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

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.

SUBMITTER: Kasprzycka P

PROVIDER: S-EPMC6873517 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Kasprzycka Paulina P Archacka Karolina K Kowalski Kamil K Mierzejewski Bartosz B Zimowska Małgorzata M Grabowska Iwona I Piotrowski Mariusz M Rafałko Milena M Ryżko Agata A Irhashava Aliksandra A Senderowski Kamil K Gołąbek Magdalena M Stremińska Władysława W Jańczyk-Ilach Katarzyna K Koblowska Marta M Iwanicka-Nowicka Roksana R Fogtman Anna A Janowski Mirosław M Walczak Piotr P Ciemerych Maria A MA Brzoska Edyta E

Stem cell research & therapy 20191121 1

<h4>Background</h4>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/ ...[more]

PMID: 31753006

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Project description:Ruxolitinib is a JAK2/JAK1 inhibitor that blocks the inflammatory JAK-STAT signaling pathway. Ruxolitinib has been demonstrated to be effective in the treatment of steroid-resistant acute graft-versus-host disease (GVHD). Ruxolitinib's effect on inflammatory cells of hematopoietic origin is known. However, its effect on nonhematopoietic cell types with immune-modulating and antigen-presenting cell competency plausibly involved in pathogenesis of GVHD has not been explored. Mesenchymal stromal cells (MSCs) are CD45- nonhematopoietic cells of the bone marrow with immune modulatory functions in vivo. MSCs' immunobiology largely depends on their responsiveness to IFNγ. We aimed to define the effect of ruxolitinib on the immunobiology of MSCs that are modulated by IFNγ. Human bone marrow derived MSCs, peripheral blood mononuclear cells (PBMCs), and primary bone marrow aspirates were analyzed for their sensitivity to ruxolitinib-mediated blocking of IFNγ-induced STAT-1 phosphorylation and downstream effector molecules, utilizing Western blot, flow cytometry, secretome analysis, and phosflow techniques. IFNγ-induced cytostatic effects on MSCs are reversed by ruxolitinib. Ruxolitinib inhibits IFNγ and secretome of activated peripheral PBMC-induced STAT-1 phosphorylation on human bone marrow derived MSCs. In addition, ruxolitinib inhibits IFNγ-induced pro-GVHD pathways on MSCs, which includes HLAABC(MHCI), HLADR(MHCII), CX3CL1, and CCL2. IFNγ-induced immunosuppressive molecules IDO and PDL-1 were also inhibited by ruxolitinib on MSCs. Comparative analysis with PBMCs has demonstrated that MSCs are as equal as to HLADR+ PBMC populations in responding to ruxolitinib-mediated inhibition of IFNγ-induced STAT-1 phosphorylation. Ex vivo analysis of human marrow aspirates has demonstrated that ruxolitinib blocks IFNγ-induced STAT-1 phosphorylation in CD45+/-HLADR+/- populations at different levels, which is depending on their sensitivity to IFNγ responsiveness. These results inform the hypothesis that ruxolitinib's immune-modulatory effects in vivo may pharmacologically involve marrow and tissue-resident MSCs. Ruxolitinib affects the immunobiology of MSCs equivalent to professional HLADR+ antigen presenting cells, which collectively mitigate GVHD.

Project description:Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of ?-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unit-fibroblasts. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a 16-week follow-up period. We found increased vascular endothelial growth factor (VEGF) levels and a decrease of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward ?-irradiation showed positive ?-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-16 weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p16) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose ?-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose ?-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.

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

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

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