Download 1 to enlarge population of stem cell-like cells from skeletal muscle

Preset TGF- 1 to enlarge population of stem cell-like cells from skeletal muscle cells of mouse
Mu, X; Ma, J; and +Li, Y
+The laboratory of Molecular Pathology, Stem Cell Research Center, Children’s Hospital of Pittsburgh of UPMC
and Department of Orthopaedic Surgery, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213
+ To whom correspondence should be addressed: [email protected]
INTRODUCTION:
In skeletal muscle, the number of muscle derived stem cells
(MDSCs) is limited and as of now, there are no current techniques that
are able to utilize all of the cells in the mature muscle fiber. A new
method based on the dedifferentiation of adult mature cells to generate
Induced Pluripotent Stem cell-like cells (iPS) would allow us to obtain a
much larger amount of autologous cells to be used in regenerative
medicine. The current project investigates the dedifferentiation process
in injured skeletal muscle and one of the potential promoters,
Transforming Growth Factor (TGF)- 1. Our previous research has
demonstrated that TGF- 1 is responsible for the regulation of both the
regeneration and fibrosis formation during the muscle healing process
(1). Also, TGF- 1 has been reported to induce dedifferentiation in
axolotls (salamanders) (2) and epithelial–mesenchymal transition (EMT)
in mammals (3). Here we characterized the expression profile of TGF- 1
after skeletal muscle injury and its possible correlation with the
dedifferentiation process in vivo by co-immunostaining of TGF- 1 and
dedifferentiation marker protein muscle segment homeobox (Msx)-1. Sphase re-entry of cell cycle-arrested cells is one critical step of the
induction of stem cell-like cells, and the possible effect of TGF- 1
treatment to S-phase re-entry and the expression of various myogenic
cell or stem cell markers were assayed in myoblasts conditionally treated
with TGF- 1. The proposed study should reveal the effectiveness of
skeletal muscle dedifferentiation into applicable progenitor cells, and
may lead to the development of future therapeutic applications.
METHODS:
Skeletal muscle cell isolation: The cells used for this research were
C2C12 and primary skeletal muscle cells, including muscle derived stem
cells (MDSC) isolated from the skeletal muscle of mice (C57BL/6J+/+).
MDSCs were purified by the preplate technique. After selection and
purification, the preplated populations (primary myoblast) muscle cells
were cultured in growth medium [DMEM supplemented with 20% Fetal
Bovine Serum (FBS), 10% Horse Serum (HS), 1% PenicillinStreptomycin antibiotics, and 0.5% chicken essential extract (CEE) ],
and incubated in 5% CO2 at 37 °C.
TGF- 1 treatment of cells: To study the role of TGF- 1 in inducing
dedifferentiation of muscle cells in vitro, various concentrations of
human recombinant (hr)TGF- 1 (0, 0.5, or 2.0 ng/mL, Sigma) were
applied to cells in serum-free DMEM for 2 hrs, and cells were then
continued to be cultured for different time periods. Differentially treated
cells were finally used for the cell cycling study (S-phase re-entry), and
for detecting the expression of Msx-1 protein and various stem cell
markers by immunocytometry or Flow Cytometry.
Immunofluorescent staining of cells or tissue sections: Cultured
cells were fixed with 4% paraformaldehyde and frozen tissue sections
were fixed with 4% formalin. After washing the cells or tissue sections
by phosphate buffer solution (PBS), 10% HS will be used to block
nonspecific background for 1 hour. The primary antibodies, TGF- 1
(Novocastra), CD34 (BD), Sca-1 (BD), myogenin (Santa Cruz), and
MSX-1 (Santa Cruz), were applied at 1 : 100~200. After fluorescent
secondary antibody staining, fluorescent microscopy (Leica
Microsystemic Inc., IL) was used to visualize all of the
immunofluorescence results and capture photographic imagines.
Propidium Iodide (PI) staining and Flow Cytometry analysis of the
cell cycling: For PI (Sigma) staining, cells were fixed by 75% ice cold
ethanol for at least 4 hrs, and then stained with PI solution (0.05 mg/mL
PI, 0.2 mg/mL RNase, and 0.1% NaCitrate, Sigma) for 30 minutes at
RT. Cell cycles of differentially treated muscle cells were then identified
by FACSCalibur flow-cytometer (Becton-Dickinson, San Jose, CA).
RESULTS:
1. More stem cells can be isolated from injured skeletal muscle
compared to noninjured muscle: The gastrocnemius muscles (GMs) of
one leg of 5 normal C57BL/6J mice (5 weeks age) were injured by a
cardiotoxin injection (2µg/leg), while the opposite leg served as a
control and was injected with PBS alone. Five days after injury, we
harvested the GMs for cell isolation by the preplate technique, and
obtained single-nucleated cells. Isolated cells were immunostained for
Sca-1 (stem cell marker) and myogenin (myogenic cell marker) to
distinguish their phenotypes. Results illustrated that the injured muscle
contains at least twice as many stem cells (Sca-1 positive/ Myogenin
negative) when compared to the non-injured muscle (Fig 1).
Fig. 1
Fig. 2
2. TGF- 1 deposition relates with Msx-1 expression in injured
muscle: Serial muscle sections (GM muscle, 4 days after muscle injury)
were stained for TGF- 1 and dedifferentiation marker Msx-1 by
immunohistochemistry, which indicated that these two proteins are
expressed in the same location. Moreover, Msx-1 expression was
observed in both myofibers and nuclei of muscle cells (Fig 2).
3. More stem cell-like cells were isolated from TGF- 1 injected
muscle compared to control muscle: Flow Cytometry results indicated
there were more stem cell-like cells (Sca1+/Cd34+) within TGF- 1
injected skeletal muscle of mice compared to control PBS injected
muscles (around 3:1).
4. More BrdU signal was detected in TGF- 1 treated C2C12:
C2C12 were treated with various concentrations of TGF- 1 for 2 hours,
and then cultured for another 12 hours with BrdU in serum free DMEM
medium to observe the cells with newly synthesized DNA (S-phase cell
cycle re-entry). TGF- 1 treatment resulted in greater BrdU incorporation
than the control, indicating more cells underwent S-phase re-entry with
TGF- 1 treatment.
5. Flow Cytometry analysis of the cell cycling during
dedifferentiation: C2C12 were treated with TGF- 1 for 2 hours and
then continuously cultured for another 4 hours in serum free DMEM. PI
staining and Flow Cytometry were also performed. Results demonstrated
that more cells re-entered S-phase (47.13% v.s. 40.95 %) and even G2/M
phase (7.17% v.s. 6.55%) in the TGF- 1 treated group, compared to the
control groups.
DISCUSSION:
The overall goal of this study is to determine that dedifferentiation
occurs in the injured skeletal muscle of mammals, where TGF- 1 is
possibly one of molecular triggers for this process. This process may
increase the total number of stem cell-like cells (iPS) available for
regenerative medicine. Here, our in vivo studies revealed the presence
of more stem cells in injured skeletal muscle of mice, which indicates a
possible correlation between the generation of more stem cells and the
dedifferentiation process, in which the function of TGF- 1 is suggested.
After conditional TGF- 1 stimulation, more myoblasts demonstrated
increased Msx-1 expression and S-phase re-entry of the cell cycle in
vitro. TGF- 1 treatment was also shown to be capable of reversing the
proliferation inhibiting effect of serum-free medium. We will further
prove the possible occurrence of dedifferentiation in vivo, and find out
how much the process of dedifferentiation and its potential mediator
TGF- 1 contribute to the stem cell proliferation in the muscle healing
process. We will also conduct transplantation of dedifferentiated muscle
cells into various mouse models to prove pluripotency of our iPS cells.
REFERENCES
1. Li Y et al. Am J Pathol. 164(3):1007-19 (2004). 2. Lévesque M et al.
PLoS ONE. 28:2(11) (2007). 3. Willis BC et al. Am J Physiol Lung Cell
Mol Physiol. 293(3):L525-34 (2007).
Paper No. 351 • 55th Annual Meeting of the Orthopaedic Research Society