Osteogenic Differentiation Capacity of Human Skeletal Muscle-Derived Progenitor Cells

<div><p>Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56⁺ and PDGFRα⁺ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56⁺ cells and PDGFRα⁺ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα⁺ cells formed bone-like tissue and showed successful engraftment, while CD56⁺ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα⁺ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα⁺ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα⁺ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα⁺ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα⁺ cells. Our results suggest that PDGFRα⁺ cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα⁺ cells.</p> </div

Immunohistological analysis of human HO sample.

<p>Human HO sample was subjected to immunofluorescent staining for PDGFRα and subsequently to H-E staining. (A) Image of H-E staining. Scale bar: 100 µm. Right panel shows high magnification image of square region in the left panel. (B) Image of PDGFRα staining. Arrows indicate PDGFRα⁺ cells surrounding ectopic bone tissue. Scale bar: 10 µm.</p

Changes in miRNA during osteogenic differentiation of PDGFRα⁺ cells.

<p>At the different time points during osteogenic differentiation of PDGFRα⁺ cells, the expressions of miRNAs indicated were quantified by qRT-PCR. Values are represented as the ratio to uninduced cells and shown as means ± s.d. of three independent preparations.</p

Identification of PDGFRα⁺ cells and CD56⁺ cells in human skeletal muscle.

<p>(A) Human skeletal muscle sections were stained with antibodies against PDGFRα (green), CD56 (red), and laminin (purple), and counterstained with DAPI (blue). Subsequently, specimens were subjected to H-E staining. Arrows indicate PDGFRα⁺ cells located in interstitial spaces of muscle tissue. Arrowheads indicate CD56⁺ cells residing beneath the basement membrane of myofibers. Scale bar: 10 µm. (B) Human muscle-derived cells were analyzed for the expression of PDGFRα and CD56 by FACS.</p

Inhibition of miRNAs during osteogenic differentiation of PDGFRα⁺ cells.

<p>PDGFRα⁺ cells were transfected with miRNA inhibitor and osteogenic differentiation was induced. PDGFRα⁺ cells transfected with control inhibitor were used as a control. Experiments were performed using cells from three independent preparations and we obtained consistent results in each experiment. Data from one representative experiment were shown. (A) Transfected fluorescein-labeled miRNA inhibitor was observed at the time points indicated. Scale bar: 50 µm. (B) Alkaline phosphatase staining was performed at the time points indicated. (C) Alizarin red S staining was performed at the time points indicated.</p

Osteogenic differentiation potential of human skeletal muscle-derived cells <i>in vitro</i>.

<p>(A) Alkaline phosphatase staining was performed at the time points indicated during osteogenic differentiation of PDGFRα⁺ cells and CD56⁺ cells. (B) Alizarin red S staining was performed at the time points indicated during osteogenic differentiation of PDGFRα⁺ cells and CD56⁺ cells. (C) Alkaline phosphatase activity of PDGFRα⁺ cells and CD56⁺ cells during osteogenic differentiation was quantified. Values are shown as means ± s.d. of ten independent preparations. *P<0.01.</p

Bioinformatics analysis of miRNA targets.

<p>Bioinformatics analysis of miRNA targets.</p

Bone-forming capacity of human skeletal muscle-derived cells <i>in vivo</i>.

<p>(A) PLGA-hydroxyapatite scaffolds containing PDGFRα⁺ cells or CD56⁺ cells were subcutaneously transplanted into immunodeficient mice. Scaffolds containing no cell were used as a control. After 8 wks, implants were removed and subjected to H-E staining. Scale bar: 50 µm. (B) Implants were stained with human lamin A/C specific antibody. Scale bar: 20 µm. (C) High magnification of PDGFRα⁺-cell transplant. Scale bar: 10 µm. (D) Human lamin-positive nuclei were counted. Number of positive cells is represented as means ± s.d., n = 15 fields from three independent implants. *P<0.01.</p

Changes in miRNAs during osteogenic differentiation of PDGFRα⁺ cells revealed by microarray analysis.

<p>Values are shown as the relative expression to uninduced cells. Day 7 and Day 14: 7 days and 14 days after osteogenic induction.</p