Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering

Abstract

Background: Volumetric muscle loss caused by trauma or after tumour surgery exceeds the natural regeneration capacity of skeletal muscle. Hence, the future goal of tissue engineering (TE) is the replacement and repair of lost muscle tissue by newly generating skeletal muscle combining different cell sources, such as myoblasts and mesenchymal stem cells (MSCs), within a three-dimensional matrix. Latest research showed that seeding skeletal muscle cells on aligned constructs enhance the formation of myotubes as well as cell alignment and may provide a further step towards the clinical application of engineered skeletal muscle. In this study the myogenic differentiation potential of MSCs upon co-cultivation with myoblasts and under stimulation with hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) was evaluated. We further analysed the behaviour of MSC-myoblast co-cultures in different 3D matrices.

Results: Primary rat myoblasts and rat MSCs were mono- and co-cultivated for 2, 7 or 14 days. The effect of different concentrations of HGF and IGF-1 alone, as well as in combination, on myogenic differentiation was analysed using microscopy, multicolour flow cytometry and real-time PCR. Furthermore, the influence of different three-dimensional culture models, such as fibrin, fibrin-collagen-I gels and parallel aligned electrospun poly-ε-caprolacton collagen-I nanofibers, on myogenic differentiation was analysed. MSCs could be successfully differentiated into the myogenic lineage both in mono- and in co-cultures independent of HGF and IGF-1 stimulation by expressing desmin, myocyte enhancer factor 2, myosin heavy chain 2 and alpha-sarcomeric actinin. An increased expression of different myogenic key markers could be observed under HGF and IGF-1 stimulation. Even though, stimulation with HGF/IGF-1 does not seem essential for sufficient myogenic differentiation. Three-dimensional cultivation in fibrin-collagen-I gels induced higher levels of myogenic differentiation compared with two-dimensional experiments. Cultivation on poly-ε-caprolacton-collagen-I nanofibers induced parallel alignment of cells and positive expression of desmin.

Conclusions: In this study, we were able to myogenically differentiate MSC upon mono- and co-cultivation with myoblasts. The addition of HGF/IGF-1 might not be essential for achieving successful myogenic differentiation. Furthermore, with the development of a biocompatible nanofiber scaffold we established the basis for further experiments aiming at the generation of functional muscle tissue.

Keywords: HGF; IGF-1; Mesenchymal stem cells; Myogenic differentiation; PCL-collagen nanofibers; Skeletal muscle tissue engineering.

Fig. 1

Expression of MEF2 and ACTN2 under different concentrations of HGF. Real-time PCR of MSC and myoblast (Mb) mono- and co-cultures under HGF stimulation as well as in unstimulated controls. Expressions are demonstrated in x-fold difference compared with unstimulated cells cultivated in basic differentiation medium (control = 1) using the 2^-ΔΔCt method. Markers are presented with mean +/- SD. a Significant and highly significant higher expression of MEF2 in co-cultures after 2 d compared with 7 d using 10, 30 and 60 ng/ml HGF. b In co-cultures, ACTN2 expression was upregulated during early stimulation compared with unstimulated control groups. 100 ng/ml HGF over 7 d induced the strongest ACTN2 expression. c Strongest MEF2 expression in MSC monocultures could be achieved with 10 ng/ml HGF after stimulation for over 7 d. d Seven-day stimulation with 10, 60 and 100 ng/ml HGF induced almost equal or higher levels of ACTN2 in MSC monocultures compared with 2 d stimulation. e-f In Mb, a dose-dependent increase in MEF2 (e) and ACTN2 (d) expression was demonstrated from 10 to 60 ng/ml HGF during early stimulation. Increased levels of MEF2 and ACTN2 under HGF during early stimulation in co-cultures and Mb monocultures compared with unstimulated controls. Mb of three different isolations were used in three independent experiments. Three replicates of each were used. (** = p ≤ 0.01). (* = p ≤ 0.05)

Fig. 2

Expression of MEF2 and ACTN2 under different concentrations of IGF-1. Real-time PCR of MSC and myoblast (Mb) mono- and co-cultures under IGF-1 stimulation as well as in unstimulated controls. Expressions are demonstrated in x-fold difference compared with unstimulated cells cultivated in basic differentiation medium (control = 1) using the 2^-ΔΔCt method. Markers are presented with mean +/- SD. a Overall higher expressions of MEF2 in co-cultures under the different IGF-1 concentrations compared with unstimulated conditions after 2 d. Significant and highly significant higher levels of MEF2 after 2 d compared with 7 d using 10 and 30 ng/ml IGF-1. b Overall higher expressions of ACTN2 in co-cultures under the different IGF-1 concentrations compared with unstimulated conditions after 2 d. c Stimulation with 60 ng/ml IGF-1 over 2 d induced the strongest upregulation of MEF2 in MSC monocultures. d Overall increased ACTN2 expression in MSC monocultures was observed during early stimulation, with highest levels under 10 ng/ml IGF-1. e-f Early stimulation with IGF-1 induced higher MEF2 (e) and ACTN2 (f) expressions in Mb monocultures compared with controls, with strongest expression under 30 ng/ml IGF-1. Increased MEF2 expression in co-cultures and Mb monocultures after 2 d compared with unstimulated controls. The highest MEF2 and ACTN2 levels were detected in Mb monocultures. Mb of three different isolations were used in three independent experiments. Three replicates of each were used. (** = p ≤ 0.01). (* = p ≤ 0.05)

Fig. 3

Expression of myogenic differentiation markers and IGFBPs under the influence of HGF and IGF-1. Real-time PCR of MSC and myoblast (Mb) mono- and co-cultures stimulated with HGF + IGF-1, HGF, IGF-1 or cultivated in unstimulated controls. Expressions are demonstrated in x-fold difference compared with Mb (=1) using the 2^-ΔΔCt method. Markers are presented with mean +/- SD. a After 2 d, the strongest DES upregulation was demonstrated in unstimulated controls. Throughout all conditions in MSCs, much higher levels of DES compared with co-cultures and Mb were observed. b After 14 d, strongest MYOG expression was detected under HGF stimulation compared with control myoblasts. MYOG could only be detected in one out of three experiments. c The highest expression of ACTN2 was observed in IGF-1 stimulated groups, both in co-cultures and MSC monocultures. d The strongest upregulation of MyHC2 in co-cultures was observed under IGF-1 stimulation. In MSC monocultures, the levels of MyHC2 remained under all conditions lower than Mb. e In co-cultures, the highest IGFBP4 levels were observed under IGF-1 stimulation. In MSC monocultures, HGF induced the strongest upregulation of IGFBP4. f In co-cultures, the highest IGFBP5 levels were observed under IGF-1 stimulation. In MSC monocultures, the expression of IGFBP5 remained lower under all conditions compared with Mb. g In both cell groups, the levels of IGFBP6 were overall lower than in control Mb. Mb of three different isolations were used in three independent experiments. Three replicates of each were used

Fig. 4

Fluorescence microscopy of MyHC2 in co-cultures. A positive staining of MyHC2, a muscle-specific major contractile protein, in MSC and Mb co-cultures under HGF stimulation for 7 d (a) and 14 d (b). Merge of DAPI (blue, nuclear staining), GFP (green, transduced MSC) and MyHC2 (red, with Alexa fluor 594 as secondary antibody). a The beginning formation of multinucleated cells could be observed under HGF stimulation. MSCs show positive expression of MyHC2 (arrows). Scale bars represent 20 μm. Magnification 400x. b The formation of multinucleated cells could be observed under HGF stimulation. It seems that MSCs are involved in the formation of multinucleated cells (arrows). Scale bars represent 50 μm. Magnification 200x

Fig. 5

Fluorescence microscopy of MEF2 in co-cultures after 14 d. A positive staining of MEF2, a co-transcriptional factor expressed during muscle differentiation, in MSC and Mb co-cultures in control groups without HGF/IGF-1 after 14 d. Merge of DAPI (blue, nuclear staining), GFP (green, transduced MSC) and MEF2 (red, with Alexa fluor 594 as secondary antibody). The red fluorescence demonstrates the characteristic perinuclear localisation of MEF2. Scale bars represent 50 μm. Magnification 200x

Fig. 6

Flow cytometry analysis of MEF2 and ACTN2 in MSC and myoblast co-cultures, MSC and myoblast monocultures and L6-myoblasts. Markers are presented with mean +/- SD. a Highly significant upregulation of MEF2 in co-cultures from 2 to 14 d of stimulation with HGF + IGF-1. Higher levels of MEF2 in MSC monocultures could be observed in stimulated and control groups compared with L6-myoblasts. The expression of MEF2 was slightly downregulated after 14 d in myoblast (Mb) monocultures. b Highly significant upregulation of ACTN2 in co-cultures both under HGF + IGF-1 and in control groups after 14 d compared with 2 d. After 2 d of cultivation, the lowest levels of ACTN2 were demonstrated in MSC monocultures. A 2.7-fold upregulation in unstimulated controls and 3.2-fold under HGF + IGF-1 was observed in MSC monocultures after 14 d. The expression of ACTN2 was downregulated in stimulated and control Mb monocultures. Higher expression of ACTN2 was observed when Mb were cultivated in control groups. (** = p ≤ 0.01). Mb of three different isolations as well as three replicates of each were used. One replicate of MSC and L6 was used

Fig. 7

Myogenic differentiation in fibrin/fibrin-collagen-I gels. Markers are presented with mean +/- SD. a Real-time PCR of MEF2 in MSC and myoblast (Mb) co-cultures cultivated in fibrin and fibrin-collagen-I gels. The expression of MEF2 was highly significantly upregulated over time in 5-mg/ml fibrin-collagen-I gels. MEF2 expression was significantly and highly significantly downregulated in other gel conditions. Expressions are shown in x-fold difference compared with co-cultures cultivated in 2D in control medium. b Real-time PCR of ACTN2 in MSC and Mb co-cultures cultivated in fibrin and fibrin-collagen-I gels. The expression of ACTN2 was highly significantly upregulated over time in 5-mg/ml fibrin-collagen I-gels. ACTN2 expression was downregulated in other conditions, except 5-mg/ml fibrin-collagen-I gels with similar expression compared with the control. Expressions are shown in x-fold difference compared with co-cultures cultivated in 2D in control medium. c Real-time PCR of different myogenic markers (DES, MEF2, MyHC2, ACTN2) and IGFBPs (IGFBP-4, -5, -6) in co-cultures cultivated in fibrin-collagen-I gels and stimulated with HGF and IGF-1 for 2 d. Expressions are demonstrated in x-fold difference compared with unstimulated cells cultivated in control (=1). Upregulation of all myogenic markers under HGF + IGF-1 stimulation compared with unstimulated controls, MyHC2 significantly. (** = p ≤ 0.01). (* = p ≤ 0.05). (# = p ≤ 0.05 compared with unstimulated controls). Mb of three different isolations as well as three replicates of each were used

Fig. 8

Cultivation of MSC-myoblast co-cultures on PCL-collagen-I nanofiber scaffolds. a-b SEM images of parallel-orientated PCL-collagen-I nanofiber scaffold cell attachments. c-d Parallel orientation of MSCs (green) on PCL-collagen-I nanofibers. e Positive staining for desmin (red) of MSC-myoblast co-cultures on PCL-collagen scaffolds. Nuclei were counterstained with DAPI (blue). Scale bars represent 200 and 100 μm. Magnifications 40x (c, e) and 100x (d)