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Establishment of myoid cells from bone marrow
Cell Biology
International
Reports,
ESTABLISHMENT Kamo,
I.*, T .,
OF MYOID Kikuchi, Furukawa,
National Institute 4-l-l Ogawahigashi, *Corresponding
Vol. 14, No. 7, July
CELLS
595
7990
FROM BONE NARROW
A., Ishii, El., S. and Ishiura,
Matsuoka, S.
of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan.
author
ABSTRACT
A peculiar adherent cell clone (R613BM) was established under muscle tissue free conditions from bone marrow of The cloned cell line was able to form a Wistar rat. myofibrils and expressed nicotinic acetylcholine receptors specific for skeletal muscles. The muscle specific characteristics have been maintained consistently for more than five years. These results suggest that bone marrow contains a precursor cell which has the potency to differentiate into muscle cells. INTRODUCTION
It is generally accepted that skeletal muscle originates from mesoderm. In many vertebrates, striated muscle fibers have also been observed in anatomical regions different from skeletal muscles, such as in the central nervous system (Freschi et al, 1979; Wier et al, and the thymus (Van de Velde and Friedman 1970; KAz8QL Little is known as to whether these myoid al, 1984). cells are of the same developmental lineage as mesodermderived skeletal muscles or have origins different from skeletal muscles. In particular, in embryonic and animals, the thymus (Stutman 1978) and central young system (McKay 1989) contain highly multipotent nerve stem cells which differentiate into organ specific cell types under the influence of each microenvironment. Bone marrow is another source of stem cells for many different types of myeloid and lymphoid cells. Through bone marrow cell culture experiments, we have observed multinucleated cells as a rare cell type. In the present study, we describe a cell cloned from such cultures which has muscle specific characteristics. MATERIALS
AND METRODS
Establishment of a bone marrow-derived myoid cell: Femur from an 8-day-old female Wistar rat (purchased from Sankyo lab. Japan) was carefully removed from neighbour0309-I
651/901070595-6/$03.00/O
@ 1990
Academic
Press
Ltd
596
Cell Biology
International
Reports,
Vol. 14, No. 7, July 1990
ing muscle tissues. The small amount of muscle tissue remaining on the surface of the femur was rubbed off with sterile gauze. The cleaned femur was soaked in a 70% ethanol solution for 3-4 seconds, passed through a burner flame, and then put immediately in a chilled culture medium (RPM1 1640 + 10% fetal calf serum). Bone marrow cells were syringed from the thoroughly cleaned femur with the culture medium; cells were washed once with the culture medium by centrifugation at 150 g for 10 min. at 4OC and resuspended in the medium at a concentration of 2 X lO'/ml. Two ml of this cell suspension was cultured in a 24 well plate (Falcon) in a humidified atmosphere of 5% COz in air at 37'C. Half of the culture medium was changed every 3-4 days. Many adherent cells and nonadherent cells, mostly macrophage lineage cells, appeared 5-7 days after the culture initiation. Non adherent cells were removed from the Cell clusters culture by flushing with a pipette. containing multinucleated cells were removed by aid of Cells were suspended at trypsin and a penicillin cup. a concentration of 4 cells/ml. in 0.2 ml. of the culture acetylcholine medium. Procedures for cell cloning, receptor (AchR) assay, and electron microscopic observation were the same as described previously (Kamo et al, specific proteins were analyzed by 1984). Muscle immunoblotting after electrophoresis in 10% polyacrylamide gel (Laemmli, 1970). RESULTS cell line R613BM was established from bone A myoid marrow of a Wistar rat. We carefully separated the femur from neighboring muscle tissue and quickly passed the cleaned femur through a burner flame to completely kill remaining muscle cells, if any. After syringing out of as thoroughly as the interior cells the femur possible, we attempted to culture the empty femur to investigate whether it gave rise to any cells. However, we failed to find any cells in the culture of the empty femur even when culture was continued for more than three months, whereas in bone marrow cell cultures adherent and non-adherent cells started to increase 5-7 days after the culture initiation. We obtained similar results in repeated experiments. These results strongindicate that our bone marrow cell preparation is 1Y free of skeletal muscle cells and our cell line originates from bone marrow cells. R613BM has been maintained in culture for more than five years without losing myogenic potency. The morphology of R613BM at a single cell stage is similar myoid to thymus-derived cell R615A and R613Ad which have been described previously (Kamo, 1984). R613BM cells fused spontaneously and formed myotubes through continuous culture (Fig. 1).
Cell Biology
International
Reports,
1. Morphology of exponentially left) and multinucleated phase cultures (6 day, right).
Fig. (2
day,
597
Vol. 74. No. 7, July 1990
growing cells
R613BM cells in stationary
contractile activity of fused forms of Spontaneous R613BM was not observed even two to three weeks after myotube formation. Dexamethazone at concentrations of 40-400 rig/ml greatly accelerated myotube formation. Electron microscopic studies indicated that myotubes of R613BM cells had developing myofibrillar elements including 2 bands (Fig. 2).
Fig. 2. Electron microscopic appearance elements of R613BM cells (8 day).
of myofibrillar
598
Cell Biology
International
Reports,
Vol. 74, No. 7, July
1990
10
I
300
600
0 . 000
Protein(pg)
AChR on R613BM cells. Culture and Fig. 3. Nicotinic conditions were the same as described (Kamo AChR assay Open circles: R613BM cell homogenate + et al, 1984). I" I alpha-bungarotoxin + PBS. Closed circles: R613BM alpha-bungarotoxin + 1000 fold cell homogenate + "'1 cold bungarotoxin.
Fig. 4. Analysis of muscle specific proteins of R613BM. As About 0.02 mg proteins were used for the analysis. anti human myosin heavy chain rabbit a first antibody IgG and anti human skeletal muscle alpha actinin rabbit system ABC kit (Vector IgG, and as a second antibody lab.) were used. A: myosin heavy chain of rat skeletal muscle homogenate. B: myosin heavy chain of R613BM rat skeletal muscle alpha homogenate (arrow). c: actinin (arrow). D: R613BM alpha actinin (arrow).
Cell Biology
International
Reports,
Vol. 14, No. 7, July
599
1990
Thy-l antigen, a marker for T cells, neural cells and muscle cells, was detected weakly on the cell surface (data not shown). AChR, another specific muscle membrane marker, was also detected on the cell surface of myotubes by specific binding of 125 I alpha-bungarotoxin (Fig 3). Skeletal muscle specific myosin heavy chains at 200 Kd and alpha actinins at 96 Kd were detected by immunoblotting (Fig. 4). The relationship between bone marrow cells and muscle precursor cells has been described. Durban and Boetmacrophages tiger (1981) found that bone marrow derived served as a target specific for avian myobalstosis virus. Yarom et al (1982) indicated that bone marrow cells accelerated muscle regeneration. Grounds (1983), however, reported that skeletal muscle precursors did not directly arise from bone marrow cells. Under conventional conditions, bone marrow cells, therefore, probably provide cellular and/or humoral circumstances accelerating the muscle regeneration rather than contributing directly to the regenerating muscle fibers. The present results suggest that bone marrow also contains stem cells which have the potency to differentiate into muscle lineage cells under unidentified conditions. In this regard, myogenic regulatory genes, such as MyoDl and myogenin genes (Sassoon, 1989) have been reported to convert non-myogenic cells into differentiated muscle cells. Several retro viruses (Lassar et al, 1989) and 5-bromo-2' -deozyuridine (Tapscott et al, 1989) affect the expression of the myogenic regulatory genes. Extracellular microenvironment is also reported to modulate the developmental expression of muscle precursors (Lipton and Schultz, 1979). Our cell line appeared to be established under these complicated conditions. REFERENCES Durban,
E.
M.
and
Boettiger,
effects of transforming avian macrophages. 78, 3600-3604.
Freischi,
J.
E.,
Parfitt,
D.
(1981).
avian RNA tumour Proc. Natl. Acad. A.
G.
and
Shain,
Differential viruses on Sci. USA.
W. G.
(1979).
Electrophysiology and pharmacology of striated muscle fibres cultured from dissociated neonatal rat pineal glands. J. Physiol. 293, l-10. Grounds, H. D. (1983). Skeletal muscle precursors do not arise from bone marrow cells. Cell and Tissue Res. 234, 713-722. Kamo,
I., (1984).
Nonaka,
I.,
Furukawa,
S.
and
Satoyoshi,
Clonal heterogeneity of thymic muscle-cell precursors. Bioscience Reports. 4, 925-932.
E.
600
Cell Biology
International
Reports,
Vol. 14, No. 7, July 1990
Laemmli,
U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685. Lassar, A. B., Thayer, M. J., Overell, R. W. and Weintraub, H. (1989). Transformation by activated ras or fos prevents myogenesis by inhibiting expression of MyoDl. Cell. 58, 659-667. Lipton, B. H. and Schultz, E. (1979). Developmental fate of skeletal muscle satellite cells. Science. 205, 1292-1294. Mckay, R. D. G. (1989). The origins of cellular diversity in the mammalian central nervous system. Cell. 58, 815-821. Sasson, D., Lyons, G., Wright, W. E., Lin, V., Lassar, A Weintraub, H. and Buckingham, W. (1989). E&ession of two myogenic regulatory factors myogenin and MyoDl during mouse embryogenesis. Nature. 341, 303-307. Stutman, 0. (1978). Intrathymic and extrathymic T cell maturation. Immunol. Rev. 42, 138-184. Tapscott, S. J., Lassar, A. B., Davis, R. L. and Weintraub, H. (1989). 5-bromo-2' -deoxyuridine blocks myogenesis by extinguishing expression of MyoDl. Science. 245, 532-536. Van der Velde, R. L. and Friedman, N. B. (1970). Thymic myoid cells and myasthenia gravis. Am. J. Pathol. 59, 347-367. Wier, Differentiation W. L. and Lennon, V. A. (1981). of skeletal muscle from dissociated optic nerve cells immunocytochemical observations. J. Neuroimmunol. 1, 61-68. Yarom, R., Meyer, S., Carmy, O., Ghidomi, B. and More, R. (1982). Enhancement of human muscle growth in diffusion chambers by bone marrow cells. Virchows Arch (Cell Pathol). 41, 17 l-180. Paper
received
18.12.89.
Revised
paper
accepted
28.3.90.
International
Reports,
ESTABLISHMENT Kamo,
I.*, T .,
OF MYOID Kikuchi, Furukawa,
National Institute 4-l-l Ogawahigashi, *Corresponding
Vol. 14, No. 7, July
CELLS
595
7990
FROM BONE NARROW
A., Ishii, El., S. and Ishiura,
Matsuoka, S.
of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan.
author
ABSTRACT
A peculiar adherent cell clone (R613BM) was established under muscle tissue free conditions from bone marrow of The cloned cell line was able to form a Wistar rat. myofibrils and expressed nicotinic acetylcholine receptors specific for skeletal muscles. The muscle specific characteristics have been maintained consistently for more than five years. These results suggest that bone marrow contains a precursor cell which has the potency to differentiate into muscle cells. INTRODUCTION
It is generally accepted that skeletal muscle originates from mesoderm. In many vertebrates, striated muscle fibers have also been observed in anatomical regions different from skeletal muscles, such as in the central nervous system (Freschi et al, 1979; Wier et al, and the thymus (Van de Velde and Friedman 1970; KAz8QL Little is known as to whether these myoid al, 1984). cells are of the same developmental lineage as mesodermderived skeletal muscles or have origins different from skeletal muscles. In particular, in embryonic and animals, the thymus (Stutman 1978) and central young system (McKay 1989) contain highly multipotent nerve stem cells which differentiate into organ specific cell types under the influence of each microenvironment. Bone marrow is another source of stem cells for many different types of myeloid and lymphoid cells. Through bone marrow cell culture experiments, we have observed multinucleated cells as a rare cell type. In the present study, we describe a cell cloned from such cultures which has muscle specific characteristics. MATERIALS
AND METRODS
Establishment of a bone marrow-derived myoid cell: Femur from an 8-day-old female Wistar rat (purchased from Sankyo lab. Japan) was carefully removed from neighbour0309-I
651/901070595-6/$03.00/O
@ 1990
Academic
Press
Ltd
596
Cell Biology
International
Reports,
Vol. 14, No. 7, July 1990
ing muscle tissues. The small amount of muscle tissue remaining on the surface of the femur was rubbed off with sterile gauze. The cleaned femur was soaked in a 70% ethanol solution for 3-4 seconds, passed through a burner flame, and then put immediately in a chilled culture medium (RPM1 1640 + 10% fetal calf serum). Bone marrow cells were syringed from the thoroughly cleaned femur with the culture medium; cells were washed once with the culture medium by centrifugation at 150 g for 10 min. at 4OC and resuspended in the medium at a concentration of 2 X lO'/ml. Two ml of this cell suspension was cultured in a 24 well plate (Falcon) in a humidified atmosphere of 5% COz in air at 37'C. Half of the culture medium was changed every 3-4 days. Many adherent cells and nonadherent cells, mostly macrophage lineage cells, appeared 5-7 days after the culture initiation. Non adherent cells were removed from the Cell clusters culture by flushing with a pipette. containing multinucleated cells were removed by aid of Cells were suspended at trypsin and a penicillin cup. a concentration of 4 cells/ml. in 0.2 ml. of the culture acetylcholine medium. Procedures for cell cloning, receptor (AchR) assay, and electron microscopic observation were the same as described previously (Kamo et al, specific proteins were analyzed by 1984). Muscle immunoblotting after electrophoresis in 10% polyacrylamide gel (Laemmli, 1970). RESULTS cell line R613BM was established from bone A myoid marrow of a Wistar rat. We carefully separated the femur from neighboring muscle tissue and quickly passed the cleaned femur through a burner flame to completely kill remaining muscle cells, if any. After syringing out of as thoroughly as the interior cells the femur possible, we attempted to culture the empty femur to investigate whether it gave rise to any cells. However, we failed to find any cells in the culture of the empty femur even when culture was continued for more than three months, whereas in bone marrow cell cultures adherent and non-adherent cells started to increase 5-7 days after the culture initiation. We obtained similar results in repeated experiments. These results strongindicate that our bone marrow cell preparation is 1Y free of skeletal muscle cells and our cell line originates from bone marrow cells. R613BM has been maintained in culture for more than five years without losing myogenic potency. The morphology of R613BM at a single cell stage is similar myoid to thymus-derived cell R615A and R613Ad which have been described previously (Kamo, 1984). R613BM cells fused spontaneously and formed myotubes through continuous culture (Fig. 1).
Cell Biology
International
Reports,
1. Morphology of exponentially left) and multinucleated phase cultures (6 day, right).
Fig. (2
day,
597
Vol. 74. No. 7, July 1990
growing cells
R613BM cells in stationary
contractile activity of fused forms of Spontaneous R613BM was not observed even two to three weeks after myotube formation. Dexamethazone at concentrations of 40-400 rig/ml greatly accelerated myotube formation. Electron microscopic studies indicated that myotubes of R613BM cells had developing myofibrillar elements including 2 bands (Fig. 2).
Fig. 2. Electron microscopic appearance elements of R613BM cells (8 day).
of myofibrillar
598
Cell Biology
International
Reports,
Vol. 74, No. 7, July
1990
10
I
300
600
0 . 000
Protein(pg)
AChR on R613BM cells. Culture and Fig. 3. Nicotinic conditions were the same as described (Kamo AChR assay Open circles: R613BM cell homogenate + et al, 1984). I" I alpha-bungarotoxin + PBS. Closed circles: R613BM alpha-bungarotoxin + 1000 fold cell homogenate + "'1 cold bungarotoxin.
Fig. 4. Analysis of muscle specific proteins of R613BM. As About 0.02 mg proteins were used for the analysis. anti human myosin heavy chain rabbit a first antibody IgG and anti human skeletal muscle alpha actinin rabbit system ABC kit (Vector IgG, and as a second antibody lab.) were used. A: myosin heavy chain of rat skeletal muscle homogenate. B: myosin heavy chain of R613BM rat skeletal muscle alpha homogenate (arrow). c: actinin (arrow). D: R613BM alpha actinin (arrow).
Cell Biology
International
Reports,
Vol. 14, No. 7, July
599
1990
Thy-l antigen, a marker for T cells, neural cells and muscle cells, was detected weakly on the cell surface (data not shown). AChR, another specific muscle membrane marker, was also detected on the cell surface of myotubes by specific binding of 125 I alpha-bungarotoxin (Fig 3). Skeletal muscle specific myosin heavy chains at 200 Kd and alpha actinins at 96 Kd were detected by immunoblotting (Fig. 4). The relationship between bone marrow cells and muscle precursor cells has been described. Durban and Boetmacrophages tiger (1981) found that bone marrow derived served as a target specific for avian myobalstosis virus. Yarom et al (1982) indicated that bone marrow cells accelerated muscle regeneration. Grounds (1983), however, reported that skeletal muscle precursors did not directly arise from bone marrow cells. Under conventional conditions, bone marrow cells, therefore, probably provide cellular and/or humoral circumstances accelerating the muscle regeneration rather than contributing directly to the regenerating muscle fibers. The present results suggest that bone marrow also contains stem cells which have the potency to differentiate into muscle lineage cells under unidentified conditions. In this regard, myogenic regulatory genes, such as MyoDl and myogenin genes (Sassoon, 1989) have been reported to convert non-myogenic cells into differentiated muscle cells. Several retro viruses (Lassar et al, 1989) and 5-bromo-2' -deozyuridine (Tapscott et al, 1989) affect the expression of the myogenic regulatory genes. Extracellular microenvironment is also reported to modulate the developmental expression of muscle precursors (Lipton and Schultz, 1979). Our cell line appeared to be established under these complicated conditions. REFERENCES Durban,
E.
M.
and
Boettiger,
effects of transforming avian macrophages. 78, 3600-3604.
Freischi,
J.
E.,
Parfitt,
D.
(1981).
avian RNA tumour Proc. Natl. Acad. A.
G.
and
Shain,
Differential viruses on Sci. USA.
W. G.
(1979).
Electrophysiology and pharmacology of striated muscle fibres cultured from dissociated neonatal rat pineal glands. J. Physiol. 293, l-10. Grounds, H. D. (1983). Skeletal muscle precursors do not arise from bone marrow cells. Cell and Tissue Res. 234, 713-722. Kamo,
I., (1984).
Nonaka,
I.,
Furukawa,
S.
and
Satoyoshi,
Clonal heterogeneity of thymic muscle-cell precursors. Bioscience Reports. 4, 925-932.
E.
600
Cell Biology
International
Reports,
Vol. 14, No. 7, July 1990
Laemmli,
U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685. Lassar, A. B., Thayer, M. J., Overell, R. W. and Weintraub, H. (1989). Transformation by activated ras or fos prevents myogenesis by inhibiting expression of MyoDl. Cell. 58, 659-667. Lipton, B. H. and Schultz, E. (1979). Developmental fate of skeletal muscle satellite cells. Science. 205, 1292-1294. Mckay, R. D. G. (1989). The origins of cellular diversity in the mammalian central nervous system. Cell. 58, 815-821. Sasson, D., Lyons, G., Wright, W. E., Lin, V., Lassar, A Weintraub, H. and Buckingham, W. (1989). E&ession of two myogenic regulatory factors myogenin and MyoDl during mouse embryogenesis. Nature. 341, 303-307. Stutman, 0. (1978). Intrathymic and extrathymic T cell maturation. Immunol. Rev. 42, 138-184. Tapscott, S. J., Lassar, A. B., Davis, R. L. and Weintraub, H. (1989). 5-bromo-2' -deoxyuridine blocks myogenesis by extinguishing expression of MyoDl. Science. 245, 532-536. Van der Velde, R. L. and Friedman, N. B. (1970). Thymic myoid cells and myasthenia gravis. Am. J. Pathol. 59, 347-367. Wier, Differentiation W. L. and Lennon, V. A. (1981). of skeletal muscle from dissociated optic nerve cells immunocytochemical observations. J. Neuroimmunol. 1, 61-68. Yarom, R., Meyer, S., Carmy, O., Ghidomi, B. and More, R. (1982). Enhancement of human muscle growth in diffusion chambers by bone marrow cells. Virchows Arch (Cell Pathol). 41, 17 l-180. Paper
received
18.12.89.
Revised
paper
accepted
28.3.90.