; the remaining two grafts contained the AA, BB but not the hybrid AB GPI isoenzyme. This implies that cells of host origin did enter the graft, but that these cells showed no evidence of myogenicity. One out of 4 grafts to which unseparated cells had been added contained AA, BB and hybrid AB GPI isoenzymes; 2 contained only BB GPI and the remaining 1 contained AA and BB GPI isoenzymes. Three out of 8 grafts to which fraction 1 muscle cells had been added contained AA, BB and hybrid AB GPI isoenzymes; 4 contained AA and BB GPI isoenzymes and the remaining 1 contained only BB GPI. None of the grafts to which fraction 2 cells had been added contained hybrid AB GPI isoenzyme; all 7 grafts contained only the AA and BB GPI isoenzymes. Three out of 7 grafts to which fraction 3 cells had been added contained AA, BB and hybrid AB GPI isoenzymes; 3 contained only AA and BB GPI isoenzymes and the remaining 1 contained only BB GPI. In whole muscle grafts, therefore, muscle ceils which were non-myogenic in vitro were also non-myogenic in vivo. Cells from fraction 3, which were more myogenic than fraction 1 in vitro, however, gave rise to no higher incidence of hybrid GPI in whole muscle grafts in vivo.
DISCUSSION
We have shown that it is possible to separate suspensions of newborn mouse muscle cells by centrifugation on discontinuous Percoll gradients into populations which, in vitro, are more myogenic and populations which are less myogenic than the original cell preparation. The in vitro myogenicity of our mouse muscle cells separated on discontinuous Percoll gradients shows some similarities to chick muscle cells sepa-
206 rated on discontinuous Ficoll (Turner 1978) or Percoll (Yablonka-Reuveni and N ameroff 1986)gradients. The gradients used by these workers separated non-myogenic cells as the least dense fraction, whereas we found that myogenic cells were present in both the least dense fraction, and in a high proportion in the densest fraction, but were absent from the intermediate fraction. It is possible that the myogenic cells in fraction 1 were in a different stage of the cell cycle from those found in fraction 3; the denser myogenic cells may, for example, have been in the S and G2 phases of the cell cycle. Proliferative myogenic cells may thus have been separated from quiescent stem cells, or from non-dividing, fusion-capable myogenic cells present in the newborn mouse muscle, but this was not further investigated. By implanting the separated muscle cell suspensions into regenerating mouse muscle grafts, we were also able to assess their myogenicity in vivo. This is the first attempt to correlate the in vivo and in vitro myogenicity of muscle cell populations. The finding of hybrid GPI was the only certain indication that the implanted cells had actually taken part in muscle fibre formation: it can only have been derived from mosaic muscle fibres, formed by the fusion of host and donor myogenic cells. Donor-type GPI could have been derived from muscle but could also have come from other cells present in the graft site. In the absence of hybrid GPI therefore, we can neither exclude nor confirm the possibility that the cell preparation in question was myogenic. As a general rule, mouse muscle cells which were myogenic in vitro were also myogenic in vivo when implanted into regenerating muscle grafts. Likewise, cells which showed no evidence of myogenicity in vitro also showed none in vivo. Implantation of a mouse muscle cell suspension which was more myogenic in vitro than unseparated cells did not give rise to a higher percentage of grafts containing hybrid isoenzyme. However, the presence or absence of hybrid GPI is a crude indicator of whether or not myogenic cells were present in a particular cell suspension. It should be possible to obtain a more precise measure of the myogenicity of an implanted muscle cell preparation by making a quantitative study of the proportions of the GPI isoenzymes present in the graft as has been attempted by Friar and Peterson (1983) and Jockusch et al. (personal communication): the proportion of hybrid GPI should be in correlation with the myogenicity of the cell preparation added to them. Such fully quantitative methods however, do require that the proportions of the isoenzymes present are not too disparate so that the reaction for each of them can be measured while it is still in the linear phase. An alternative method of assessing myogenicity is by a dilution assay: the more myogenic a cell preparation is, the fewer cells would need to be implanted into grafts to produce a detectable amount of hybrid GPI. Approximately half of the nuclei in skeletal muscle belong to non-muscle cells and tissue culture studies (Sanderson et al. 1986) have shown that muscle fibroblasts are necessary for the formation of basal lamina. We are now using a model system whereby we can make a new muscle in vivo by grafting a scaffolding of freeze-killed muscle and repopulating it with muscle precursor cells (Morgan et al. 1987), to investigate the interaction of non-myogenic cells and myogenic cells, separated on discontinuous Percoll gradients, to the formation of new muscle in vivo. Our preliminary findings suggest that implanted muscle fibroblasts play a helper role in the formation of new muscle in freeze-killed grafts.
207 ACKNOWLEDGEMENTS
I wish to thank Dr. T. A. Partridge for his advice and criticism and Mr. R. Barnett and the Department of Medical Illustration for photography. This work was supported by the Muscular Dystrophy Group of Great Britain.
REFERENCES Billingham, R.E., L. Brant, and P.B. Medawar (1956) Quantitative studies on tissue transplantation immunity III: Actively acquired tolerance. Phil. R. Soc. Lond. Series B Biol. Series, 239: 357-424. Curtis, A. S. G. (1960) Area and volume measurements by random sampling methods. Med. Biol. Illustr., 10: 261-266. Frai L P. M. and A. C. Peterson (1983) The nuclear-cytoplasmic relationship in 'mosaic' skeletal muscle fibres from mouse chimaeras. Exp. Cell Res., 145: 167-178. Hauschka, S.D. and N.K. White (1972) Studies ofmyogenesis in vitro. In: B.Q. Banker, R.J. Pryzybylski, J.P. Van der Meulen and M. Victor (Eds.), Research in Muscle Development and the Muscle Spindle, Excerpta Medica, Amsterdam, pp. 53-71. Konigsberg, I. R. (1979) Skeletal myoblasts in culture. Methods Enzymol., 58:511-527. Morgan, J.E., G.R. Coulton and T.A. Partridge (1987) Muscle precursor cells invade and repopulate freeze-killed muscles. J. Muscle Res. Cell Motil., 8: 386-396. Morris, G.E. and R.J. Cole (1972) Cell fusion and differentiation in cultured chick muscle cells. Exp. Cell Res., 75: 191-199. Paul, J. (1972). In: Cell and Tissue Culture, Churchill Livingstone, Edinburgh, pp. 73-74. Partridge, T. A., M. Grounds and J.C. Sloper (1978) Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature (Lond.), 273: 306-308. Puri, E.C. and D.C. Turner (1978) Serum-free medium allows chick myogenic cells to be cultivated in suspension and separated from attached fibroblasts. Exp. Cell. Res., 115: 159-173. Sanderson, R.D., J.M. Fitch, T.R. Lisenmayer and R. Mayne (1986) Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro. J. Cell Biol., 102: 740-747. Seed, J. and S.D. Hauschka (1984) Temporal separation of the migration of distinct myogenic precursor populations into the developing chick wing bud. Dev. Biol., 106: 389-393. Shainberg, A., G. Yagil and D. Yaffe (1971) Alterations of enzymatic activities during muscle differentiation in vitro Dev. Biol., 25: 1-29. Turner, D.C. (1978) Differentiation in cultures derived from embryonic chicken muscle. The postmitotic, fusion-capable myoblast as a distinct cell type. Differentiation, 10: 81-93. Watt, D.J., K. Lambert, J. E. Morgan, T.A. Partridge and J. C. Sloper (1982) Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse. J. Neurol. Sci., 57: 319-331. Watt, D.J., J.E. Morgan and T.A. Partridge (1984) Use of mononuclear muscle precursor cells to insert allogeneic genes into growing mouse muscles. Muscle Nerve, 7: 741-750. Watt, D.J., J. E. Morgan, M. A. Clifford and T.A. Partridge (1987) The movement of muscle precursor cells between adjacent regenerating muscles in the mouse. Anat. Embryol., 175: 527-536. White, N.K., P.H. Bonnet, D.R. Nelson and S.D. Hauschka (1975) Clonal analysis of vertebrate myogenesis. Dev. Biol., 44: 346-361. Yablonka-Reuveni, Z. and M. Nameroff (1986) Analysis of skeletal muscle cell populations using density centrifugation J. Cell Biol., 103: 120a. Yaffe, D. (1968) Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. U.S.A., 61: 477-483.