Alpha 6 integrin is important for myogenic stem
Karlijn J. Wilschuta,⁎, Helena T.A. van Tola, Ger J.A. Arkesteijnb,
Henk P. Haagsmanb, Bernard A.J. Roelena
aDepartment of Farm Animal Health, Faculty of Veterinary Medicine, Yalelaan 104, 3584 CM, Utrecht University, Utrecht,
bDepartment of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht,
Received 7 December 2010; received in revised form 17 April 2011; accepted 2 May 2011
Available online 10 May 2011
muscle tissue. We show the presence of at least two types of stem cells in porcine muscle: those that express α6 integrin and
those that lack expression of this integrin type. By flow cytometry, we could select for myogenic stem cell populations
expressing the neural cell adhesion molecule in the presence and absence of α6 integrin. The expression of α6 integrin showed
an advantage in the formation of myotubes, possibly by an improved cell fusion capacity. This notion was strengthened by qRT-
PCR analysis showing sustained PAX7, MYF5 and DESMIN expression and a strong myogenic differentiation capacity of this stem
cell population. Selective inhibition of α6 integrin function, both by blocking antibodies and RNA interference, showed the
importance of α6 integrin in myogenic differentiation of muscle stem cells. It is concluded that α6 integrin expression can be
used as biomarker to select for highly myogenic cell populations in muscle tissue.
© 2011 Elsevier B.V. All rights reserved.
A muscle progenitor cell population, other than muscle satellite cells, can be isolated and purified from porcine
Skeletal muscle contains a population of stem cells to
support postnatal muscle growth and regeneration. These
cells, characterized as muscle satellite cells, adopt a
position between the basal lamina and plasma membrane
of muscle fibers and possess a self-renewing capacity to
sustain the resident stem cell pool (Montarras et al., 2005).
The characteristic PAX7 expression by satellite cells is
important to maintain postnatal self-renewal and myogenic
commitment until activation upon damage or during devel-
opment occurs (Seale et al., 2000; McKinnell et al., 2008;
Relaix et al., 2005; Oustanina et al., 2004). Here, cells start
to proliferate and differentiate into myoblasts, a process
coordinated by the muscle regulatory factors (MRFs) Myf5 and
MyoD (Buckingham et al., 2003; Rudnicki et al., 1993;
Tajbakhsh, 2005). The second differentiation step occurs
after myoblast alignment, initiating myoblast fusion resulting
in the formation of myotubes. The MRF Myogenin controls
⁎ Corresponding author at: Cardiovascular Research Institute,
Department of Pediatrics, University of California San Francisco, 513
USA. Fax: +1 415 514 0235.
E-mail addresses: email@example.com (K.J. Wilschut),
firstname.lastname@example.org (H.T.A. van Tol), email@example.com
(G.J.A. Arkesteijn), firstname.lastname@example.org (H.P. Haagsman),
email@example.com (B.A.J. Roelen).
1873-5061/$ - see front matter © 2011 Elsevier B.V. All rights reserved.
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Stem Cell Research (2011) 7, 112–123
2002; Alapat et al., 2009).
Integrins function as receptors important for adhesion to
extracellular matrix (ECM) proteins or to other cells. They
are composed of α- (18 types) and β- (8 types) subunits that
can form 24 distinct heterodimers of which the composition
dictates ligand specificity (Hemler, 1999; Hynes, 2002; van
der Flier and Sonnenberg, 2001). In particular the integrin
β1 has a role in regulating muscle integrity and is essential
for myoblast adhesion to the ECM (Menko and Boettiger,
1987). During muscle development various integrin subunits
are expressed including α1, α4, α5, α6, α7, αV, β1 and β3.
They play roles in mediating modulation of cell proliferation,
differentiation, migration, polarity, and motility triggering
calcium influx and are involved in apoptosis (Hynes, 2002;
Danen and Sonnenberg, 2003; Gullberg et al., 1998). The
the myotendinous and neuromuscular junction of the muscle
fiberand is required for musclemaintenance (Bao etal., 1993;
Hayashi et al., 1998). In a study with quail embryonic muscle,
α5 integrin in myoblasts was demonstrated to stimulate
proliferation, whereas α6 integrin mediated differentia-
tion (Sastry et al., 1996). This suggests distinct functions
for α-integrins in regulating myogenesis from proliferation
towards terminal differentiation.
Functions of α6 integrin (α6ITG) have been studied using
a GoH3 neutralizing antibody directed against α6ITG. It is
suggested that α6β1 is involved in adhesion to the E8-cell-
binding site of laminin in non-muscle cells, whereas in
muscle cells, not α6β1, but a different β1 integrin-series
binds laminin (von der Mark et al., 1991; Sonnenberg et al.,
1990). Alpha 6 integrin is expressed during early
mouse development at the stages of laminin containing
basement formation, which remains during embryogenesis
(Hierck et al., 1993). Here, delamination of Myf5 expres-
sing muscle progenitor cells formed laminin-rich myotome
mediated by α6β1 integrin expression (Bajanca et al.,
2004, 2006). Previous results showed that sustained α6ITG
expression of muscle stem cells cultured on Matrigel
surface coating was correlated with a high myogenic
differentiation in vitro, suggesting a role for α6ITG during
muscle stem cell differentiation (Wilschut et al., 2010).
Muscle tissue contains a heterogeneous population of
muscle progenitor cells (Zammit et al., 2006; Kuang et al.,
2007; Cerletti et al., 2008). Selection for specific
progenitor cells can be important to enrich for homoge-
neous cell populations to study function and differentia-
tion potentials. Indeed, in several studies integrins as
well as other membrane associated biomarkers were
used to select for specific stem cell populations (Cerletti
et al., 2008; Webster et al., 1988; Sherwood et al., 2004;
Tamaki et al., 2002; Zheng et al., 2007). In this respect,
expression of the neuronal cell adhesion molecule (NCAM)
by satellite cells has been used to mark a myogenic cell
population (Capkovic et al., 2008; Mesires and Doumit,
In this study, muscle stem cells isolated from the pig
were used. We used flow cytometry to sort for muscle stem
cells based on biomarker expression of NCAM and α6ITG.
These cells were examined for their myogenic differentia-
tion capacity revealing a potential function of α6ITG during
α6ITG expression levels
To specifically localize cells expressing α6ITG and to detect
muscle satellite cells by PAX7 expression, cryosections of
porcine semitendinosus muscle were stained with antibodies
against PAX7, α6ITG and Laminin. Two types of satellite cells
could be distinguished; (1) PAX7-expressing (satellite) cells
that lacked α6ITG expression (Fig. 1D; arrowhead) and (2)
PAX7-expressing cells (satellite) that co-expressed α6ITG
(Fig. 1D; arrow). Additionally, α6ITG expressing cells that
lacked PAX7 expression were observed (Fig. 1D; asterisks).
Thus, the identification of at least two different satellite cell
types indicates the heterogeneity of satellite cells in muscle.
Furthermore, α6ITG expressing cells were observed adjacent
to the extracellular matrix component laminin, indicating
the presence of these cells in laminin rich muscle areas
2.2. Selection for α6ITG expression results in highly
myogenic committed stem cell population
Isolated porcine primary muscle stem cells were expanded
on Matrigel-coated flasks. Myogenic stem cell populations
were selected by flow cytometry based on antibodies
directed against NCAM (expressed by satellite cells) and
α6ITG. The FITC- and PE-conjugated isotype control anti-
bodies were used to gate the background. Three cell
populations were sorted individually (Fig. 2); (1) NCAM+/
α6ITG+population (22%), (2) NCAM+/ α6ITG−cells (8%) and
(3) NCAM-/ α6ITG−cells (3%) (double negative for α6ITG and
NCAM), most likely representing non-myogenic cells.
All other experiments were conducted using laminin- and
Matrigel-coated plates functioning as substrates containing
α6ITG engaging ligands (Sonnenberg et al., 1990). The
myogenic differentiation capacity of the sorted NCAM+/
α6ITG+cell population was demonstrated by detection of
DESMIN and MYHC expression (Fig. 3A) revealed the high
myogenic character of this cell population and was also
indicated by formation of (striated) myotubes after 4 days of
of α6ITG confirmed cell sorting efficiency. Cells expressing
α6ITG formed large multinucleated myotubes while cells
lacking α6ITG expression only displayed alignment of the cells
without fusion (Fig. 3B). Myotube formation was never
observed in NCAM+cells lacking α6ITG expression (data not
levels were determined relative to those of unsorted primary
muscle cells. The NCAM+/ α6ITG+population showed a
significantly (pb0.001) higher expression of A6ITG mRNA
compared to the expression of the NCAM+/ α6ITG−sorted
tions (Fig. 3C). Interestingly, the NCAM+/ α6ITG−population
expressed significantly higherlevels of A7ITG mRNA, whereas,
the NCAM−/ α6ITG−population, expressed significantly
(pb0.001) higher levels of A5ITG mRNA. The B1ITG mRNA
levels were similar between the two NCAM+populations.
113 Alpha 6 integrin is important for myogenic stem cell differentiation
Examination of muscle related gene expression revealed
that both NCAM+populations comprised a high PAX7
expression with a significantly (pb0.05) higher MYF5
expression in α6ITG+cells (Fig. 3C). MYF5 is important for
initiation of muscle differentiation, suggesting a stronger
myogenic potential of the α6ITG+cells. However, DESMIN
expression was significantly (pb0.05) higher in α6ITG−cells,
indicating a strong myogenic feature despite the lower MYF5
expression. The NCAM-/ α6ITG−cell population showed lower
expression levels of MYF5 and DESMIN, which are important
during myogenesis, demonstrating the non-myogenic feature
of these cells. Morphology of both NCAM+populations showed
populations corresponding to specific antibody staining as indicated. NCAM+cells were visualized by the PE-channel and the α6ITG+
cells were visualized in FITC-channel. The NCAM+/ α6ITG+population represented 22%, NCAM+/ α6ITG−cells 8% and NCAM-/ α6ITG−3%
of the total population. B. Negative isotype control represents non-specific background staining.
Flow cytometric analysis of primary muscle stem cells for NCAM and α6ITG expression. A. Gates represent sorted cell
Cell type # Percentage
PAX7+ cells 41 12%
α6ITG+/ PAX7+ 24
Nuclei 344 100%
A. Localization of α6ITG expression with anti-α6ITG antibodies (GoH3, red). B. Satellite cells are identified by PAX7 expression using
anti-PAX7 antibodies (PAX7, green). C, G. Visualization of nuclei by DAPI (blue). D. Merged channels showing three different cell
populations; α6ITG expressing satellite cells (arrows), satellite cells lacking α6ITG expression (arrowheads) and unidentified muscle
cells with α6ITG expression (asterisks). Scale bars represent 20 μm. E. Localization of α6ITG expression with anti-α6ITG antibodies
(GoH3, green). F. Visualization of the extracellular matrix component laminin (red). H. Merged channels identifying the sub-laminar
position of α6ITG positive cells. I. Determination of frequency of different cell types. Scale bars represent 50 μm.
Immunofluorescence of PAX7 and α6ITG expression in transverse cryosections of semitendinosus muscle tissue.
114K.J. Wilschut et al.
bi- and triangular shaped cells with several pseudopodia.
compared to the α6ITG-cells (Fig. 4A).
The difference between the two populations (NCAM+/
α6ITG+/−) concerning their growth rate was monitored by
the determination of cell numbers. Cells lacking α6ITG
expression had a 3-fold higher expansion rate compared to
α6ITG+cells as indicated at day 7 of proliferation (Fig. 4B).
In differentiation medium, NCAM+/ α6ITG−cells sustained a
high proliferative activity suggesting no initiation of
2.3. Alpha 6 integrin is required for robust myogenic
To examine and compare the myogenic differentiation
capacity of the NCAM+/ α6ITG+/−cell populations the
expression of genes involved in myogenesis was measured by
qRT-PCR during differentiation. Here, both NCAM+/ α6ITG+
and NCAM+/ α6ITG−populations showed approximately equal
PAX7 mRNA levels directly after sorting. Initiation of differen-
tiation resulted in a drop of PAX7 expression in both
populations, whereas PAX7 expression recovered in α6ITG+
cells significantly towards α6ITG−cells during differentiation
(Fig. 5A). Levels of MYF5 mRNA, coding for the transcriptional
initiator of muscle stem cell differentiation, remained
significantly higher (pb0.01) in α6ITG+cells compared to
α6ITG−cells after sorting. This indicates the low myogenic
orientation of these cells when lacking α6ITG expression
(Fig. 5B). In the α6ITG−cells a decrease in DESMIN expression
was observed (Fig. 5C). The expression of intermediated
muscle filament protein DESMIN sustained in α6ITG+cells
(pb0.001), while DESMIN in α6ITG−cells reduced considerably
after several days of differentiation. Furthermore, the
morphological myotube formation by α6ITG+cells was corre-
lated to the remarkable increase of MYHC expression (compo-
resulted in a significant difference in MYHC expression
between the populations at day 4 of differentiation as
supported morphologically (data not shown). Furthermore,
the α6ITG expression determined over time during 4 days of
differentiation showed that both cell populations undergo an
increase in α6ITG expression at the time point of differenti-
ation (Fig. 5E). During differentiation, the cells depleted for
α6ITG show a decrease in expression, whereas a sustained
increased gene expression is observed in the α6ITG+cells.
2.4. Blocking of α6ITG inhibits myogenic differentia-
tion of primary muscle stem cells
Primary muscle stem cells were cultured on laminin-coated
cover slips. The α6ITG function was blocked with anti-α6ITG
antibody (GoH3) incubation for 7 days (Sonnenberg et al.,
1988). The antibody binding to the cells was visualized by
immunofluorescence (Fig. 6A). Blocking of α6ITG function
did not interfere with the adhesion of myogenic cells to the
cover slips and myogenic cell division. This was indicated by
the expression of DESMIN both in the presence or absence of
the blocking antibodies (Fig. 6B). The negative staining of
isotype control antibodies indicated antibody specificity
(Fig. 6C). Blocking of the α6ITG function by antibodies
resulted in the inhibition of primary muscle stem cell fusion
into myotubes after exposure to differentiation conditions
for 6 days. Cells lacking GoH3 antibody treatment were able
to form myotubes suggesting an important function of α6ITG
during myogenic differentiation (Fig. 6D).
2.5. RNAi mediated knockdown of α6ITG results in
inhibition of differentiation
The role of α6ITG in muscle stem cell differentiation was
further examined by transient downregulation using siRNA in
proliferating primary muscle stem cells. Transfection with
siRNA targeted against α6ITG expression resulted in sig-
nificant decreased expression of 90% after 24 h (Fig. 7A;
transfection. Control siRNA (mock) did not result in a
significant downregulation of α6ITG expression. Protein
detection using immunoblotting indicated the downregula-
tion of α6ITG till 72 h compared to mock control (Fig. 7B).
MYOGENIN expression was not detected during the first 96 h
in both groups indicating the proliferative stage of the cells.
After96 hMYOGENIN expressionwasupregulatedinthemock
control group, but remained low in cells with downregulated
α6ITG expression suggesting inhibition of terminal myogenic
differentiation in these cells. Indeed, cells with down-
even after prolonged time, whereas large myotubes were
observed in the mock control group (Fig. 7D). In addition, the
expression of MYHC as detected with qRT-PCR was signifi-
cantly decreased after α6ITG downregulation (Fig. 7C).
Expression levels of MYF5, MYOD and DESMIN were not
significantly different between the cells with downregulated
α6ITG and mock control cells (data not shown).
In muscle tissue, satellite cells are heterogeneous as
demonstrated by a distinction in α6ITG expression (Fig. 1).
Laminin is expressed along the muscle fiber within the
extracellular matrix enclosing the muscle fiber, where the
laminin ligands cause α6/β1 integrin heterodimers receptor
binding (Sonnenberg et al., 1990; Hall et al., 1990; Aumailley
et al., 1990; Le Bellego et al., 2002). The localization of
α6ITG integrin expressing satellite cells with laminin-rich
extracellular matrix enclosing the muscle fiber emphasizes
this important ligand binding interaction (Fig. 1H). Previ-
ously, we observed that increased levels of α6ITG expression
in primary muscle stem cells were correlated with a better
myogenic differentiation capacity (Wilschut et al., 2010).
Therefore, we further investigated the role of α6ITG during
myogenesis by selecting and sorting for NCAM expressing
cells with or without α6ITG expression from isolated primary
muscle stem cell culture. NCAM is involved in myoblast fusion
and is expressed by satellite cells that are associated with
myogenic cell commitment (Capkovic et al., 2008; Charlton
et al., 2000). Thereby we could distinguish between
myogenic and non-myogenic cells (Webster et al., 1988;
Capkovic et al., 2008; Mesires and Doumit, 2002; Blanton
et al., 1999; Krauss et al., 2005; Covault and Sanes, 1986).
Three cell populations were obtained; muscle stem cells
positive for α6ITG expression, muscle stem cells negative for
115Alpha 6 integrin is important for myogenic stem cell differentiation
α6ITG expression and the non-myogenic cells expressing
neither NCAM nor α6ITG (Fig. 2). Here, the α6ITG positive
myogenic stem cells showed a robust myogenic differentia-
tion capacity as indicated by the expression of MYOGENIN,
DESMIN and MYHC. Interestingly, it was shown that expres-
sion of α6ITG expression is required for myogenic differen-
tiation into myotubes. The α6ITG−cells showed cell
elongation and alignment, but no myoblast fusion (Fig. 3).
The myogenic commitment of the cell populations was
further examined by determination of mRNA expression
levels. PAX7 expression was similar between the two
myogenic NCAM+cell populations after isolation indicating
that they both related to satellite cells. Interestingly, both
α6ITG+and α6ITG−cells expressed the MRFs MYF5 and
DESMIN, indicating the myogenic commitment of both cell
types. It has been shown that in murine muscle tissue,
myogenic progenitor cells are subdivided in Pax7 expressing
satellite cells lacking Myf5 and committed satellite cells co-
116K.J. Wilschut et al.
expressing Myf5 (Kuang et al., 2007). The hierarchy is based
on the apical–basal position of the satellite cells towards the
muscle fiber. Satellite cells identified as Pax7+/ Myf5−with
an orientation towards the basal lamina (basal-position)
were demonstrated to express α7β1 integrins. Satellite cells
with an apical orientated position towards the plasma
membrane of the myofiber expressed Myf5 (Kuang et al.,
2007). These Myf5+cells were indicated as a committed
phenotype due to the loss of contact with the basal lamina
and extracellular environment or niche, which is important
in sustaining stem cell identity. Interestingly, in our
experiments the α6ITG−cells expressed significantly higher
levels of A7ITG and lower levels of MYF5 compared to α6ITG+
cells. This suggests that α6ITG-cells could have a basal
orientated position and not yet directed into the myogenic
program. This indicates that these cells are uncommitted
early muscle precursor cells whereas the α6ITG+cells could
be the myogenic initiated myogenic progenitor cells. The
non-myogenic cells lacking NCAM expression showed signif-
icant higher levels of A5ITG, which is the receptor for
fibronectin in fibroblasts (Huveneers et al., 2008).
Examination of the proliferation capacity of the two
populations revealed that α6ITG−cells proliferated at a
higher rate than α6ITG+cells. The α6ITG+cells stopped
proliferation and formed myotubes by cell fusion, while
α6ITG−cells continued cell proliferation until confluence
was reached (Fig. 4). The high proliferation capacity of
α6ITG−cells is consistent with the involvement of α6 integrin
in the negative regulation of cell growth (Sastry et al., 1996).
The differentiation capacity revealed a diminished myogenic
commitment by cells lacking α6ITG expression compared to
the α6ITG positive cells. Whether the α6ITG negative cells
will become less proliferative and more myogenic when
α6ITG is introduced into these cells for instance by over-
expression, or whether the α6ITG negative cells have already
entered different differentiation pathways is currently
Monoclonal antibodies blocked α6ITG expression on
primary muscle stem cells and prohibited myoblast fusion
without affecting proliferation or the expression of muscle-
specific genes. This indicates a direct inhibition of cell fusion
and differentiation. Downregulation of α6ITG mRNA expres-
sion using siRNA oligonucleotides confirmed the role α6ITG in
Besides muscle satellite cells, several muscle progenitor
cells have been identified so far among which are blood-
vessel associated myo-endothelial cells (Zheng et al., 2007;
Crisan et al., 2008), meso-angioblasts (Minasi et al., 2002)
and muscle-derived stem cells (MDSCs) (Qu-Petersen et al.,
2002). Interestingly, the MDSCs with the highest in vivo
regeneration capacity are the most proliferative in vitro,
similar to the α6ITG−population (Qu-Petersen et al., 2002).
Early preplate (EP) cells, characterized as late myogenic
precursors in vitro (Qu-Petersen et al., 2002), are compara-
ble with the myogenic α6ITG+population. Differences in
α6ITG expression could attribute to the in vitro and in vivo
potentials and α6ITG can therefore be a useful target to
Days of cell culture (prol + diff)
Growth curve (cell counts)
alpha6 + cells
alpha6 - cells
with amore flattenedappearance for α6ITGexpressing cells.Scale
bar is 50 μm. B. Growth curves represent cell amount during
proliferating and differentiating. Cells that express α6ITG exhibit a
lower proliferation rate (●) compared to cells without α6ITG
expression (■. (*pb0.0001). Experimental outcome represents
three biological replicates.
Cell culture. A. Both NCAM+/ α6ITG+and NCAM+/
NCAM+/ α6ITG–and NCAM–/ α6ITG–cells relative to unsorted primary muscle cells. A. Myogenic differentiation capacity of α6ITG+
cells after 4 days of induction. MYOGENIN (left, in green) detection on terminal differentiated muscle stem cells. DESMIN (middle, in
green) detection represents formation of large multinucleated myotubes (arrow). Formation of MYHC (right, in green) expressing
striated myotubes (arrow head). DNA is visible in blue, scale bar represents 20 μm. B. Immunofluorescent cell staining with anti-α6ITG
antibodies (GoH3) on cytospins of the sorted cell populations (NCAM+/ α6ITG+, left upper panels; NCAM+/ α6ITG–, left lower panels)
visualizes the expression of α6ITG in the α6ITG+cells (red). After 2 days of inducing differentiation α6ITG+cells formed myotubes
(arrow; upper right panel), whereas cells lacking α6ITG remained aligned showing no cell fusion (lower right panel). Nuclei are
visualized with DAPI staining (blue). Scale bar is 50 μm. C. The A6ITG was expressed at significantly higher levels in α6ITG+cells, A5ITG
was expressed in NCAM−/ α6ITG−(non-myogenic cells) at significantly higher levels, while A7ITG was significantly higher expressed in
α6ITG−cells. Similar expression levels of B1ITG were detected between both α6ITG+and α6ITG–cell but the non-myogenic cells
expressed significantly higher levels of B1ITG. Expression levels of PAX7 were detected in α6ITG+and α6ITG–cells which were
significantly higher compared to non-myogenic cells. A significantly higher MYF5 expression level was detected in α6ITG+cells, while
expression of DESMIN was significantly higher in α6ITG−cells. Experiment is performed in three biological replicates, in which the qRT-
PCR analysis is run as technical triplicates. Asterisks denote significant differences in gene expression between the sorted population
(NCAM+/ α6ITG+, NCAM+/ α6ITG−and NCAM-/ α6ITG-) (ns=no significance, * pb0.05, * pb0.01, *** pb0.001).
Characterization of sorted cell populations. Gene expression levels were determined by qRT-PCR on NCAM+/ α6ITG+,
117Alpha 6 integrin is important for myogenic stem cell differentiation
improve muscle regeneration. Studying similarities and
differences between the α6ITG positive and α6ITG negative
cell populations such as characterizing differentiation
potential and in vivo exposure would attribute to a
presumable role for α6ITGs on in vivo engraftment, survival
and self-renewal. Discrimination between behavioral in vivo
cell fate would indicate a preference to re-enter a quiescent
satellite cell position, which could be determined by cell
location towards the muscle fiber and Sprouty expression
(Shea et al., 2010), or a more differentiated characteristic
by fusion with myotubes showing a more myogenic regener-
Here, two types of stem cells have been identified in
porcine muscle: those that express α6ITG and those that lack
advantage in the formation of myotubes, possibly by an
improved cell fusion capacity. Whether the cells that lack
α6ITG will eventually start to express this integrin or whether
Relative to sorted α α6ITG+ cells E
Relative to sorted α α6+ cells
Relative to sorted α α6+ cells
Diff d0 Diff d2Diff d2
relative to α6ITG+cells at day 0 (prol d0). A. PAX7 expression. B. MYF5 expression. C. DESMIN expression. D. MYHC fast type
expression. E. Alpha6ITG expression. Experiment is performed in three biological replicates, in which the qRT-PCR analysis is run as
technical triplicates. Asterisks denote significant differences in gene expression at the specific time point between the sorted
population NCAM+/ α6ITG+and NCAM+/ α6ITG−(** pb0.01, *** pb0.001).
Differentiation capacity of NCAM+/ α6ITG+and NCAM+/ α6ITG-cells. Expression levels during differentiation (diff) are
118K.J. Wilschut et al.
antibodies directed against α6ITG. Lower row depicts primary muscle cells without GoH3 antibody treatment. A. Detection of GoH3
antibodies on primary muscle stem cells using Alexa568-labeled secondary antibody against rat-IgGs. B. Visualization of DESMIN
expression indicates adhesion of dividing myoblasts (arrows) on the cover slips. C. Isotype antibody incubations served as negative
control for aspecific antibody binding. Nuclei were stained with DAPI (blue), scale bars represent 20 μm. D. No myotubes were
observed in GoH3 treated primary muscle stem cells after 6 days of differentiation, while cells lacking GoH3 antibody treatment
formed myotubes in culture. Scale bars represent 50 μm. Experimental outcome represents three biological replicates.
Inhibition of α6ITG function using the rat-GoH3 antibodies. Upper row depicts primary muscle stem cells treated with GoH3
RNA expression levels of α6ITG relative to expression levels directly after transfection (0 h) determined by qRT-PCR. Efficient
downregulation of α6ITG has been obtained by using ITGA6 siRNA. B. Protein levels determined by western blotting confirm silencing
of α6ITG during 96 h post transfection. Myogenic determination was observed in the mock group by MYOGENIN (MYOG) expression.
C. Quantitative RT-PCR confirmed myogenic differentiation of cells transfected with control siRNA by increased levels of MYHC
expression in time. D. Morphological differentiation (myotube formation, arrows) was observed in the mock control group (M), which
was very poor in cells with downregulated α6ITG expression (I). BACT=beta actin. MYHC=myosin heavy chain 2. Scale bars represent
50 μm. (* pb0.01). Experiment is performed in three biological replicates.
Inhibitory effects of α6ITG downregulation on myogenic differentiation capacity of primary muscle stem cells. A. Messenger
119 Alpha 6 integrin is important for myogenic stem cell differentiation
these cells have a different role in skeletal muscle is not
known. The functions of integrins can be different in the
various stem cell types predisposed by the extracellular
environment and developmental progression. It has, for
instance, been established that α6/β1 integrins are markers
of neural stem cells, and that expression of these integrins
are a different type of cells that require fusion of progenitor
cells during maturation. We propose that α6ITG is particularly
important to establish fusion of the muscle progenitor cells
important for differentiation of skeletal muscle.
In this study, it is proposed that α6ITG could serve as a
biomarker to select for highly myogenic stem cells that may be
used in experimental stem cell therapy. It should however be
investigated whether cells expressing α6ITG lead to better
engraftment than α6ITG negative cells, since successful
engraftment encompasses more than in vitro differentiation
4. Materials and methods
4.1. Isolation of porcine primary muscle stem cells
Porcine primary muscle stem cells were isolated from
semitendinosus muscle of euthanized (intracardial injection
of 0.15 ml/kg T61) piglets (hybrid York boars; 3 months of
age). The dissected muscle parts were minced with scalpels
after removal of adipose and connective tissues. Minced
muscle tissue (50 g in total) was centrifuged (5 min, 2000 g) in
phosphate-buffered saline (PBS, Braun, Melsungen, Germany)
containing 1% HEPES and a cocktail of anti-bacterial agents
(50 μg/ml gentamycin, 1% antibiotic-antimycotic mix and
250 ng/ml fungizone (all from Sigma ST Louis, MO), referred
to as PBS+-H. Muscle tissue parts were digested in 100 ml
preheated 1 mg/ml protease solution (from Streptomyces
griseus,Sigma,STLouis,MO)inPBS+-Hfor60 minutesat37 °C
under repeated shaking as described previously (Doumit and
Merkel, 1992; Gharaibeh et al., 2008). To further homogenize
the tissue, trituration with 5 ml pipettes was performed.
Undigested larger muscle parts were collected by modest
centrifugation(5 minat200 g)andstoredonice.Supernatant,
containingsingle cells, was washed twice (10 min at 2000 g) in
serum (FBS, Invitrogen) and 50 μg/ml gentamycin]. The cells
were filtered with cell strainers (pore size 70 μm, BD Falcon,
Erembodegem,Belgium)and subsequently pelleted(10 minat
cells from the remaining undigested muscle parts was
performed by a 1 hour incubation with 0.15% w/v collagenase
XI (Sigma) in DMEM-HG with 1% HEPES and 5% FBS. During
pipettes. Finally, the fully digested tissue was filtered (70 μm
cell strainer), washed in GM and pelleted (10 min at 2000 g).
To shock the erythrocytes both cell pellets were pooled in
20 ml cold hypotonic buffer (0.2 M NH4Cl, 13 mM KHCO3, pH
7.4) for 10 min on ice and centrifuged in cold PBS+-H (5 min at
1000 g). Cells were taken up in GM, filtered through a 40 μm
cell strainer (BD Falcon) to remove small tissue debris and
subsequently collected by pelleting (5 min at 700 g). Cells
were pre-plated in GM in addition of antibiotic–antimycotic
mix and fungizone in uncoated T175 flasks (Corning Life
Sciences, Amsterdam, The Netherlands) for the exclusion of
fast-adhering fibroblasts during 1 h (37 °C, 5% CO2). Cells that
had not adhered were collected and stored in liquid nitrogen
until further use (Qu etal., 1998).All steps wereperformed at
room temperature (RT), unless otherwise mentioned.
4.2. Muscle stem cell proliferation and
For cell expansion primary muscle stem cells were cultured
in 1 mg/ml Matrigel (Matrigel™ Basement Membrane Matrix;
mouse tumor; phenol-red free, BD Bioscience, Bedford, MA)
coated flasks (Sigma) in GM containing 5 ng/ml human basic
fibroblast growth factor (bFGF; Sigma). Differentiation of
the muscle stem cells was performed in differentiation
medium [DM; DMEM-HG, 2% horse serum (HS, Invitrogen) with
50 μg/ml gentamycin] on laminin-coated (1 μg/ml) Lumox
dishes (Ø 35 mm; Greiner Bio-One, Frickenhausen, Germany)
or on laminin-coated 6-wells plates (Greiner Bio-One).
4.3. FACS sorting
Indirect cell staining with primary mouse antibodies against
NCAM (5.1H11; 1:200 dilution; Developmental Studies
Hybridoma Bank, Iowa City, IA) and rat antibodies against
α6ITG (GoH3; 2.5 μg/ml; BD) was used to select for specific
cell populations by flow cytometry using a VantageTM SE
flowcytometer (BD). Fluorescent conjugated secondary
goat-anti mouse-PE and goat-anti rat-FITC antibodies were
used to localize the primary antibodies. Isotype control
antibodies (rat-IgG2 and mouse-IgG1; both 1:10 dilution;
DakoCytomation, Glostrup, Denmark) were used to gate the
fluorescence threshold for antibody specificity based on the
visualization of nonspecific binding by the primary anti-
bodies. Data were analyzed using FlowJo software (Oregon
corporations, Ashland, OR).
4.4. Indirect immunofluorescence
After sorting by flow cytometry, cell populations were spun
onto glass cover slides for 5 min at 800 rpm in 0.5% BSA in PBS
using the Shandon Cytospin 4 (Thermo Scientific, Breda, The
cells were cultured for 6 days in GM on 1 μg/cm2laminin-
coated (murine sarcoma; Sigma) glass cover slips and on
laminin-coatedLumox dishesfor 4 days in DM. Cells werefixed
in cold methanol (2 min at −20 °C) for α6ITG staining. To stain
DESMIN, MYOGENIN and Myosin heavy chain (MYHC) cells were
fixed in 4% paraformaldehyde (PFA; Electron Microscopy
in 0.5% Triton X-100 for 10 min. Snap-frozen muscle tissue was
cryosectioned (5 μm), fixed in 4% PFA (15 min) and incubated
for 30 min in blocking buffer [2% normal goat serum (DakoCy-
tomation), 1% Bovine Serum Albumin (BSA; Roth, Karlsruhe,
Germany), 0.1% fish gelatin (Sigma), 0.1% Triton X-100, 0.05%
Tween-20 in PBS] to avoid aspecific antibody binding.
Subsequently, cells were incubated with primary antibodies
α6ITG (rat, clone GoH3; 10 μg/ml; BD), DESMIN (mouse, clone
120K.J. Wilschut et al.
D33; 10 μg/ml; DakoCytomation), MYOGENIN (mouse, clone
PAX7 (mouse, clone PAX7; 10 μg/ml; R&D systems, MN) and
Laminin (rabbit; 1:25; Sigma) in blocking buffer for 1 h. After
washing in PBS with 0.05% Tween-20 (PBST; 3 times; 5 min)
slides were incubated with Alexa488-labeled or Alexa568-
labeled goat anti-mouse/ anti-rat/ anti-rabbit IgG (1:200;
counterstaining with DAPI (Invitrogen) to visualize the nuclei.
Slides were dehydrated in an increasing alcohol series (70%,
FluorSave (Calbiochem, Darmstadt, Germany) and covered
4 °C at full speed prior to use. All steps were performed at RT.
performed on a Leica DMRE fluorescence microscope.
4.5. Inhibition of α6ITG expression
On laminin expanded primary muscle stem cells were seeded
at a density of 3000 cells/cm2into a laminin coated 12-wells
plate. Blocking of α6ITG expression on primary muscle stem
cells was performed in presence of the blocking antibody
GoH3 (GoH3; 2.5 μg/ml; BD). After 8 days of cell culturing in
GM differentiation was induced during 6 days in DM.
Proliferation and differentiation medium was refreshed
every third day with addition of GoH3 antibody.
Expression of α6ITG in primary muscle stem cells was
downregulated by RNAi mediated knockdown. Cells were
transfected with α6ITG siRNA duplex oligoribonuleotides
(5′-GAAACGUGCUGUUCCAUAA-3′) using electroporation (1
time at 160 V, 70 ms pulse length; ECM 830 Electroporation
System, Harvard Apparatus, Kent, UK). Universal control
siRNA duplex oligoribonuleotides were used as a nonspecific
control (mock) (all purchased from Eurogentec, Maastricht,
The Netherlands). Cells were transfected with 500 pmol
siRNA per 1×105cells and seeded onto Matrigel-coated
plates in a density of 5000 cells/cm2in GM. After 48 h
myogenic differentiation was induced by medium replace-
ment for DM. Cells were collected for RNA and protein
isolation at 0 h to 120 h of culturing.
4.6. Quantification of gene expression
RNA isolation using a RNeasy Mini Kit (Qiagen, Valencia, CA)
was either performed from directly sorted cell populations or
from cell cultures which were first trypsinized (0.25%
Trypsin-EDTA, Invitrogen), pelleted by centrifugation
(5 min at 300 g) and stored at −80 °C before RNA isolation.
Additionally, a second DNase treatment was performed on
RNA samples by incubation with 2 μl DNase (Qiagen) for
25 min at 37 °C, followed by inactivation for 10 min at 70 °C.
The cDNA was generated using Superscript™ III First-strand
Synthesis System (Invitrogen) and quantitative real-time RT-
PCR (qRT-PCR) performed on cDNA (1 μl) using an iCycler
with iQ™ SYBR® Green supermix (both from BIO-RAD,
Hercules, CA). A 12.5 pmol primer concentration per 25 μl
reaction amplified cDNA after a denaturation step of 3 min at
95 °C in a 40 cycle protocol [30 s at 95 °C, 20 s 51–63 °C
(Table 1 Wilschut et al., 2010; Wilschut et al., 2008; Kuijk
et al., 2007), 30 s at 72 °C and subsequently 77 repeats of
15 s with a 0.5 °C increase in temperature after every repeat
starting at60 °C].Primer specificity was confirmed by product
sequencing. For relative gene expression quantification sam-
ples were normalized against mRNA levels of reference genes
Table 1Overview of primers used for quantitative RT-PCR.
Gene Primer sequence 5′→3′ Amplicon sizeTa (°C)GenBank accession no.
ITGA5 F: GCCTGCAAAGATCTGTCCTC
215 bp58XM_001925252 (Wilschut et al., 2010)
ITGA6365 bp54NM_001109981 (Wilschut et al., 2010)
ITGA7132 bp 60XM_001916598 (Wilschut et al., 2010)
ITGB1304 bp 53NM_213968
PAX7155 bp55 AY653213 (Wilschut et al., 2008)
MYF5 160 bp55Y17154 (Wilschut et al., 2010)
DESMIN162 bp 55AF363284 (Wilschut et al., 2010)
MYHC-2179 bp60NM_214136 (Wilschut et al., 2008)
PGK1 126 bp56.4AY677198 (Kuijk et al., 2007)
UBQ186 bp51M18159 (Kuijk et al., 2007)
GAPDH219 bp51AF017079 (Kuijk et al., 2007)
121Alpha 6 integrin is important for myogenic stem cell differentiation
Ltd., Southampton, UK) (Vandesompele et al., 2002). Gene
expression levels of siRNA-transfected cells and mock-trans-
fected cells were normalized against PGK1 expression levels
and plottedrelatively to expression levels determined directly
after transfection (0 h) (Fig. 7). Results were analyzed using
GraphPad prism in a two-way ANOVA with a Bonferroni post-
140 mM NaCl, 50 mM Tris–HCL pH7.5, 1 mM EDTA, 0.1% Triton
X-100, 10% glycerol and protease inhibitor cocktail 1
tablet/50 ml lysis buffer (Roche Applied Sciences, Almere,
DC protein assay (Bio-Rad, Veenendaal, The Netherlands).
Equal amounts (3.5 μg) of reduced (5 min at 95 °C) protein
lysate was separated onto 12% Tris–HCl PAGE gels and blotted
to Trans-Blot nitrocellulose transfer membranes (Bio-Rad).
Membranes were blocked in 5% Blotting Grade Blocker non-fat
primary antibodies against ITGA6 (rabbit polyclonal, 1:1000;
Cell Signaling Technology, Danvers, MA), MYOGENIN (F5D,
mouse,2 μg/ml,BD) andBACT(rabbitpolyclonal;1:1000;Cell
Signaling Technology). Secondary incubation for 1 h with
horseradish peroxidase-conjugated anti-mouse/rabbit IgG
(1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) was
followed by SuperSignal West Dura Extended Duration Sub-
strate (Pierce, ThermoScientific, Erembodegem, Belgium)
after which the blots were exposed to autoradiography film
(Kodak, Rochester, NY).
Author disclosure statement
No potential conflicts of interest.
This work was supported by a SenterNovem grant from the
Ministery of Economic Affairs. The NCAM antibody (5.1H11)
developed by H.M. Blau and F.S. Walsh was obtained from
the Developmental Studies Hybridoma Bank developed under
the auspices of the NICHD and maintained by The University
of Iowa, Department of Biological Sciences, Iowa City, IA
52242. KW was responsible for the design, experimental
procedures and was the primary author for the manuscript.
HT largely contributed to the RNAi silencing procedures and
GA supported the flow cytometric procedures. HH supervised
the design and analysis. BR supervised the design, analysis
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123Alpha 6 integrin is important for myogenic stem cell differentiation