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. 2019 Nov 21;10(1):343.
doi: 10.1186/s13287-019-1444-1.

The factors present in regenerating muscles impact bone marrow-derived mesenchymal stromal/stem cell fusion with myoblasts

Paulina Kasprzycka  1 , Karolina Archacka  1 , Kamil Kowalski  1 , Bartosz Mierzejewski  1 , Małgorzata Zimowska  1 , Iwona Grabowska  1 , Mariusz Piotrowski  1 , Milena Rafałko  1 , Agata Ryżko  1 , Aliksandra Irhashava  1 , Kamil Senderowski  1 , Magdalena Gołąbek  1 , Władysława Stremińska  1 , Katarzyna Jańczyk-Ilach  1 , Marta Koblowska  2 , Roksana Iwanicka-Nowicka  2   3 , Anna Fogtman  3 , Mirosław Janowski  4   5   6 , Piotr Walczak  4   7   8 , Maria A Ciemerych  1 , Edyta Brzoska  9
Affiliations

The factors present in regenerating muscles impact bone marrow-derived mesenchymal stromal/stem cell fusion with myoblasts

Paulina Kasprzycka et al. Stem Cell Res Ther. .

Abstract

Background: Satellite cells, a population of unipotent stem cells attached to muscle fibers, determine the excellent regenerative capability of injured skeletal muscles. Myogenic potential is also exhibited by other cell populations, which exist in the skeletal muscles or come from other niches. Mesenchymal stromal/stem cells inhabiting the bone marrow do not spontaneously differentiate into muscle cells, but there is some evidence that they are capable to follow the myogenic program and/or fuse with myoblasts.

Methods: In the present study we analyzed whether IGF-1, IL-4, IL-6, and SDF-1 could impact human and porcine bone marrow-derived mesenchymal stromal/stem cells (hBM-MSCs and pBM-MSCs) and induce expression of myogenic regulatory factors, skeletal muscle-specific structural, and adhesion proteins. Moreover, we investigated whether these factors could induce both types of BM-MSCs to fuse with myoblasts. IGF-1, IL-4, IL-6, and SDF-1 were selected on the basis of their role in embryonic myogenesis as well as skeletal muscle regeneration.

Results: We found that hBM-MSCs and pBM-MSCs cultured in vitro in the presence of IGF-1, IL-4, IL-6, or SDF-1 did not upregulate myogenic regulatory factors. Consequently, we confirmed the lack of their naïve myogenic potential. However, we noticed that IL-4 and IL-6 impacted proliferation and IL-4, IL-6, and SDF-1 improved migration of hBM-MSCs. IL-4 treatment resulted in the significant increase in the level of mRNA encoding CD9, NCAM, VCAM, and m-cadherin, i.e., proteins engaged in cell fusion during myotube formation. Additionally, the CD9 expression level was also driven by IGF-1 treatment. Furthermore, the pre-treatment of hBM-MSCs either with IGF-1, IL-4, or SDF-1 and treatment of pBM-MSCs either with IGF-1 or IL-4 increased the efficacy of hybrid myotube formation between these cells and C2C12 myoblasts.

Conclusions: To conclude, our study revealed that treatment with IGF-1, IL-4, IL-6, or SDF-1 affects BM-MSC interaction with myoblasts; however, it does not directly promote myogenic differentiation of these cells.

Keywords: BM-MSC; Fusion; IGF-1; IL-4; IL-6; Myogenic differentiation; SDF-1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Expression of IGF-1, IL-4, IL-6, and SDF-1 and their receptors in human BM-MSCs (hBM-MSCs) non-treated (NT) and treated with either IGF-1, IL-4, IL-6, or SDF-1 cultured in the proliferating medium (PM) or differentiating medium (DM) - qRT-PCR analysis. a The experimental design. b Expression of mesenchymal stromal/stem cell markers in non-treated (NT) hBM-MSCs cultured in PM or DM for 7 days (n = 3). c Expression of cytokines and their receptors in hBM-MSCs non-treated (NT) or 7 days treated with either IGF-1 or IL-4 or IL-6 or SDF-1 cultured in PM (n = 3). d Expression of cytokines and their receptors in hBM-MSCs non-treated (NT) or 7 days treated with either IGF-1 or IL-4 or IL-6 or SDF-1 cultured in DM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 2
Fig. 2
Proliferation and migration of human BM-MSCs (hBM-MSCs) treated or pre-treated with IGF-1, IL-4, IL-6, or SDF-1. a The experimental design. b The number of hBM-MSCs non-treated (NT) or treated with either IGF-1, IL-4, IL-6, or SDF-1 and cultured in the proliferating medium (PM) for 7 days (n = 3). c The number of hBM-MSCs non-treated (NT) or treated with either IGF-1, IL-4, IL-6, or SDF-1 and cultured in the differentiating medium (DM) for 7 days (n = 3). d The number of hBM-MSCs pre-treated with either IGF-1, IL-4, IL-6, or SDF-1 for 3 days and cultured in the proliferating medium (PM), analyzed after 1, 3, and 7 days (n = 3). e The migration of hBM-MSCs non-treated (NT) and treated either with IGF-1, IL-4, IL-6, or SDF-1 cultured in the proliferating medium (PM) and analyzed after 6 h and 12 h (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 3
Fig. 3
The IGF-1, IL-4, IL-6, or SDF-1 impact on adhesion and structural protein expression in human BM-MSCs (hBM-MSCs). a The expression of ADAM9, CD9, NCAM, VCAM, CDH15 (m-cahderin), desmin, and MYH3 (muscle embryonic myosin heavy chain 3) in hBM-MSCs non-treated (NT) or treated with either IGF-1, IL-4, IL-6, or SDF-1, cultured for 7 days in the proliferating medium (PM) (n = 3). b The expression of mRNAs encoding ADAM9, CD9, NCAM, VCAM, m-cadherin (CDH15), desmin, and MYH3 in hBM-MSCs non-treated (NT) or treated with either IGF-1, IL-4, IL-6, or SDF-1, cultured for 7 days in the differentiating medium (DM) (n = 3). c The expression of ADAM9, CD9, and CDH15 in hBM-MSCs non-treated (NT) or pre-treated with either IGF-1, IL-4, IL-6, or SDF-1 for 3 days, then cultured in the proliferating medium (PM) and analyzed after 1, 3, and 7 days (n = 3); *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 4
Fig. 4
The IGF-1, IL-4, IL-6, or SDF-1 impact on MRF and structural protein expression in co-cultures of human BM-MSCs (hBM-MSCs) and mouse C2C12 myoblasts. a The experiment design. b The expression of human ADAM9, CD9, CDH15 (m-cadherin), VCAM, and MYH3 (muscle embryonic myosin heavy chain 3) in hBM-MSCs non-treated (NT) and treated either with IGF-1, IL-4, IL-6, or SDF-1 co-cultured with C2C12 for 7 days in the proliferating medium (PM) (n = 3). c The expression of human ADAM9, CD9, CDH15 (m-cadherin), VCAM, and MYH3 (muscle embryonic myosin heavy chain 3) in hBM-MSCs non-treated (NT) or treated with either IGF-1, IL-4, IL-6, or SDF-1 co-cultured with C2C12 myoblasts for 7 days in the differentiating medium (DM) (n = 3). d The expression of mouse Adam9, Myog, and Myh3 mRNA in C2C12 myoblasts non-treated (NT) and treated with either IGF-1, IL-4, IL-6, or SDF-1 co-cultured with hBM-MSCs for 7 days in the proliferating medium (PM) (n = 3). e The expression of mouse Adam9, Myog, and Myh3 mRNA in C2C12 myoblasts non-treated (NT) and treated with either IGF-1, IL-4, IL-6, or SDF-1 co-cultured with hBM-MSCs for 7 days in the differentiating medium (DM) (n = 3). f The expression of human ADAM9, CD9, and CDH15 (m-cadherin) in hBM-MSCs pre-treated for 3 days with either IGF-1, IL-4, IL-6, or SDF-1 and then co-cultured with C2C12 myoblasts in the proliferating medium (PM) for 7 days (n = 3); *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 5
Fig. 5
The fusion of non-treated (NT) and IGF-1-, IL-4-, IL-6-, or SDF-1-treated or pre-treated human and pig BM-MSCs (hBM-MSCs and pBM-MSCs) with C2C12 myoblasts. a The percentage of hybrid myotubes in co-culture of treated hBM-MSCs and C12C12 myoblasts in the proliferating medium (PM) for 7 days (n = 3). b The percentage of hybrid myotubes and the fusion index in co-culture of treated hBM-MSCs and C12C12 myoblasts in the differentiating medium (DM) for 7 days (n = 3). c The percentage of hybrid myotubes and fusion index in co-culture of hBM-MSCs and C2C12 myoblasts pre-treated for 3 days in control (without selected factor supplementation) proliferating medium (PM) for 7 days (n = 3). d The percentage of hybrid myotubes and fusion index in co-culture of non-treated (NT) or treated pBM-MSCs and C2C12 myoblasts in the differentiating medium (DM) after 7 days (n = 3). e hBM-MSC localization in myotubes in co-cultures with C2C12 myoblasts after 7 days of IGF-1, IL-4, IL-6, or SDF-1 treatment in PM and DM. Scale bar 50 μm. Blue—cell nuclei, red—skeletal myosin, green—human cell nuclei. *p < 0.05, **p < 0.01, ***p < 0.005
Fig. 6
Fig. 6
The changes in human BM-MSC (hBM-MSCs) transcriptome after IL-4 treatment. Human BM-MSCs were cultured for 48 h in the presence of IL-4 and then analyzed. a The changes in the level of transcripts between IL-4-treated and non-treated hBM-MSCs. b Gene networks created by interposing the results onto database of Ingenuity containing information about the gene function with the use of Ingenuity Pathway Analysis tool (red—upregulation, green—downregulation)
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