Biomed Res Int. 2014;2014:129048. doi: 10.1155/2014/129048.

Adipose tissue-derived stem cell secreted IGF-1 protects myoblasts from the negative effect of myostatin.

Gehmert S1, Wenzel C2, Loibl M3, Brockhoff G4, Huber M5, Krutsch W5, Nerlich M5, Gosau M6, Klein S7, Schreml S8, Prantl L7, Gehmert S9.
  • 1Applied Stem Cell Research Center, University Medical Center Regensburg, 93053 Regensburg, Germany ; Department of Trauma Surgery, Center of Plastic and Hand Surgery, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 2Applied Stem Cell Research Center, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 3Applied Stem Cell Research Center, University Medical Center Regensburg, 93053 Regensburg, Germany ; Department of Trauma Surgery, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 4Department of Obstetrics and Gynecology, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 5Department of Trauma Surgery, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 6Department of Cranio-Maxillofacial Surgery, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 7Department of Trauma Surgery, Center of Plastic and Hand Surgery, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 8Department of Dermatology, University Medical Center Regensburg, 93053 Regensburg, Germany.
  • 9Applied Stem Cell Research Center, University Medical Center Regensburg, 93053 Regensburg, Germany ; Department of Obstetrics and Gynecology, University Medical Center Regensburg, 93053 Regensburg, Germany.

 

Abstract

Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

PMID: 24575400

 

Supplement:

Duchenne Muscular Dystrophy is one of the most common single gene disorders found in the developed world. Glucocorticoids are currently the best treatment option available to patients suffering from DMD. However, the exact way how glucocorticoids work is not completely understood and requires future research. TGF-β protein has been shown to be elavated in dystrophic muscles. Particularly myostatin, a TGF-β family member, acts as a negative regulator with regard to muscle growth and differentiation. Thus, it is hypothesized that myostatin plays a destructive role in the degeneration of dystrophic muscles by interaction with the IGF-1 signaling pathway. In addition, existing evidence indicates that administered IGF-1 is able to ameliorate the inhibitory effect of myostatin on muscle growth. Since the differentiation and fusion potential as well as the paracrine action of adipose tissue derived mesenchymal stem cells (ASCs) were shown in various studies, the therapeutic possibility for Duchenne Muscular Dystrophy has emerged.

 

SG fig1

Figure 1: Healthy ASCs and DMD myoblasts were co-cultured and subjected to fusion media that provoke myoblasts to form multinucleated structures. After 4 days, multinucleated myotubes were observed with DAPI and GFP-labeled nuclei as well with red cell membranes. ASCs culture subjected to the same fusion media did not contain any myotubes. This result clearly shows that healthy ASCs contribute to the generation of myotube formation when co-cultured with myoblasts lacking the dystrophin expression. Scale bar, 50 μm

 

 

However, it is unclear if ASCs promote proliferation and differentiation and prevent apoptosis in myoblasts during myostatin exposure by secretion of cytokines, particularly IGF-1.

Based on our own data and research expertise with ASCs we hypothesized that paracrine factors released by ASCs, especially IGF-1, have a positive effect on proliferation, viability, apoptosis and differentiation of murine myoblasts under myostatin influence.

The present results show that IGF-1 secreted by stem cells improves the proliferation and viability of myostatin-treated myoblasts. Blocking IGF-1 in CM of wildtype ASCs and IGF-1 receptor of C2C12 cells using polyclonal neutralization antibodies resulted in significant less proliferation. Co-culture experiments with wildtype ASCs and ASCs silenced for IGF-1 showed a significant decline of myoblasts’ viability. Furthermore, the IGF-1 impact on myostatin-exposed myoblasts was demonstrated by restoring the protein level of the muscle differentiation marker MyoD and higher levels in MyoD mRNA expression.

The current work extends previous observations in cardiac myoblasts [1] and indicates that IGF-1 secreted by ASCs improves the catabolic effect of myostatin on myoblasts.

A recent study confirms the apoptotic effect of myostatin, revealing the anti-apoptotic role of IGF-1 in myostatin-treated murine myoblasts. These results are in line with similar findings describing IGF-1 in a conditioned medium of ASCs as an apoptosis protection factor for cerebellar granule neurons under serum and K+-deprivation [2].

The present study underlines the previously described results of stem cell benefits without fusion or transdifferentiation. The fact that ASCs are easy to harvest and to expand makes these stem cells a convincing therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases. However, further studies are necessary to investigate the long-term benefit.

 

SG fig2

Figure 2: Differentiation of DAPI stained DMD myblasts into myotubes was induced by fusion medium and revealed the lack of dystrophin expression by immunofluorescence (top row). ASCs kept in fusion medium did not show myotube formation as well as no dystrophin expression (middle row). Co-culture of DMD myoblast and ASCs showed myotube formation and the expression of dystrophin (bottom row). This indicates that ASCs are able to restore missing dystrophin expression of DMD myoblasts. Scale bar, 50 μm.

 

References:

  1. Sadat S, Gehmert S, Song Y-H, et al. The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF. BIOCHEM. BIOPHYS. RES. COMMUN. 2007;363(3):674–679
  2. Wei X, Zhao L, Zhong J, et al. Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis. NEUROSCI. LETT. 2009;462(1):76–79.

 

Acknowledgments

This work was supported by the German Research Foun- dation (DFG) within the funding program Open Access Publishing. The project was funded by the University Hospital Regensburg within the ReForM-A-program (Sebastian Gehmert).

 

SG fig3Contact:

Sebastian Gehmert, M.D.

Orthopaedic Department, University of Basel, Spitalstrasse 21, 4031 Basel, Switzerland.

s.gehmert@gmail.com

 

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