Elsevier

Experimental Cell Research

Volume 300, Issue 2, 1 November 2004, Pages 418-426
Experimental Cell Research

Signals from damaged but not undamaged skeletal muscle induce myogenic differentiation of rat bone-marrow-derived mesenchymal stem cells

https://doi.org/10.1016/j.yexcr.2004.07.017Get rights and content

Abstract

The regenerative capacity of skeletal muscle has been usually attributed to resident satellite cells, which, upon activation by local or distant stimuli, initiate a myogenic differentiation program. Although recent studies have revealed that bone-marrow-derived progenitor cells may also participate in regenerative myogenesis, the signals and mechanisms involved in this process have not been elucidated. This study was designed to investigate whether signals from injured rat skeletal muscle were competent to induce a program of myogenic differentiation in expanded cultures of rat bone-marrow-derived mesenchymal stem cells (MSC). We observed that the incubation of MSC with a conditioned medium prepared from chemically damaged but not undamaged muscle resulted in a time-dependent change from fibroblast-like into elongated multinucleated cells, a transient increase in the number of MyoD positive cells, and the subsequent onset of myogenin, α-actinin, and myosin heavy chain expression. These results show that damaged rat skeletal muscle is endowed with the capacity to induce myogenic differentiation of bone-marrow-derived mesenchymal progenitors.

Introduction

In the adult organism, skeletal muscle has the ability to repair and regenerate itself after injury. This property is ascribed to satellite cells, a subpopulation of undifferentiated mononuclear cells that surround muscle fibers. Most satellite cells are mitotically quiescent and are activated to re-enter the cell cycle, proliferate, undergo terminal differentiation, and fuse into muscle fibers in response to environmental cues such as injury [1], [2], [3]. In addition to satellite cells, other progenitor cells known to be present in skeletal muscle may also be involved in muscle regeneration [4], [5]. Recent data has shown the presence of a side population (SP) of progenitors replenished by bone marrow stem cells in skeletal muscle [6]. In addition, a non-SP population of stem cells with a mesenchymal phenotype has been reported to be present in the adult skeletal muscle environment [6], [7], [8]. Bone-marrow-derived mesenchymal stem cells (MSC) have the potential to differentiate into various mesenchymal lineages including bone, cartilage, adipose, stroma, and muscle in response to nonphysiological stimuli [9], [10], [11], [12]. Furthermore, in vivo studies have shown that bone-marrow-derived progenitors differentiate into myofibers [13], [14], [15], and may also contribute to the replenishment of the satellite cell compartment and thus to muscle regeneration [15], [16].

Progenitor cells that contribute to muscle repair appear to enter a program that is analogous to that displayed during the embryonic development of skeletal muscle [17], [18]. At a molecular level, myogenesis is controlled by a family of myogenic regulatory factors (MRFs), which includes MyoD, Myf5, myogenin, and MRF4 that are expressed with a well-defined time course [18], [19], [20].

Muscle repair and functional restoration from progenitor cells require a suitable environment to promote the process of myogenesis, which comprises cell commitment, proliferation, fusion, and terminal maturation. It has been proposed that factors released from injured muscle provide the signals that contribute to the establishment of a favorable microenvironment to initiate the regeneration process [2], [21], [22]. However, it is not known whether local signals released after damage are specific for satellite cells or whether they also promote the myogenic commitment and differentiation of MSC. The studies reported here were performed to investigate if experimentally damaged muscle in vivo releases factor(s) capable of inducing the myogenic differentiation of bone marrow MSC in vitro.

Section snippets

MSC isolation and culture

Bone marrow was harvested from femurs and tibias of 5-week-old male Wistar rats [11]. Briefly, marrow plugs resuspended in α-minimum essential medium (MEM) were desegregated, centrifuged, and the resulting cell pellet was resuspended in α-MEM containing 10% fetal bovine serum (FBS, Gibco Invitrogen) (complete medium). Nucleated cells (5 × 107) were seeded into 100-mm culture dishes and incubated at 37°C for 3 days. Then, nonadherent cells were eliminated and the monolayer of adherent cells was

Differentiation potential of the rMSC

In complete culture medium (10% FBS), rMSC grow as a morphologically homogenous population of tightly adherent fibroblast-like cells (Fig. 1A), with a population doubling time of approximately 27 h and exhibiting contact inhibition of cell growth after reaching confluence (Fig. 2A). When rMSC are incubated in culture medium containing 0.5% FBS, cells do not proliferate, but remain viable and adherent for at least 10 days (Fig. 2A).

The capability of rMSC to differentiate into the myogenic

Discussion

In addition to satellite cells, bone-marrow-derived progenitors have been implicated in skeletal muscle regeneration [4], [13], [14]. Recent data has shown that the commitment and differentiation of bone-marrow-derived progenitors into multinucleated muscle fiber increases after muscular stress or injury [15], [16]. Thus, it seems that signals released or produced by an injured muscle not only trigger the myogenic differentiation of satellite cells [21], [22], [27], but bone-marrow-derived stem

Acknowledgments

We thank Dr. Enrique Jaimovich (Universidad de Chile, Santiago, Chile) for the generous donation of antibodies, and Dr. Miguel Allende for critical reading of the manuscript. We also acknowledge Marisol Blanco for help with RT-PCR studies. The antibody MF-20 developed by D.A. Fischmann was obtained from the Developmental Studies Hybridoma Bank developed under Department of Biological Sciences, Iowa City, IA 52242. This work was supported by grant FONDECYT Chile No. 1010566 and by grant PG/58/02

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