Schwann Cells Promote Myogenic Differentiation of Myoblasts and Adipogenic Mesenchymal Stromal Cells on Poly-ɛ-Caprolactone-Collagen I-Nanofibers

Abstract

For the purpose of skeletal muscle tissue engineering, different cell types have been investigated regarding their myogenic differentiation potential, including co-cultured myoblasts and adipogenic mesenchymal stromal cells (Mb/ADSC). As neural cells enhance synaptic junction formation, the aim of this study was to co-culture Schwann cells (SCs) with Mb/ADSC on biocompatible electrospun aligned poly-ε-polycaprolacton (PCL)-collagen I-nanofibers. It was hypothesized that SCs, as part of the peripheral nervous system, promote the myogenic differentiation of Mb/ADSC co-cultures. Mb/ADSC were compared to Mb/ADSC/SC regarding their capacity for myogenic differentiation via immunofluorescent staining and gene expression of myogenic markers. Mb/ADSC/SC showed more myotubes after 28 days of differentiation (p ≤ 0.05). After 28 days of differentiation on electrospun aligned PCL-collagen I-nanofibers, gene expression of myosin heavy chains (MYH2) and myogenin (MYOG) was upregulated in Mb/ADSC/SC compared to Mb/ADSC (p ≤ 0.01 and p ≤ 0.05, respectively). Immunofluorescent staining for MHC showed highly aligned multinucleated cells as possible myotube formation in Mb/ADSC/SC. In conclusion, SCs promote myogenic differentiation of Mb/ADSC. The co-culture of primary Mb/ADSC/SC on PCL-collagen I-nanofibers serves as a physiological model for skeletal muscle tissue engineering, applicable to future clinical applications.

Keywords: ADSC; Schwann cells; mesenchymal stem cells; myoblasts; myogenic differentiation; nanofibers.

Figure 1

(A) Fluorescence microscopy of desmin-positive (red) human primary myoblasts (Mb) in pre-plate 3 (PP3) and passage 4 (P4). Merge with DAPI (blue) shows that nearly all cells were desmin-positive. Insert shows human primary fibroblasts in P9 negative for desmin. Scale bar 100 µm applies to insert as well as to main image. (B) Fluorescence microscopy of Mbs in PP3 and P5 after seven days of myogenic differentiation. Merge with DAPI (blue) shows that cells were desmin-positive (red) and formed multinucleated cells, indicating myotube formation. Scale bar 50 µm.

Figure 2

Characterization of human adipose derived stem cells (ADSC). ADSCs in P4 were differentiated into osteocytes (A), adipocytes (B), and chondrocytes (C). Calcium deposits were stained with Alizarin Red S (A). Lipid vacuoles were visualized with oil red O staining (B). Proteoglycans of chondrogenic pellets were detected with Alcian blue staining (C). Inserts represent ADSC, cultured in proliferation medium as negative controls. Scale bars 200 µm (A,B) and 100 µm (C) refer to inserts as well as to main images.

Figure 3

Fluorescence microscopy of human Schwann cells (SC) in P5. Cells were positive for glial fibrillary acidic protein (GFAP) (red) (A), S100 (green) (B), and p75 (red) (C). Inserts represent human Mbs (blue = DAPI) as negative controls. Scale bars 100 µm apply to inserts as well as to main images.

Figure 4

Optimal ratio of Mb/ADSC/SC determined with creatine kinase (CK) activity. Values are presented in mean ± standard deviation. CK activity increased over time within 1:1:1 (# p < 0.05, repeated measures ANOVA with Tukey’s correction for multiple comparisons) and 1:1:0.5 (## p < 0.01, repeated measures ANOVA with Tukey’s correction for multiple comparisons). After 14 days, CK activity was lower in 1:1:0.25 groups compared to 1:1:0.5 groups (** p < 0.01, one way ANOVA with Tukey’s correction for multiple comparisons). Based on those results, Mb/ADSC/SC were used at a ratio of 1:1:0.5 for further experiments.

Figure 5

Myotube formation after 2D myogenic differentiation of Mb/ADSC vs. Mb/ADSC/SC. Values are presented in mean ± standard deviation. (A) Fluorescence microscopy of 2D co-cultures shows myosine heavy chain (MHC) positive (red) multinucleated (DAPI = blue) cells after 7, 14, and 28 days of myogenic differentiation. Scale bar 100 µm. (B) Myotube fusion index (MFI = nuclei in myotubes/total nuclei) was higher in Mb/ADSC/SC after 28 days (* p < 0.05, unpaired t-test). Myotube maturation index (MMI = myotubes with five or more nuclei/total myotubes) did not differ between Mb/ADSC and Mb/ADSC/SC during all time periods.

Figure 6

SEM image of electrospun aligned PCL-collagen I-nanofibers. Scale bar 2 µm.

Figure 7

Cell viability on PCL-collagen I-nanofibers determined with wst-8-assay. Absorbance at a wave length of 450 nm is expressed as mean ± standard deviation. Cell viability decreased from 14 to 28 days in Mb/ADSC (# p < 0.05, repeated measures ANOVA with Tukey‘s multiple comparison test).

Figure 8

Cell status on PCL-collagen I-nanofibers determined with live dead assay. (A) After 0, 14, and 28 days of myogenic differentiation, live cells are demonstrated in blue, apoptotic cells in green, and necrotic cells in red. Scale bar 100 µm. (B) Apoptotic index (AI) and necrotic index (NI) did not differ between Mb/ADSC and Mb/ADSC/SC (unpaired t-test). AI decreased from 0 days to 14 days (# p < 0.05, repeated measures ANOVA with Tukey’s correction for multiple comparisons) and increased from 14 days to 28 days (# p < 0.05, repeated measures ANOVA with Tukey’s correction for multiple comparisons) for Mb/ADSC/SC.

Figure 9

Gene expression of ACTA1 (A), MYH 2 (B), and MYOG (C) in Mb/ADSC vs. Mb/ADSC/SC after 28 days of myogenic differentiation on PCL-collagen I-nanoscaffolds. Expressions are demonstrated in x-fold difference compared with RNA from human muscle tissue using the 2^−ΔΔCt method. GAPDH was used as housekeeping gene in co-cultures and muscle tissue. MYOG and MYH2 were upregulated in Mb/ADSC/SC (69.97-fold ± 33.09-fold and 4.169-fold ± 0.756-fold, respectively) compared to Mb/ADSC (8.404-fold ± 4.8-fold and 1.038-fold ± 0.396-fold, respectively). * p < 0.05 (unpaired t-test), ** p < 0.001 (unpaired t-test).

Figure 10

Myogenic differentiation and cell configuration after 28 days of myogenic differentiation of Mb/ADSC (A) and Mb/ADSC/SC (B) on PCL-collagen I-nanofibers. Fluorescence microcopy shows MHC positive (red) cells with clear evidence of highly aligned multinucleated (DAPI = blue) cells as possible myotube formation (white arrows point to nuclei in myotubes) in Mb/ADSC/SC. Scale bar 100 µm.