MBP-FGF2-Immobilized Matrix Maintains Self-Renewal and Myogenic Differentiation Potential of Skeletal Muscle Stem Cells

Abstract

The robust capacity of skeletal muscle stem cells (SkMSCs, or satellite cells) to regenerate into new muscles in vivo has offered promising therapeutic options for the treatment of degenerative muscle diseases. However, the practical use of SkMSCs to treat muscle diseases is limited, owing to their inability to expand in vitro under defined cultivation conditions without loss of engraftment efficiency. To develop an optimal cultivation condition for SkMSCs, we investigated the behavior of SkMSCs on synthetic maltose-binding protein (MBP)-fibroblast growth factor 2 (FGF2)-immobilized matrix in vitro. We found that the chemically well-defined, xeno-free MBP-FGF2-immobilized matrix effectively supports SkMSC growth without reducing their differentiation potential in vitro. Our data highlights the possible application of the MBP-FGF2 matrix for SkMSC expansion in vitro.

Keywords: MBP-FGF2; Myogenic differentiation; Satellite cell; Self-renewal; Skeletal muscle stem cell.

Fig. 1

MBP-FGF2-immobilized matrix can support the activation of SkMSCs. (A) Immunofluorescence staining images for the expression of myogenic markers, MyoD and myogenin (MYOG), in quiescent SkMSCs grown on Matrigel or MBP-FGF2 matrix for 48 h in growth medium. Cell nuclei were counterstained with DAPI. Scale bar, 50 μm. (B) Quantitation of MyoD- and MYOG-positive cells. Marker-positive cells are presented as the percentage of total cell number from triplicate samples. Error bars indicate standard error of the mean (SEM). NS, no statistical significance.

Fig. 2

SkMSCs competently proliferate on the MBP-FGF2-immobilized matrix. (A) Phase-contrast microscopic images of proliferating SkMSCs cultured on Matrigel or MBP-FGF2 matrix-coated surface with or without FGF2 supplementation in the growth medium for 4 days. Scale bar, 50 μm. (B, C) Determination of live SkMSC density with a modified MTS assay. The MTS assay was performed daily during the time course of 4 days. SkMSCs were cultured on BSA-, MBP-FGF2-, or Matrigel-coated 96-well plates in the absence (B) or presence (C) of soluble FGF2 (5 ng/mL) as indicated. Viable cell density was determined from the optical density (O.D.) at 450 nm wavelength. Experiments were performed in triplicates. Data represent normalized mean values with SEM.

Fig. 3

SkMSCs maintain robust myogenic potential on MBP-FGF2-mmobilized matrix. (A) Immunofluorescence staining images for MyHC expression in the SkMSCs cultured on Matrigel- or MBP-FGF2 (50 μg/mL)-coated surfaces at differentiation day 3. Cell nuclei were counterstained with DAPI. Scale bar, 50 μm. (B) To calculate differentiation indices after 3 days, MyHC-positive cell nuclei were counted and expressed as the percentage of total cell nuclei. (C) Fusion indices were shown as a distribution of MyHC-positive cells based on cell nucleus number; mono, 2~5, and more than 5 nuclei. Experiments were performed in triplicates. Error bars indicate SEM. NS, no statistical significance.

Fig. 4

Focal adhesion kinase expression is detected in the SkMSCs cultured on the MBP-FGF2-immobilized matrix. Confocal microscopic images of immunofluorescence staining of vinculin (red) and F-actin (green). SkMSCs were cultured on MBP-GF2 matrix (50 μg/mL) and Matrigel for 48 h in growth medium. White arrows indicate the cytoplasmic area overlapped between vinculin and F-actin expression. Scale bar, 10 μm.