Evidence for a resident subset of cells with SP phenotype in the C2C12 myogenic line: a tool to explore muscle stem cell biology Rachid Benchaouir, a Philippe Rameau, a Charles Decraene, b Patrick Dreyfus, a David Israeli, a Genevie `ve Pie ´tu, b Olivier Danos, a and Luis Garcia a, * a Genethon-Centre National de la Recherche Scientifique, UMR 8115, 91002 Evry cedex, France b CEA/Evry, Service de Ge ´nomique Fonctionnelle, LGRH, 91057 Evry cedex, France Received 26 June 2003, revised version received 6 November 2003 Abstract Muscle satellite cells are heterogeneous and present functional disparities, some of them behaving as multipotent stem cells. Yet their phenotype is obscure and their isolation remains elusive. The ability to purify stem cells from a wide variety of tissues by using Hoechst 33342 staining/FACS methods has permitted access to this category of cells (side population, or SP) in a manner independent of antibodies. Here, we show that the C2C12 myogenic line comprises a minor population of cells with SP phenotype. These cells are growth-arrested and delayed in their ability to differentiate. Dye efflux in C2C12-derived SPs is likely mediated by mdr1a, whose overexpression results in increased dedifferentiation. Interestingly, growth-arrested SPs rapidly appear in purified MP populations, thus suggesting a dynamic equilibrium among different states of differentiation. Finally, transcriptional profiling of C2C12-derived SP and MP cells corroborates the many similarities of SP to stem cells. D 2003 Elsevier Inc. All rights reserved. Keywords: Muscle; C2C12; SP phenotype; mdr1; Stem cell Introduction Until recently, muscle precursors cells were identified in adult skeletal muscle solely by positional criteria since they were thought to reside exclusively beneath the basal lamina surrounding each muscle fiber [1]. These cells, also termed satellite cells and usually mitotically quiescent unless acti- vated, are involved in muscle postnatal growth and repair in response to injuries [2,3]. Their determination and differen- tiation depends on MyoD and Myf-5, two muscle-specific basic helix-loop-helix transcription factors [4]. Whereas MyoD is expressed in activated muscle precursor cells, Myf-5 is subject to periodic fluctuations during the cell cycle and is absent in muscle precursor cells undergoing mitosis or in which the differentiation program has been initiated [5]. The maintenance or safeguarding of the satellite cell com- partment depends on the transcription factor Pax7 since this compartment is absent in Pax7 À/À mice [6]. Muscle precursor cells are also phenotypically heterogeneous [7–10], and a subset of them may represent true stem cells [11–16]. The isolation of muscle stem cells has proven difficult since whole muscle naturally contains cells of different origins. Cell sorting techniques based on Hoechst 33342 exclusion have shown that skeletal muscle contains a subset of cells (SP) with phenotypic markers of stem cells [17–19]. However, further studies have shown that many of these cells belong to hematopoietic and vascular compartments [20– 23]. Thus, paradoxically, whole muscle is probably not the most appropriate source of muscle stem cells to be charac- terized. The biochemical and molecular characterization of these cells require both reliable markers to guarantee cell origin and an adequate number of cells to perform the study. At present, genuine muscle stem cell markers are not avail- able, and conditions for stem cell amplification are unknown. A way to circumvent these limitations would be to use appropriate cell lines that display a patent cell diversity mimicking the lineage cell hierarchy observed in vivo [24]. Such a diversity inside a cell line has been previously documented for the muscle cell line C2C12 [5]. In particular, 0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2003.11.005 * Corresponding author. Genethon-Centre National de la Recherche Scientifique, UMR 8115, 1 bis rue de l’Internationale, 91002 Evry cedex, France. Fax: +33-1-60-77-86-98. E-mail address: garcia@genethon.fr (L. Garcia). www.elsevier.com/locate/yexcr Experimental Cell Research 294 (2004) 254 – 268