Cai et al. BMC Molecular and Cell Biology(2023) 24:18
https://doi.org/10.1186/s12860-023-00478-1

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Myogenic differentiation of human myoblasts
and Mesenchymal stromal cells under
GDF11 on Poly-ɛ-caprolactone-collagen
I-Polyethylene-nanofibers
Aijia Cai1*, Paul Schneider1, Zeng-Ming Zheng1, Justus P. Beier2, Marcus Himmler3, Dirk W. Schubert3,
Volker Weisbach4, Raymund E. Horch1 and Andreas Arkudas1
Abstract
Background For the purpose of skeletal muscle engineering, primary myoblasts (Mb) and adipogenic mesenchymal
stem cells (ADSC) can be co-cultured and myogenically differentiated. Electrospun composite nanofiber scaffolds
represent suitable matrices for tissue engineering of skeletal muscle, combining both biocompatibility and stability
Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoringskeletal muscle function in aging mice, some studies have also described a harming effect of GDF11. Therefore, theaim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolactone (PCL)collagen I-polyethylene oxide (PEO)-nanofibers.
Results Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D)on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, orserum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic
differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity.
Immunofluorescence staining showed myosine heavy chain expression in all groups after 28 days of differentiation
without any clear evidence of more or less pronounced expression in either group. Gene expression of myosine heavy
chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone.
Conclusions This is the first study analyzing the effect of GDF11 on myogenic differentiation of Mb and ADSC
co-cultures under serum-free conditions. The results of this study show that PCL-collagen I-PEO-nanofibers represent
a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote
myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidenceof a harming effect.
Keywords Myogenic differentiation, Nanoscaffolds, Electrospun composite nanofibers, poly-ε-caprolacton (PCL),
Collagen, Polyethylene oxide (PEO), ADSC, Myoblasts, GDF11, Myostatin*Correspondence:
Aijia Cai
Aijia.cai@uk-erlangen.de
Full list of author information is available at the end of the article© The Author(s) 2023, corrected publication 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Background
Muscle regeneration is orchestrated by muscle satellite cells, adult stem cells, located in a niche between thebasal lamina and the sarcolemma. Those satellite cells
from a pool of myogenically committed cells, the myoblasts, which fuse and differentiate into multinucleatedmyotubes and eventually into myofibers [1, 2]. In theevent of volumetric muscle loss, the natural regeneration capacity of skeletal muscle tissue is exceeded [3]. To
overcome the issue of donor site morbidity when using
autologous muscle tissue to reconstruct the resulting
defect, tissue engineering approaches for creating functional skeletal muscle tissue have been investigated thoroughly [3–6]. Since skeletal muscle represents a complex
tissue with hierarchically organized fibers, a matrix,
mimicking those properties is needed [7]. A prior study
demonstrated that poly-ε-caprolacton (PCL)-collagen I
nanofibers, electrospun with acetic acid combined withultrasonic treatment as a benign solvent system, served
as a suitable platform for those tissue engineering purposes due to their parallel alignment, stability, and biocompatibility [4, 8, 9]. However, high density packed
fibers might result in poor cell infiltration [10]. One
method to improve cell permeability into the scaffolds is
to integrate polyethylene oxide (PEO) as sacrificial fibers
to increase porosity of nanoscaffolds [11, 12].
In prior studies, Mb were co-cultured with mesenchymal stromal cells (MSC) for the purpose of tissue engineering means. Unlike Mb, MSC can be easily expanded
without losing their differentiation ability [4, 9, 13].
In this context, MSC from bone marrow (BMSCs) orfrom adipose tissue (ADSC) were co-cultured with primary Mb three-dimensionally (3D) on PCL-collagen
I-nanoscaffolds and myogenically differentiated. ADSC
seemed superior in terms of proliferation and cell viability compared to BMSC [4]. To eliminate potential inconsistencies and safety concerns, a serum-free medium for
myogenic differentiation was established. All in all, theestablishment of this biocompatible system has beena crucial step in terms of future translational applications. However, gene expression of late myogenic markers like myosin heavy chain (MHC) was still higher after
long term stimulation with donor horse serum (DHS)
compared to serum-free differentiation [4]. Thus, theoptimization of a serum-free medium for myogenic differentiation is crucial in terms of clinical translation ofmodels of skeletal muscle tissue engineering.
Growth differentiation factor 11 (GDF11), a memberof the transforming growth factor (TGF)-beta superfamily [14], has shown beneficial effects on skeletal muscle
regeneration, restoring satellite cell regenerative function
in aged muscle cells and mice [15] and was identified asa rejuvenation factor in heterochronic parabiosis experiments [16, 17]. It was suggested that GDF11 declines with

Page 2 of 12age, and that restoration of systemic GDF11 to „youthful“ levels is beneficial for several age-related conditions[18]. Sinha et al. were able to show that supplementationof GDF11 reversed functional impairments and restored
genomic integrity in aged muscle stem cells and mice,suggesting rejuvenating effects of GDF11 on aging skeletal muscle. The authors concluded that GDF11 acts both
directly and indirectly to restore satellite cell regenerative
function [15]. Contrary to those results, others showedthat GDF11 inhibited muscle regeneration and decreased
satellite cell expansion in mice [19] and that GDF11
exposure in mice induced whole body wasting and profound loss of function in cardiac and skeletal muscle [20].
All in all, conflicting results as to the effect of GDF11 onmuscle regeneration exist.
In order to better understand the effect of GDF11
on myogenic differentiation, the aim of this study wasto analyze its effect on ADSCs co-cultured with Mb
on PCL-collagen I-PEO-nanofibers under serum-free
conditions.

Results
Human myoblast and ADSC characterization

Human skeletal Mb in passage 6 (P6) showed > 95% positive staining for the muscle-specific marker desmin. After
7 days of myogenic differentiation, there was evidence ofmultinucleated myotube formation (Fig. 1a and b).
Extensive ADSC characterization has been performed
in a prior study [21]. Briefly, they were successfully differentiated into the chondrogenic, osteogenic, and adipogenic lineage and showed positive expression for CD90,
CD73, and CD105 while negative MSC markers CD34,
CD11b, CD19, CD45, HLA-DR, CD31, CD271, and
SSEA-4 (stage-specific embryonic antigen-4) were notexpressed in both passage 3 (P3) and passage 6 (P6) in a
subsequent FACS analysis [21]. This phenotype is characteristic for MSC [22].
Optimal GDF11 concentration for myogenic differentiationof mb and ADSC co-cultures

Co-cultures of Mb and ADSC were seeded as monolayers and stimulated with serum free differentiation
medium supplemented with 3 different concentrationsof GDF11: 25 ng/ml, 0.1 µg/ml, and 0.5 µg/ml. After
3 days of myogenic differentiation, there was decreasing creatine kinase (CK) activity with increasing GDF11
concentration (Fig. 2a). Statistical analysis showed significant differences between all groups with highly significant differences between 25 ng/ml GDF11 and allother GDF11 concentrations, including serum-free
media without addition of GDF11 (p < 0.001). There
was no difference between serum-free media and 500
ng/ml GDF11 (p = 0.4). After 7 days, pairwise comparisons showed a significant decrease of CK activity afterCai et al. BMC Molecular and Cell Biology(2023) 24:18

Fig. 1 (a) Mb in P6 showed > 95% positive staining for the muscle-specific marker desmin. Human primary fibroblasts in P9 served as negative
control (insert); (b) Mb in P6 showed formation of multinucleated myotubes (exemplarily marked with white arrows) after 7 days of myogenic
differentiation

serum-free differentiation (p = 0.0037) and with additionof 100 ng/ml GDF11 (p = 0.0071), but not with 25 ng/
ml GDF11 (p = 0.11) and 500 ng/ml GDF11 (p = 0.25),
although there was a tendency towards lower CK activity
for both concentrations. Similarly, desmin staining after
7 days of myogenic differentiation showed formation ofmultinucleated myotubes under serum-free + 25 ng/ml
GDF11 and serum-free + 100 ng/ml (Fig. 2b). Myotube
fusion index (MFI) was highest for serum- free + 25 ng/
ml GDF11 (0.159) while MFI was 0.078, 0.071, and 0.034
for serum-free + 100 ng/ml GDF11, serum-free alone,and serum-free + 500 ng/ml GDF11, respectively. Basedon those results, a GDF11 concentration of 25 ng/ml waschosen for further experiments.
Characterization of PCL-collagen I-PEO-nanoscaffolds

SEM images showed aligned PCL-collagen I-PEOnanofibers (Fig. 3). Mean diameter of nanofibers was
233 ± 116 nm. Concerning fiber orientation, 68.27% ofPage 3 of 12

Fig. 2 (a) CK activity after 3 and 7 days of myogenic differentiation of
2D co-cultures of Mb and ADSC under serum-free conditions (0 ng/ml
GDF11). Multiple comparisons between different groups within the sametime period were performed with one-way ANOVA with Tukey‘s multiple
comparisons test or Friedman test with Dunn‘s correction as appropriate (n = 3). Levels of significance were * p < 0.05, ** p < 0.01, *** p < 0.001.
Paired t-test was used to compare same groups over different time periods (3 days vs. 7 days) (n = 3). Level of significance was ## p < 0.01; (b)Desmin staining of co-cultures of Mb and ADSC after 7 days of differentiation under serum-free (top left), serum-free + 25 ng/ml GDF11 (top right),
serum-free + 100 ng/ml GDF11 (bottom left), and serum-free + 500 ng/ml
GDF11 (bottom right) differentiation

all fibers were within ± 4.6°, while 95.45% of all fibers
were within ± 9.2° of main fiber orientation. Singlefiber tensile test revealed an Elastic Young’s modulus of
21.3 ± 7.3 MPa for PCL-collagen I-PEO-bundles.
Cell viability on PCL-collagen I-PEO-nanoscaffolds

Cell viability of 3D co-cultures of Mb and ADSC on
PCL-collagen I-PEO-nanoscaffolds was assessed after 7,
14, and 28 days of myogenic differentiation. While there
was no difference between group 1 (serum-free) andgroup 2 (serum-free + GDF-11) (p = 0.952), cell viability
was higher in group 3 (standard) compared to group 1(p = 0.048) and group 2 (p = 0.034). With increasing differentiation time, this difference became more pronounced:
After 14 days, cell viability of group 3 increased highly
significantly compared to group 1 (p = 0.010) and group

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Page 4 of 12

Myogenic differentiation on PCL-collagen I-PEOnanoscaffolds

Fig. 3 SEM image of aligned PCL-collagen I-PEO-nanofibers. Scale bar
10 μm

2 (p = 0.008). There was no difference between group
1 and 2 (p = 0.984). After 28 days, cell viability also differed between group 3 and group 1 in favor of group 3(p = 0.001) and between group 3 and group 2 in favorof group 3 (p = 0.0003). Over time, cell viability did notincrease within groups except within 7 to 14 days forgroup 1 (p = 0.030). There was a trend towards higher
cell viability after 28 days compared to 7 days for group 3(p = 0.080) (Fig. 4).

CK assay showed no difference between groups after 7
days of myogenic differentiation (p = 0.439). After 14 daysand 28 days, CK activity highly significantly increasedin group 3 (standard) compared to group 1 (serum-free)(p = 0.001) and group 2 (serum-free + GDF11) (p = 0.001).
There was no difference between group 1 and group 2during all time periods (p > 0.999 after 7 days, p = 0.986
after 14 days, p = 0.991 after 28 days). Over time, CK
activity did not change within groups, except in group3, where CK activity increased from 7 days to 28 days
(p = 0.031) (Fig. 5).
With scanning electron microscopy (SEM) images,
configuration of attached cells on PCL-collagen I-PEOnanoscaffolds could be analyzed (Fig. 6, Supplementary
Figures S1-3). After 28 days of myogenic differentiation,
co-cultures of Mb and ADSCs showed parallel alignmenton the scaffolds under all three differentiation conditions. While in group 2 (serum-free medium + 25 ng/
ml GDF11) and group 3 (standard), cells covered almost
the entire surface of the scaffold, the cells in group 1(serum-free) were not as densely packed. Fluorescencemicroscopy showed a similar trend after 28 days of serum
free, serum free + GDF11, and standard myogenic differentiation. All groups led to positive expression of myosin heavy chain (MHC) of differentiated Mb and ADSC
co-cultures compared to negative control (Fig. 6). Fluorescent intensity ratio of MHC positive cells to diamidine-phenylindole-dihydrochloride (DAPI) was 0.989 forgroup 1, 1.045 for groups 2, and 0.957 for group 3.

Fig. 4 Wst-8-assay after 7, 14, and 28 days of myogenic differentiation of 3D co-cultures of Mb and ADSC on PCL-collagen I-PEO-nanofibers. Viability isshown as percentage of control (absorbance of cell-seeded scaffolds after 7 days of proliferation). Multiple comparisons between different groups within
the same time period were performed with one-way ANOVA with Tukey‘s multiple comparisons test (n = 3). Levels of significance were * p < 0.05, **p < 0.01, *** p < 0.001. Repeated measures ANOVA with Tukey‘s multiple comparisons test was used to compare same groups over different time periods(n = 3). Level of significance was # p < 0.05

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Fig. 5 CK assay after 7, 14, and 28 days of myogenic differentiation of 3D
co-cultures of Mb and ADSC on PCL-collagen I-PEO-nanofibers. Multiple
comparisons between different groups within the same time period were
performed with one-way ANOVA with Tukey‘s multiple comparisons testor Kruskal-Wallis test with Dunn‘s correction as appropriate (n = 3). Levelof significance was *** p < 0.001. Repeated measures ANOVA with Tukey‘s
multiple comparisons test was used to compare same groups over different time periods (n = 3). Level of significance was # p < 0.05

Gene expression of the skeletal muscle motor protein MYH2 (myosin heavy chain 2) increased after 28
days of serum-free differentiation with GDF11 (group 2)
compared to serum-free differentiation alone (group 1)(p = 0.021) while there was neither a difference between
group 1 and group 3 (standard) (p = 0.229) nor between
group 2 and group 3 (p = 0.206). There was no change
in the cytoskeletal protein ACTA1 (skeletal alpha actin)
expression between either group (group 1 vs. group 2:
p = 0.303, group 1 vs. group 3: 0.890, group 2 vs. group 3:
p = 1.000) (Fig. 7).

Discussion
The findings of this study implicate that Mb and ADSC
co-cultures showed adequate viability and adherenceon PCL-collagen I-PEO-nanofibers. Myogenic differentiation over a period of 28 days led to the expression of
myogenic key markers and parallel arrangement along
the aligned nanofibers. A low concentration of 25 ng/
ml GDF11 led to positive MHC expression as shown by
immunostaining and enhanced MYH2 gene expressionof Mb and ADSC co-cultures on PCL-collagen I-PEOnanoscaffolds at least under serum-free differentiation
conditions. MYH2 and ACTA1 are both terminal myogenic markers guiding sarcomere formation [9]. On theother hand, GDF11 was not beneficial in terms of cell viability and myogenic differentiation, assessed by CK activity and even inhibited those parameters when compared
to standard serum-containing differentiation.
The role GDF11 has on skeletal muscle regeneration iscontroversial as both beneficial as well as harming effects
have been described [19, 20, 23]. This might be explained
by GDF11 being closely related to GDF8 or myostatin,
which is a potent inhibitor of skeletal muscle growthPage 5 of 12[24]. Both myostatin and GDF11 act via the activin typeII receptor, activating the Smad2-/Smad3-complex, andthereby mediating downstream signaling [25, 26]. The
effect of GDF11 on MSC has also been studied intensely.
For instance, GDF11 inhibited MSC to differentiate into
the adipogenic lineage via the TGF-beta signaling pathway [27]. GDF11 also inhibited osteoblast and chondrogenic differentiation of bone marrow-derived MSC [28,29]. The influence of GDF11 on myogenic differentiation
capacity of MSC let alone co-cultures of primary Mb and
ADSC on PCL-collagen I-PEO-nanoscaffolds has notbeen investigated so far.
The results of the present study reflect a similar trend,
but they do not clearly support the previously mentioned
studies. CK activity, which has been used as a biochemical marker for direct measurement of myogenic differentiation [20, 30], decreased over time after induction ofserum-free myogenic differentiation, which is in accordance to a previous study [4]. As described in the aforementioned study, decrease in CK activity could have been
evoked by a decline in growth factors by using serumfree medium [4]. Additional GDF11 did not lead to anincrease in CK activity in the present study. When using
GDF11 at a high concentration of 500 ng/ml, CK activity even decreased over time, which could be explainedby an inhibiting effect of GDF11 on myogenic differentiation. More recent findings support this theory and demonstrate that GDF11 is not the long-sought rejuvenating
factor as opposed to the theory of Sinha et al. [15, 31].
Egerman et al. showed that previously used reagents todetect GDF11 were nonspecific and that GDF11 rather
increases with age and inhibits muscle regeneration andmyoblast differentiation [19].
On the other hand, no difference in GDF11 concentration could be detected via enzyme-linked immunosorbent assay between plasma of younger compared to older
individuals [32]. In a recent study, low concentrations of 1
ng/ml and 10 ng/ml of GDF11 have not led to a decrease
in myogenic differentiation but rather to an increase innuclear density and myotube length and width of different myogenic cells [33]. Similarly, in the present study, theaddition of GDF11 did not lead to a decrease of CK activity and even upregulated MYH2 compared to serum-freedifferentiation alone. However, both groups were still not
as stimulating as the standard serum-containing differentiation medium. As described in a previous study, myogenic differentiation of MSC necessitates growth factors,
which might not be contained in Ultroser® G [4]. Other
growth factors besides GDF11 might be necessary for an
increase of myogenic differentiation to the level of stimulation with serum-containing differentiation medium, forexample insulin-like growth factor-1 [9, 34] or humanepidermal growth factor [35]. At least, GDF11 did notlead to a significant decrease of myogenic key markers as

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Page 6 of 12

Fig. 6 Myosin heavy chain (MHC) immunofluorescent staining (red = MHC, blue = DAPI) of co-cultures of Mb and ADSC on PCL-collagen I-PEO-nanofibers. Co-cultures were myogenically differentiated for 28 days in serum-free media (up right), serum-free media + GDF11 (down left), or standard serumcontaining media (up left). Fibroblasts were allowed to proliferate on nanofibers for 1 week and served as negative control (down right). Scanningelectron microscopy (SEM), showing configuration of attached cells on PCL-collagen I-PEO-nanofibers are represented as inserts in the corresponding
fluorescent microscopy image

Fig. 7 Gene expression of the late myogenic markers myosine heavy
chain (MYH2) and actin alpha skeletal muscle (ACTA1) in co-cultures of Mb
and ADSC after 28 days of myogenic differentiation. Expressions are demonstrated in x-fold difference compared with RNA from human muscle
tissue using the 2−ΔΔCt method. GAPDH was used as housekeeping genein co-cultures and muscle tissue. Comparisons between groups were performed with one-way ANOVA with Tukey‘s multiple comparisons test or
Kruskal-Wallis test with Dunn‘s correction as appropriate (n = 3). Level ofsignificance was * p < 0.05

opposed to the findings by Egerman et al. [19]. The effectof GDF11 in combination with the standard serum-containing medium remains unclear as this combination was
not tested in the present study since the main goal was totest the effect of GDF11 independent of potential growth
factors of unknown composition as contained in serum.
Electrospun PCL-collagen I-nanoscaffolds have provento be a suitable and biocompatible matrix for longterm differentiation of co-cultures of Mb and ADSC
[4]. The integration of PEO sacrificial fibers is supposed
to increase porosity of nanoscaffolds, facilitating cell
influx [11, 12]. Unfortunately, we did not compare the
newly established PCL-collagen I-PEO-nanoscaffolds to
PCL-collagen I-nanoscaffolds. Thus, conclusions abouta more efficient myogenic induction of either of thoseblended fiber types cannot be made. Yet, we were able
to demonstrate positive MHC expression in this settingof 28 days of myogenic differentiation. A prior analysis

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

of 3D vascularization of PCL-collagen I-nanofiber scaffolds versus PCL-collagen I-PEO-nanofiber scaffolds inthe arteriovenous loop model of the rat showed better
integration into the host organism for the PEO-blended
fibers [12]. However, the PCL-collagen I-PEO-nanofibers
in that study were randomly arranged. A more recent
study, which had been completed simultaneously to thecurrent study, compared aligned PCL-collagen I-nanofiber scaffolds to aligned PCL-collagen I-PEO-nanofiber
scaffolds, which were similar to the scaffolds used in
the current study, in an in vivo vessel loop model. Surprisingly, the mentioned study revealed higher amountof vascularization and angiogenesis for PCL-collagen
I-nanofibers compared to PCL-collagen I-PEO-nanofibers. The authors concluded that pores could have led
to a certain degree of loss of mechanical stability of PCLcollagen I-PEO-nanofibers compared to PCL-collagen
I-nanofibers, fostering tissue ingrowth [36]. Thus, further
comparison between those two scaffolds types is a prerequisite for future studies before this 3D model can betranslated into clinical applications.
There are several limitations of this study. First, standard deviations were sometimes high which might be
explained by the limited sample size and the use of primary human cells of different subjects. The purchased
myoblasts were all from the quadriceps muscle andhuman subjects were all male to limit heterogeneity.
Other factors like age are known to influence the degreeof myotube formation [37, 38]. Furthermore, maintenance and myogenic differentiation of primary cells isolated from adult muscle tissue is a larger challenge thanculturing immortal cell lines like C2C12 with a rapid proliferative and differentiation capacity [39]. However, celllines are not suitable for establishing a model of skeletal
muscle engineering, which can be translated into clinicalapplication since they do not represent the characteristics of skeletal muscle as accurately as primary myoblasts
do [39]. Second, the 3D structure of the cell-seedednanoscaffolds impeded taking clear immunofluorescentpictures with a fluorescence microscope. Configurationof attached cells could only be detected via SEM, but
myotube formation could not be seen by this method.
This could also be ascribed to the properties of the PCLcollagen I-PEO_nanofibers for the reasons describedabove. Nevertheless, confocal microcopy should be used
for future studies involving cell-seeded 3D nanoscaffolds[39]. After all, a clear difference could be shown between
probes and negative control. Mb monocultures were notanalyzed, so it is uncertain what effect GDF11 has on Mb
in our experimental setting. On the other hand, Mb alone
might not be promising for engineering large-scale tissue,
which can eventually be applied in the patient since they
have a limited proliferation capacity [7, 40, 41]. In several
studies, Mb and ADSC co-cultures were differentiatedPage 7 of 12

into the myogenic lineage [4, 42, 43]. Gehmert et al. wereable to show that ADSC secreted IGF-1 that protected
myoblasts from negative effects of myostatin [4, 42, 43].
After exposure to ADSC-conditioned medium, myostatin
treated myoblasts showed less apoptosis and more proliferation as well as higher expression of MyoD [4, 42,43], a marker of myogenic commitment [4, 42, 43]. Oki
et al. found similar results and discovered that decorin,a dermatan sulfate proteoglycan and known inhibitor of
TGFbeta1, was secreted by ADSCs, protecting myoblasts
against fibrosis [4, 42, 43]. The protective effects of ADSC
might have competed with the supposedly negative
effects of GDF11, which led to the presented ambiguous
effects of GDF11 on Mb and ADSC co-cultures. Anotherbenefit of ADSC is their relatively easy and safe harvesting method and their high expansion capacity, making
them an attractive cell source for co-culture with primary
myoblasts [4, 42, 43]. In the current study as well as inprior studies, ADSC in higher passage (P6) displayed thesame characteristics as ADSC in lower passage (P3) [4][21]. Thus, ADSC in P6 were used for experiments. This
has an impact on later clinical translation since a small
amount of harvested tissue results in abundant number of cells. Specifically in cases of muscular diseases as
Duchenne muscular dystrophy where myoblasts cannot be isolated due to depletion of the satellite cell pool
ADSC would be the sole cell source for skeletal muscle
tissue engineering purposes [44]. Thus, an ADSC monoculture system is of particular interest for future studies.
Given the results of the present study, we propose that
PCL-collagen I-PEO-nanofibers are a viable option for
myogenic differentiation of Mb and ADSC co-cultures.
In this setting, GDF11 alone might be promoting myogenic differentiation of Mb and ADSC co-cultures underserum-free conditions. We were not able to prove aharming effect of the myostatin homologue as proposedby recent studies [4, 42, 43]. A combination of GDF11
with other proteins, particularly those secreted by ADSC
should be analyzed in terms of the effect on myogenesis under serum-free conditions as serum-free + GDF11
stimulation was not beneficial on cell viability compared
to standard serum-containing myogenic differentiation.
Regardless of the effects of GDF11, this biocompatible 3D
model, consisting of primary Mb and ADSC co-cultured
on PCL-collagen I-PEO-nanofibers can serve as a platform for skeletal muscle tissue engineering, which can betransferred to further in vivo and translational research.
Other nanofiber scaffolds like PCL-collagen I-nanofibers
are also viable options and should be directly comparedto PCL-collagen I-PEO-nanofibers in terms of myogenic
differentiation of Mb and ADSC.

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Conclusion
We have established electrospun aligned PCL-collagen
I-PEO-nanofiber scaffolds as a novel biocompatible
matrix for skeletal muscle tissue engineering purposes.
Mb and ADSC showed adequate cell adherence, viability,
and myogenic differentiation when co-cultured on thosematrices. In this setting of serum-free myogenic differentiation, GDF11 had a promoting effect on myogenic
differentiation of Mb and ADSC co-cultures, although
this serum-free differentiation system was still inferior toserum-containing differentiation.
Methods
Human cell culture and characterization

Human Mb were purchased from Lonza (Walkersville,MD) and cultured in SkBMTM-2 Basal Medium with
supplements (catalog #CC-3246 and #CC-3244, all from
Lonza). Cells from quadriceps muscle from three different adult male donors (catalog #CC-2580, HSMM
33,406/Lot# 650,386, HSMM 30,551/Lot# 583,849,
HSMM 33,607/Lot# 655,307) were passaged up to P6.
Desmin immunofluorescence (ab8470, Abcam, Cambridge, UK) as described in detail under “Immunofluorescence” was used to confirm myogenic characteristicsof the Mb. Human primary fibroblasts (HFIB-D, cryo,
provitro AG, Berlin, Germany) in passage 9 (P9) served
as negative control. To verify myotube formation capacity, Mb of P6 were stained for desmin after 7 days of myogenic differentiation induced by myogenic differentiation
medium, containing DMEM/Ham’s F12 + 2% donor horse
serum (DHS) + 1% L-Glutamin + 1% Penicillin/Streptomycin (P/S) (all from Biochrom GmbH, Berlin, Germany) + 0,4 µg/ml dexamethasone (Sigma Aldrich, St.
Louis, Missouri, USA), 1 ng/ml basic fibroblast growth
factor (bFGF) (Peprotech, Hamburg, Germany).
ADSC were isolated from human adipose tissue andcharacterized in P3 and in P6 as described previously[21]. Tissue collection was approved by the local Ethics
Committee (approval number 424_18 B) in accordancewith the World Medical Association Declaration of Helsinki and informed consent was obtained.
For each experiment, myoblasts from the three different donors were co-cultured with the ADSC in P6 at aratio of 1:1 (n = 3). Since ADSC in P6 displayed the samecharacteristics as ADSC in P3 the higher passage of theboth was used.
Determination of optimal GDF11 concentration

Co-cultures of Mb and ADSC (n = 3) were seeded asmonolayers in a ratio of 1:1 in 6-well culture plates at adensity of 3 × 105 cells in expansion medium containing DMEM/Ham’s F 12, 10% FCS, 1% L-Glutamin, 1%
P/S (all from Biochrom GmbH). After 48 h, medium
was replaced by serum free differentiation medium,

Page 8 of 12

containing DMEM/Ham’s F12 + 0.2% Ultroser® G (Cytogen GmbH, Wetzlar, Germany) as previously described[4]. Three different concentrations of GDF11 (LifeSpan
BioSciences, Seattle, WA) were added to the serum freedifferentiation medium: 25 ng/ml, 0.1 µg/ml, and 0.5 µg/
ml. The choice for the different concentrations was basedon concentrations used for in vitro experiments reported
in the literature [15, 19, 45]. Medium including fresh
GDF11 was changed every 2–3 days. After 3 and 7 days,
CK activity was colorimetrically determined (Abcam) as
previously described [4]. The amount of nicotinamide
adenine dinucleotide (NADH) generated by CK wasdetermined photometrically at 450 nm with Thermo Scientific ™ Multiskan™ GO during minute 16–20 of reaction
time since after 20 min, the activity of the samples wasfound to have reached a plateau. For further experiments,
GDF11 in a concentration of 25 ng/ml was used. To visualize myogenic potential of GDF11 in the chosen concentration, Mb and ADSC were differentiated with serum
free differentiation medium + 25 ng/ml GDF11 for 7 days
and immunostained for desmin as described in detailunder „Immunofluorescence“. Myotube fusion index(MFI) was calculated semi-automatically via ImageJ 1.53e(National Institutes of Health, Bethesda, MD, USA) as
described previously [21].
Electrospinning of PCL-collagen I-PEO-nanofibers and 3D
cell culture

PCL-collagen I-PEO-nanofibers were produced by electrospinning [36]. Briefly, PCL (80.000 g/mol, Sigma
Aldrich) was blended with bovine collagen type I (Symatese, Lyon, France) in a ratio of 2:1 at a 12% (w/v) solution,using acetic acid (90% v/v in dH2O, Carl Roth GmbH,
Karlsruhe, Germany) as solvent. Parallel PCL-collagen
I-nanofibers were electrospun on a standard electrospinning machine onto parallel metal rods on a custommade rotating drum (15 kV, 15 cm, 1 ml/h, 50 rpm). PEO(concentration 10% (w/v), molecular weight: 900.000 g/
mol, Sigma Aldrich) nanofibers were similarly spun
(14 kV, 13 cm, 1 ml/h, 50 rpm). The single-fiber tensile
test was performed on 5 different PCL-collagen I-PEOfiber bundles as described by Munawar and Schubert[46]. The aligned PCL-collagen I and PEO-fibers werecollected in alternate layers on plastic rings with 10 mmdiameter (Minusheet carrier, Minucells and Minutissue
Vertriebs GmbH, Bad Abbach, Germany). The area ofthe resulting scaffolds measured approximately 0,8 cm2.
Scanning electron microscopy (SEM) images were taken
as described below (“scanning electron microscopy”) andused for characterization of unseeded scaffolds. Fiber
diameter was measured using ImageJ (National Institutesof Health, Bethesda, MD, USA, Version 1.53e). Fiberorientation was analyzed using the OrientationJ plugin(ImageJ) on 5 different images of the scaffolds taken in

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Table 1 Myogenic differentiation mediaGroup1) serum-free2) serum-free + GDF11
3) standard

Contains DMEM/Ham’s F12 + 1%
L-Glutamin + 1% P/S + 0.4 µg/ml
dexamethason + 1 ng/ml bFGF +0.2% Ultroser® G
0.2% Ultroser® G + 25 ng/ml GDF11
2% DHS

10,000x magnification. This resulted in an orientation
distribution histogram for each image. For cell seeding,scaffolds were sterilized in 70% ethanol, washed with PBS
afterwards [36] and placed into 24 well-plates while they
were soaked in DMEM/Ham’s F12 for approximately 1 h
at 37 °C. ADSC and Mb were seeded with 100 µL thickened medium containing expansion medium and dissolved methyl cellulose (Sigma Aldrich) on PCL-collagen
I-PEO-nanoscaffolds at 3 × 105 cells in a ratio of 1:1. After
7 days of proliferation in expansion medium, differentiation was induced and continued for 28 days. As negative
control for immunofluorescence staining, fibroblastsin P9 were seeded onto the nanoscaffolds as describedabove and allowed to proliferate in DMEM + 10% fetal
calf serum (FCS) + 1% P/S (all from Biochrom GmbH) for
7 days.
Myogenic differentiation conditions

To confirm myogenic differentiation potential of Mb in
P6, monolayers were seeded in 48-wells at a cell densityof 20.000 and allowed to proliferate in expansion medium
for 2 days until cells reached confluence. Afterwards, differentiation was induced by switching to standard differentiation medium, containing 2% donor horse serum(DHS, Biochrom GmbH) (Table 1). Co-cultures of ADSC
and Mb were seeded similarly into 48-wells and wereallowed to differentiate with serum free differentiation
medium + 25 ng/ml GDF11 (Table 1) for 7 days. Differentiation was continued for 7 days prior to desmin staining
as described in detail under “Immunofluorescence”.
For three-dimensional (3D) co-cultures, ADSC and
Mb were seeded onto PCL-collagen I-PEO-scaffolds andallowed to proliferate for 7 days as described above. After
proliferation, co-cultures were myogenically differentiated under three different conditions: (1) DMEM/Ham’s
F12 + 0.2% Ultroser® G + 1% L-Glutamin + 1% P/S + 0.4 µg/
ml dexamethason + 1 ng/ml bFGF (Peprotech, Hamburg, Germany) (serum-free medium), (2) serum-free
medium + 25 ng/ml GDF11, (3) DMEM/Ham’s F12 + 2%
DHS + 1% L-Glutamin + 1% P/S + 0.4 µg/ml dexamethason + 1 ng/ml bFGF (standard differentiation medium,
containing serum). For every experiment, 3 scaffolds pergroup were analyzed (n = 3). Mb from one donor (out of 3
in total) were seeded in co-culture with the ADSC onto
one scaffold per group.

Page 9 of 12

Table 2 Primer sequences
MYH2
ACTA1
GAPDH

Forward primer
GGGCCTTTCAAGAGGGACACCACAATGTGCGACGAAGACG
TCCACCCATGGCAAATTCCA

Reverse primer
TGCGCTCCCTTTCAGACTTTCTCTCTTGCTCTGAGCCTCG
TTCCCGTTCTCAGCCTTGAC

Cell viability and creatine kinase activity on PCL-collagen
I-PEO-nanoscaffolds

3D co-cultures (n = 3) were allowed to proliferate for 7
days and subsequently to myogenically differentiate for7, 14, and 28 days. After each time period, water-soluble
tetrazolium salt (wst)-8-assay (Promokine, Promocell
GmbH, Heidelberg, Germany) of the seeded scaffolds
was performed as described previously [4]. Absorbancewas measured at 450 nm with Photometer Thermo Scientific™ Multiskan™ GO to assess cell viability. Percentage of
viability was shown after normalization of absorbance ofeach differentiation period to control (proliferation only).
Afterwards, CK activity was determined as describedabove (n = 3).
RNA isolation and quantitative PCR analysis

In 3D co-cultures (n = 3), the gene expression rate of thelate myogenic markers MYH2 (myosin heavy chain 2)
and ACTA1 (skeletal alpha actin) was analyzed as previously described [4]. As housekeeping gene, GAPDH(glyceraldehyde-3-phosphate dehydrogenase) was used
as internal control. RNA of the samples was extractedusing Trizol (Life Technologies, Carlsbad, CA, USA)and chloroform [21]. RNA was reverse-transcribed into
cDNA using a QuantiTect Reverse Transcription Kit anda Sensiscript Reverse Transcription Kit (both from Qiagen GmbH). cDNA was amplified through quantitative
real-time PCR using SsoAdvanced Universal SYBR GreenPCR Supermix (Bio-Rad, Hercules, CA, USA) and Light
Cycler (Bio-Rad CFX96 Touch™). Evaluation of gene
expression was performed using the 2−ΔΔCt method. RNA
from human muscle tissue served as control sample. The
primer sequences used are given in Table 2.
Immunofluorescence

Mb in P6, myogenically differentiated Mb in P6, as well as
differentiated co-cultures of ADSC and Mb were desminstained as previously described [4]. Briefly, cells were
fixed with formaldehyde (Carl Roth GmbH), washed, andblocked in PBS with 1.5% FCS and 0.25% TritonX (CarlRoth GmbH) for one hour at room temperature. Cells
were incubated with desmin primary antibody (ab8470,
Abcam) at 0.5 µg/ml for one hour at room temperature.
ADSC and Mb were 3D co-cultured on PCL-collagen
I-PEO-nanoscaffolds (n = 3) for 7 days before expansion medium was switched to differentiation medium(serum-free, serum-free + GDF11, standard). After 4
weeks, scaffolds were fixed, washed, blocked, and stained

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

with anti-fast myosin skeletal heavy chain (MHC) antibody (ab91506, Abcam) at 5 µg/ml for one hour at room
temperature.
Alexa Fluor 594 goat anti-mouse IgG1 cross-adsorbed
secondary antibody (A-21,125, Thermofisher Scientific Inc.) was used as secondary antibody at 4 µg/ml for
30 min at room temperature for desmin stained cells andAlexa fluor 594 goat anti-rabbit IgG (H + L) (Thermofisher Scientific Inc.) was used as secondary antibody for
MHC stained cells at the same conditions. After counterstaining with DAPI 1 µg/ml (Thermofisher Scientific Inc.)for 5 min, cells were subsequently analyzed and digitallyphotographed with a fluorescence microscope (IX83,
cellSens, software, Olympus, Hamburg, Germany).
Human primary fibroblasts in P9 served as negative
control.
Fluorescent intensity of MHC stained seeded nanoscaffolds was determined as a ratio of mean grey area of
inverted blue channel and mean grey area of inverted redchannel via ImageJ 1.53e (National Institutes of Health,Bethesda, MD, USA).
Scanning electron microscopy

After immunofluorescence analysis of the seeded scaffolds after 28 days of myogenic differentiation, microstructural analysis of the scaffolds was performed using
an Auriga Fib-scanning electron microscope (SEM)(Zeiss, Oberkochen, Germany) as described previously[4]. Probes were sputter-coated with gold using a Q150T
Turbo-pumped Sputter Coater (Quorum Technologies
Inc., Guelph, Canada).
Statistical analysis

Shapiro-Wilk test was used to test data normality. Statistical analysis was performed with one-way analysis ofvariance (ANOVA) with Tukey’s multiple comparisonstest or Kruskal-Wallis test with Dunn’s correction for
multiple comparisons, as appropriate. Repeated measures ANOVA with Tukey’s multiple comparisons test
was used for comparisons between matched variables atdifferent time points for normally distributed data, otherwise Friedman test with Dunn’s correction for multiple
comparisons was used. Pairwise comparison was doneusing paired t-test or Mann-Whitney test, as appropriate.
Statistical analysis was performed using GraphPad Prism
version 8.3 (La Jolla, CA, USA). A p-value ≤ 0.05 was considered statistically significant.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00478-1.
Additional file 1 Scanning electron microscopy (SEM) of Mb and ADSC
co-cultures on PCL-collagen I-PEO-nanofibers after 4 weeks of myogenic
differentiation in standard serum-containing medium

Page 10 of 12

Additional file 2 Scanning electron microscopy (SEM) of Mb and ADSC
co-cultures on PCL-collagen I-PEO-nanofibers after 4 weeks of myogenic
differentiation in serum-free medium
Additional file 3 Scanning electron microscopy (SEM) of Mb and ADSC
co-cultures on PCL-collagen I-PEO-nanofibers after 4 weeks of myogenic
differentiation in serum-free medium + 25 ng/ml GDF11
Additional file 4
Acknowledgements
The present work was performed in fulfillment of the requirements for
obtaining the degree “Dr. med.” For PS. We would like to thank Andrea Beckand Stefan Fleischer for their excellent technical support.
Author contributions
AC designed the study, acquired funding, analysed and interpreted the data,
and was a major contributor in writing the manuscript. PS and ZZ performed
cell isolations, cell culture experiments, and immunocytochemistry. ZZ
performed qPCR. MH carried out the electrospinning process and performed
scanning electron miscroscopy and fiber characterization. DWS participatedin the electrospinning process. VW performed FACS analysis of ADSC. JPB and
REH contributed to study design and interpretation of data. AA contributed
to funding acquisition, study design, study coordination, interpretation of thedata and helped to draft the manuscript. All authors have seen and agreed to
the final submitted version of the paper.
Funding
Open Access funding enabled and organized by Projekt DEAL and by
Deutsche Forschungsgemeinschaft and Friedrich-Alexander-Universität
Erlangen-Nürnberg within the funding programme “Open Access Publication
Funding“. This study was funded by the ELAN-Fond (15-10-07-1-Cai) of theFriedrich-Alexander University of Erlangen-Nürnberg, and the Deutsche
Forschungsgemeinschaft (DFG, CA 2301/1–1). Funding was used for
production of scaffolds and cell culture experiments.
Data Availability
The datasets used and/or analysed during the current study are available fromthe corresponding author on reasonable request, qPCR-data are provided as
supplementary file.

Declarations
Ethics approval and consent to participate
Tissue collection was approved by the institutional ethics committee of theFriedrich-Alexander-University of Erlangen-Nuremberg (approval number
424_18 B). Informed consent was obtained from all subjects involved in the
study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Plastic and Hand Surgery, Laboratory for Tissue
Engineering and Regenerative Medicine, University Hospital of Erlangen,Friedrich-Alexander University of Erlangen-Nürnberg (FAU),
91054 Erlangen, Germany
2
Department of Plastic Surgery, Hand Surgery - Burn Center, University
Hospital RWTH Aachen, 52074 Aachen, Germany
3Institute of Polymer Materials, Department of Materials Science andEngineering, University of Erlangen-Nürnberg (FAU), 91058 Erlangen,Germany
4
Department of Transfusion Medicine, University Hospital of Erlangen,Friedrich-Alexander- University Erlangen-Nürnberg (FAU), 91054 Erlangen,Germany
Received: 19 February 2023 / Accepted: 27 April 2023

Cai et al. BMC Molecular and Cell Biology(2023) 24:18

Page 11 of 12

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