BMP4 MIGHT INDUCE DEDIFFERENTIATION OF MYOGENIC CELLS TOWARD AN OSTEOGENIC
LINEAGE
*Olshanski, A; *Li, G H; *Peng, H; +*Huard, J
+*Growth and Development Laboratory, Children’s Hospital of Pittsburgh and Department of Orthopaedic Surgery, University of Pittsburgh,
Pittsburgh, PA
jhuard@pitt.edu
INTRODUCTION
Previous studies by members of our laboratory have demonstrated
that muscle-derived stem cells (MDSCs) have osteogenic potential and
can form bone when genetically engineered to express bone
morphogenetic protein (BMP) (1). Specifically, we have shown that
cells expressing BMP4 can elicit ectopic bone formation when
implanted intramuscularly into the hind limb of a mouse (2). These
findings prompted us to investigate the cell source behind this bone
formation. We hypothesized that the mature muscle fibers from the host
animal dedifferentiate (that is, revert from a terminally differentiated
state to a more primitive progenitor-like state) and then redifferentiate
toward an osteogenic lineage and contribute to intramuscular bone
formation. The study reported here was designed to evaluate the effects
of BMP4 on C2C12 cells and myotubes. We used molecular techniques
to specifically label terminally differentiated myotubes and assess
whether BMP4 induces the dedifferentiation of myogenic cells.
Figure 2:
a
Floxed
ROSA26 3’
locus LTR
b
pClCre
CMV-P
PGK-CD-PGK-PURO
LoxP
Cre
5’
LTR
LTR
Figure 3. β-gal expression in
myotubes formed by co-culture of
MDSC-Cre cells and MDC-Loxβgeo cells. Note the absence of βgal expression in mononucleated
cells (arrowheads). DAPI stain
shows multiple nuclei in β-gal+
myotube (arrows).
RESULTS
In our preliminary studies, we cultured C2C12 cells in low-serum
conditions to promote myotube formation. If maintained for 3–5 days,
this culture system induces approximately 80% of the cells to
differentiate into multinucleated myotubes, whereas the other 20%
remain as mononucleated cells. The addition of BMP4 to the cultures
resulted in the disappearance of myotubes and an increase in the number
of mononucleated cells (Figure 1).
To examine this phenomenon more closely, we first purified our
myotube cultures by treating them with β-D-arabinofuranoside (aracytosine) in the presence of 2% FBS to kill residual mononucleated,
dividing cells; we then passed the remaining myotubes gently through a
100-µm sieve. Next, we replated the myotubes and treated them with
BMP4. When treated with BMP4, the myotubes disappeared. In contrast,
parallel cultures of C2C12 cells proliferated in the presence of BMP4
(data not shown).
Dedifferentiation did not seem to be a major factor in the
aforementioned studies; however, because it was virtually impossible to
monitor every myotube in each culture, we developed a system to
specifically label terminally differentiated myotubes and track their fate
after BMP4 treatment. Two populations of muscle-derived myogenic
cells were used for these experiments. One population, MDC-Lox-βgeo,
was isolated from 129-Gt(ROSA)26Sortm1Sho transgenic mice; these cells
have the floxed ROSA26 locus (3) encompassing a gene that encodes for
the β-galactosidase and neomycin resistance genes (βgeo) (Figure 2a).
The other population, MDC-Cre, comprises muscle-derived cells
(MDCs) isolated from normal C57B6J mice (Jackson Laboratory); we
transfected these cells with a Cre-expressing plasmid (pClCre) (Figure
2b).
MDC-Cre and MDC-Lox-βgeo cells were co-cultured and stimulated
to fuse or differentiate in low-serum conditions. Resultant cultures were
stained with LacZ. Upon fusion of the 2 cell types, Cre cleavage of the
β-geo
LoxP
Lox sites in the Lox-βgeo cells induced β-galactosidase (β-gal)
expression and neomycin resistance in the resultant myotubes (Figure 3).
We then stimulated cultures with BMP4 to induce dedifferentiation.
We expected any cells derived from a dedifferentiated myotube to
remain β-gal-positive. We treated parallel cultures (such as those found
in Figure 3) with G418 (1 mg/ml) for 6 days and with BMP4 (200
ng/ml) for 6 days. Double-staining for β-gal and alkaline phosphatase
(ALP), an early osteogenic marker, revealed some double-positive
mononucleated cells (Figure 4).
METHODS
Myogenic cells were stimulated to fuse and form myotubes in the
presence of 2% horse serum (HS) in DMEM. Myotube cultures were
maintained in 0.5% HS. BMP4 concentration in all experiments was 200
ng/ml.
Figure 1: Top row: C2C12 myotubes kept in
0.5% HS DMEM. Bottom row: C2C12 myotubes
treated with BMP4 (200 ng/ml) in 0.5% HS
DMEM.
STOP
LoxP
Figure 4: Dedifferentiation
of myotubes after BMP4
stimulation. Some of the
dedifferentiated cells (βgal+) also differentiated
toward the osteogenic
lineage, as indicated by the
expression of ALP (arrow).
DISCUSSION
Our results suggest that BMP4 can have several effects on myotubes
and myogenic cells: myotubes can die while mononucleated cells
proliferate, myotubes can dedifferentiate into mononucleated cells, or a
combination of these events can occur. Our use of the Cre-Lox system
provided genetic rather than morphologic evidence of the effect of
BMP4 on myotubes. Results from the first set of experiments suggested
that most myotubes died when exposed to BMP4. However, the second
set of experiments generated striking evidence of myogenic cell
dedifferentiation and redifferentiation into osteogenic cells. Although
such events might be rare, they could explain ectopic bone formation.
Other researchers have induced dedifferentiation of mouse myotubes by
using protein extracted from the regenerating limb tissue of newts (4),
which likely contains growth factors. The results of our study suggests
that BMP4 expression from genetically engineered cells that are
implanted into intramuscular pockets in mice might cause
dedifferentiation of mature muscle fibers and subsequent bone formation
by redifferentiated osteoprogenitor cells.
ACKNOWLEDGMENTS
We wish to thank Ryan Sauder for editorial assistance. This work was
supported by funding from the Henry J. Mankin Endowed Chair for
Orthopaedic Research at the University of Pittsburgh, the William F. and
Jean W. Donaldson Chair at Children’s Hospital of Pittsburgh, the
Hirtzel Foundation, and a grant awarded to Dr. Johnny Huard from the
National Institutes of Health (R03 AR050201).
REFERENCES
1. V. Wright et al., Mol Ther 6, 169–78 (Aug, 2002) 2. H. Peng et al., J
Clin Invest 110, 751–9 (Sept 15, 2002). 3. X. Mao et al., Proc Natl Acad
Sci USA 96, 5037–42 (April 1999). 4. CJ. McGann et al., Proc Natl
Acad Sci USA 98, 13699–704 (Nov 2001).
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 1883