Research Article: Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

Date Published: January 1, 2010

Publisher: Public Library of Science

Author(s): Carlo A. Rossi, Michela Pozzobon, Andrea Ditadi, Karolina Archacka, Annalisa Gastaldello, Marta Sanna, Chiara Franzin, Alberto Malerba, Gabriella Milan, Mara Cananzi, Stefano Schiaffino, Michelangelo Campanella, Roberto Vettor, Paolo De Coppi, Alejandro Lucia. http://doi.org/10.1371/journal.pone.0008523

Abstract: Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

Partial Text: Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle [1]. They were originally characterized on the basis of their unique position in mature skeletal muscle: closely juxtaposed to the surface of myofibers such that the basal lamina surrounding the SCs and its associated myofiber is a continuous [2], [3]. SCs are mitotically quiescent and activated in response to diverse stimuli, including stretching, injury and electrical stimulation [4], [5], [6]. The descendants of activated SCs, called myogenic precursor cells (MPCs), undergo multiple rounds of cell division before fusing with new or existing myofibers. Although the total number of quiescent SCs decreases with age [7], it remains constant over repeated cycles of degeneration and regeneration thus indicating that the steady-state SCs population is maintained by self-renewal [8], [9], [10], [11]. Therefore, SCs fulfill the criteria of adult stem cells and are distinct from MPCs as underlined by biological and biochemical criteria [2], [12]. Initially, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype [2], whilst subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible [13]. Recently, it was also shown that both human and porcine SCs can differentiate under appropriate stimuli into mature adipocytes [14], [15]. However, even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is most likely that SCs are not all multipotent stem cells [16]. Thus, evidences for diversities within the myogenic compartment have been described both in vitro and in vivo[17], [18]. Alternative sensitivity to high-dose irradiation revealed that at least two populations of SCs are present [19]: they are distinguishable by proliferative and myogenic capacities [20] with a proportion that varies according to the age [21]. Similarly, after bupivacain injection, two SCs subpopulations get activated: committed myogenic precursors and “stem” satellite cells [22], [23], [24]. Intrinsic heterogeneity was indeed evident when the activating sequence of myogenic regulatory factors (MRFs) was exploited [24]. Among others, Myf5 expression has led to the existence of hierarchical subpopulations of SCs [16;17;25]. In particular, SCs have been shown to be composed of about 10% stem cells (Pax7+/Myf5−) and 90% committed myogenic progenitors (Pax7+/Myf5+) [16]. More recently, variation in the expression of various non-specific myogenic markers such as nestin [26], CXCR-4 and b1-integrin [27], and ABCG2 and Syndecan-4 [28] have also been described. Despite the evident heterogeneity, the phenotypical characteristics of these subpopulations were hard to elucidate because their behavior in vitro has been difficult to investigate. Using a new experimental maneuver that permits clear and correct isolation of SCs from the fiber of origin, we report, for the first time, that two subpopulations of SCs coexist in fixed proportions on the single fiber: the low proliferative (LPC) and the high proliferative clones (HPC) which show alternative myogenic potential in vitro retained also in vivo. Intriguingly, although the HPC give spontaneously rise to adipocytes their myogenic potential can be boosted if co-cultured with LPC. Besides assessing the regenerative and proliferative potentials of SCs, we also investigated functional cellular markers attributable to mitochondrial function. Thus, we exploited the mitochondrial membrane potential (ΔΨm) [29], the pathways of ATP production and the rate of Reactive Oxygen Species (ROS) generation discovering that LPC and HPC remarkably differ in every of these parameters accounting for differences in basal cell signaling and metabolism. In this way, we do not just provide experimental evidences for different populations of SCs but also indications for readouts that may lead to applied studies of regenerative medicine.

SCs and their heterogeneity have been widely investigated and various reports already described differences in their proliferative and myogenic potentials [20], [21], [22], [23]. Here, we confirmed such differences and showed for the first time that in rat myofibers there is a correlation between SCs proliferation and differentiation potential similarly to that previously reported for other stem cells. Using an innovative technique for single muscle fiber culture in suspension, adapted from embryoid-bodies cultures [30] we observed that not only SCs proliferate at different rates [52] but also that the ratio between clones with high (HPC) and low (LPC) proliferative rate was fixed at single fiber level. Moreover, after mechanical dissociation and cloning through limiting dilution, it was still possible to distinguish an identical ratio of LPC and HPC. Notably, differences in proliferation mirror differences in functional parameters such as mitochondrial membrane potential (ΔΨm), ATP balance and ROS generation. HPC compared to the LPC proved to have a more glycolytic phenotype characterized by increased ΔΨm, reduced mitochondrial generation of ATP and higher rate of ROS production. Abnormal rising of the ΔΨm at resting conditions defines impairments in the H+ transport through the Electron Respiratory Chain (ERC) thus acting as a parameter to assess mitochondrial activity [34]. Since this is normalized via pharmacological inhibition of the terminal enzyme of the ERC (the F1Fo-ATPsynthase) (see Fig. 2b), the oxidative phosphorylation of the HPC can be considered underperforming compared to that of the LPC. Therefore, the mitochondrial ATP generation is also reduced and addition of oligomycin able to promote a mild reduction of intracellular ATP in HPC compared to LPC (Fig. 2c). Alternative efficiency of the mitochondrial coupling was not the only distinctive feature between the two populations since HPC presented also a remarkable higher level of ROS production compared to LPC. ROS is a signal associated to cell differentiation into adipocytes [40] and HPC which spontaneously differentiate into adipocytes (Fig. 3b) show plausibly for this reason an increased rate of basal ROS production.

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http://doi.org/10.1371/journal.pone.0008523

 

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