Soluble factors from neuronal cultures induce a specific proliferation and resistance to apoptosis of cognate mouse skeletal muscle precursor cells

Neuroscience Letters 407 (2006) 20–25

Soluble factors from neuronal cultures induce a specific proliferation and resistance to apoptosis of cognate mouse skeletal muscle precursor cells Maude Pelletier a,b , Julien Rossignol b,c,d , Lisa Oliver a,b , Maryvonne Zampieri e,f , Josiane Fontaine-P´erus e,f , Franc¸ois M. Vallette a,b,∗ , Laurent Lescaudron b,c,d,f a

INSERM UMR 601, 9, Quai Moncousu, Nantes, France Universit´e de Nantes, UFR de M´edecine, Nantes, France c INSERM UMR 643, Nantes, France d ITERT, Institut de Transplantation et de Recherche en Transplantation, CHU, Nantes, France e CNRS UMR 6204, Nantes, France f Universit´ e de Nantes, UFR des Sciences et des Techniques, Nantes, France b

Received 11 April 2006; received in revised form 7 June 2006; accepted 7 June 2006

Abstract The mechanisms or the physiological events, which control the regeneration of skeletal muscle through muscle precursor cell multiplication and differentiation, are still largely unknown. To address the question of the involvement of neurons in this process, skeletal muscle progenitors were grown in the presence of conditioned media obtained from 3-day-old cultures of embryonic neurons (derived from either the dorsal or the ventral region of 11-day-old mouse embryos) or media conditioned with satellite cells. Strikingly, only satellite cells cultured in medium conditioned from ventral embryonic neurons exhibited increased proliferation, as well as resistance to staurosporine (STS)-induced apoptosis. Our results suggest the existence of specific anti-apoptogenic neural soluble signals, which could be involved in skeletal muscle regeneration pathways. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Muscle precursor cells; Expansion; Survival; Neuron

Programmed cell death has been long recognized as essential for the construction of functional nervous system and neuromuscular degenerative disease [29] but most researches have focused on neuron cells death while little is known about muscle cell death aetiology and mechanisms [31]. Skeletal muscles has been considered as an ideal target for cell-mediated therapy and represent a paradigm in the stem cell/precursor cell research, as they possess a remarkable capacity to regenerate even in adults. Their regeneration is mainly attributed to mpc (satellite cells) [17]. They can also arise from different origins as it has been demonstrated that adult bone marrow derived cells can differentiate into skeletal myotubes, contribute to skeletal Abbreviations: mpc, muscle precursor cells; BMP, bone morphogenetic protein; BrdU, bromodeoxyuridine; Shh, sonic hedgehog; STS, staurosporine ∗ Corresponding author at: UMR INSERM 601, 9, Quai Moncousu, Nantes, France. Fax: +33 2 40 08 40 81. E-mail address: [email protected] (F.M. Vallette). 0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.06.076

muscle regeneration after muscle injury [9] and thus constitute a reservoir for muscle cells progenitors [7]. Tissue-specific signals from stroma are determinant for the cell survival, differentiation and proliferation of both adult stem cells and their progeny [30]. Another important feature of muscle regeneration is the capacity of mpc to evade apoptosis in both normal and hostile environments, which trigger their activation, proliferation and fusion into adult muscle fibre [13]. Macrophages, the main stromal cell type observed at site of muscle regeneration, release chemotactic factors which stimulate mpc and rescue them from apoptosis by direct contacts [4]. Little is available on the existence of neural factors which could also participate in the progenitor cell maintenance [21]. We intended to undergo the first step of this kind of study by demonstrating the existence of such factors. In this work, we show that conditioned medium derived from neural cells specifically enhanced mpc proliferation and resistance to apoptosis. Our results could provide the basis for the elaboration of future

M. Pelletier et al. / Neuroscience Letters 407 (2006) 20–25

strategies for mpc transplantation as a treatment for muscular dystrophies. Isolation of mpc was performed using 15 days old RJOrl Swiss mice and for neuronal cells isolation using E-11 days old RJOrl Swiss mouse embryos. Experiments were carried out in accordance with the European Communities Council Directives. Mpc were isolated from Pectoralis muscles according to Creuzet et al. [6] at day 15 of postnatal development. Pectoralis muscles excised under sterile conditions were dissected to remove blood vessels, fat, and connective tissues. After three washes in Ca2+ - and Mg2+ -free Dulbecco’s phosphate-buffered saline, muscle fragments were dissociated with 0.06% Pronase E (Sigma), Ham’s F12, 10 mM Hepes, NaOH (pH 7.3), and 20% fetal calf serum (FCS, Invitrogen) for 1 h at 37 ◦ C. The mixture was then triturated mechanically for 15 min to further disperse the mpc. After rapid decantation, the supernatant was homogenized in an equal volume of Dulbecco’s Minimum Essential Medium (DMEM, Invitrogen) to stop enzymatic digestion. The cells were then centrifuged for 20 min at 300 × g and the pellet was finally rinsed and resuspended in DMEM-20% FCS. 5 × 104 cells/ml were plated alone or with neuronal cells at on collagen pre-coated culture dishes (Iwaki) and grown in DMEM20% FCS in a humidified 95% air/5% CO2 atmosphere. After 3 days of culture, the media were removed and filtered (0.22 ␮m filter) for immediate use. Under a binocular magnifier, E-11 days old mouse embryos were dissected and a micro scalpel was used to surgically separate the neural tube from the somitic mesenchyma and notochord. Then, it cephalic and very caudal portions were discarded in order to only keep the truncal part of the tube corresponding to the future spinal cord. Then, all nerves were excised from the neural tube which was carefully then cut in half under high magnification using a microscapel [10] in order to isolate the ventral part (containing skeletal muscle matching neurons, i.e. motor neurons) from the dorsal part. The two parts were transferred into DMEM with 20% FCS and mechanically dissociated by repeated pipetting through a syringe [16]. The neural cells were collected by centrifugation at 200 × g for 10 min, resuspended in DMEM-20% FCS and 5 × 104 cells/ml were plated in Petri dishes containing mpc and cultured for 3 days. We used E-11-day-old tissue as devoid of any astrocytes [19]. Freshly isolated mpc were cultured in conditioned medium obtained from culture media of mpc cultures alone or from mpc plus neuronal cells from either the dorsal or the ventral regions of the neural tube. The cultures were analyzed after 3 days in the conditioned media for analysis. The cells fixed with 95% ethanol then treated for 30 min with 5% Triton X-100 were incubated overnight with an antidesmin (Biomakor, Israel, 1/100) or anti-NF-70KD antibodies (Sigma, 1/1000) then with fluorescein or rhodamine-conjugated goat anti-rabbit (desmin) or anti-mouse (NF-70KD) antibodies (Southern Biotechnology Associates Inc., 1/100) for 1 h at room temperature. Co-cultures were labeled with GFAP antibodies (Dakopatt, 1/500) then with fluorescein conjugated antibodies in order to visualize astrocytes. All cultures were counterstained with Hoechst 33342 in order to label cell nucleus. To investigate the presence or the absence of macrophages in our culture conditions, we used Mac-1 antibodies (Cedar

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Lane) diluted 1/100. As a control, the same staining conditions were performed on mouse peritoneal macrophage exudates [23]. After several washes, cultures of macrophages alone or of mpc with dorsal or ventral neurons were incubated for 1 h with an rhodamine-conjugated antibody (Southern Biotechnology Associates Inc., 1/100) and then counterstained with Hoechst 33342. Negative controls omitting primary antibodies were also performed. To determine cell proliferation, 3-day cultures were incubated for 17 h with BrdU then fixed with 95% ethanol for 10 min. BrdU incorporated into DNA was visualized by indirect enzymatic immunostaining [23]. We used an antibody to BrdU (Sigma, BU 33, 1/200), and a second antibody conjugated with horseradish peroxidase (Southern Biotechnology Associates Inc., 1/100). An average of 24 microscopic fields always centered in the left corner of 4 wells Lab-Tek culture dishes were used for the three culture conditions. The reproducibility and reliability of this method has been shown to be excellent [23] as shown by the small standard of the mean values observed in that previous study as well as in the present one. The number of BrdU-positive mpc per field (i.e. cells presenting a clearly visible dark brown stained nucleus) was expressed as the mean ± S.E.M. [23]. A Kruskal–Wallis test was performed to determine if there were any statistical differences among the three culture conditions. Post hoc comparisons on data were then performed using the Dunn’s multiple comparison test. In addition, the number of BrdU-positive cells was expressed as a percentage of the total number of cells visible in the field in order to be consistent. After 3 days of culture, 2 ␮M staurosporine (STS) was added to cells. Sixteen hours later, cells were recuperated by scraping and added to an equal volume of lysis buffer (1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM Na-Vanadate and a protease inhibitor cocktail from Boehringer Mannheim, 1378996; 1 ␮g/ml). Neurons from dorsal and ventral regions of the neural tubes were removed from E-11 mice embryos as described earlier [10]. Neuronal cells were co-cultured with mpc for 3 days and then the conditioned media were removed. Freshly isolated mpc were cultured for 3 days in conditioned media from neurons or in conditioned medium from mpc only. As shown in Fig. 1 (A versus B), some morphological differences were observed between mpc grown in ventral neuron-conditioned medium and those cultured under control conditions (i.e. mpc-conditioned media). Under control conditions, some of the desmin-positive mpc were elongated (i.e. bipolar) as compared to most of the round-shaped (i.e. apolar) mpc observed in ventral neuron-conditioned media cultures. These differences were not visible in dorsal neuronconditioned media cultures (data not shown). Note that most of the cells were labeled with the desmin antibody suggesting that the cultures contained at least 95% muscle progenitor cells (Fig. 1A and B). Morphologically ventral neurons have larger cell bodies and longer processes as compared to neurons originating from the dorsal part of the neural tube as shown in the NF-70KD stained cultures (Fig. 1C versus D). No astrocytes or macrophage were noticed in our conditioned-media cultures (Fig. 1E and F). The data suggest that under our specific culture conditions, the only

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M. Pelletier et al. / Neuroscience Letters 407 (2006) 20–25

Fig. 1. Expression of endogenous desmin in 3-day-old primary cultures of muscle precursor cells from 15-day-old muscle grown in conditioned media with mpc alone (A) or plus ventral neurons (B). Under control conditions (A), most of the desmin-positive mpc (anti-desmin antibody) had a bipolar shape as compared to most of the round shaped cells grown in ventral neuron-conditioned media (B). In DMEM-20% FCS culture medium, ventral neurons (C) or dorsal neurons (D) are visible using an anti-neurofilament 70KD antibody but in similar culture conditions, no astrocytes (E) were present (anti-GFAP antibody and Hoechst 33342 to label cell nuclei). In addition, in (F) no macrophages were visible in 1-day-old culture of mpc grown with ventral neurons (anti-Mac-1 antibody and Hoechst 33342). In the contrary, as a control of the Mac-1 antibody, a 1-day-old culture of macrophages showed numerous Mac-1 positive cells (G). In (H), same picture than (G) and counterstained with Hoechst 33342. Scale bars: 70 ␮m.

cells growing in the culture dish presented either a myogenic phenotype (being desmin-positive) or a neuronal one (being NF70KD labeled) as shown in Fig. 1. It appeared (Table 1) that the mitogenic influence of the ventral neuron-conditioned medium on muscle precursor cells was about 2.4-fold as compared to control conditions.

Table 1 Average number of BrdU-positive myonuclei per field (mean ± S.E.M.) in 3day-old satellite cell cultures Culture conditions

Mean ± S.E.M.

Percent from total number of cells

(1) CM with satellite cells alone (2) CM with dorsal neurons (3) CM with ventral neurons Percent changea

21.6 ± 1.2 17.1 ± 1.2 53.2 ± 3.9* 146

33 28 60

Calculated as [(3) − (1)]/(1) × 100, corresponding to a 2.4-fold. P < 0.001 (compared with CM with satellite cells alone, Dunn’s statistical test). a

*

As determined in the BrdU assay (Fig. 2A–C), the presence of medium conditioned from ventral neurons stimulated DNA synthesis since more mononucleated BrdU-positive mpc were observed as compared to conditioned media from dorsal neurons or from mpc alone. On the basis of these observations, it was concluded that ventral neuron-conditioned medium could stimulate the proliferation of mpc in vitro. We also tested the resistance to apoptosis by analyzing DNA condensation and measure of the activity of caspase, a family of enzymes specific of this cell death programme. Analyses of caspase activities were done directly on the lysed cells collected every 2 h over a period of 16 h and the DEVDase activity was measured using the CaspACE Assay (G7220, Promega) as described previously [26]. Caspase-6 (Ac-VEIDAMC), caspase-8 (Ac-IETD-AMC), and caspase-9 substrate (Ac-LEHD-AMC) were obtained from Bachem (France). Their activities were measured as for caspase-3. The caspase activities are represented by a regression graph, which shows the relationship between the fluorescence and incubation time and are normalized by the total protein amount in the culture dish.

M. Pelletier et al. / Neuroscience Letters 407 (2006) 20–25

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Fig. 2. Table 1 shows the average number of BrdU-positive myonuclei in 3-day-old mpc cultures under the three culture conditions. A significant increase in myonuclei number is observed when the mpc were grown in conditioned media plus ventral neurons as compared to mpc grown alone. In the presence of conditioned medium with ventral neurons, 60% of the cells incorporated the BrdU as compared to about 1/3 of the cells when they were grown in conditioned media with mpc alone or plus dorsal neurons. In (A), (B), (C) are shown the qualitative effects of conditioned media from mpc alone (A) on mpc number using BrdU staining as compared to cells grown in conditioned media plus dorsal neurons (B) or plus ventral neurons (C) (arrows: BrdU-positive cells; scale bar: 80 ␮m).

Apoptosis was also determined by the observation of DNA condensation in control and apoptotic cells incubated for 30 min. at 37 ◦ C with Hoechst 33342 (20 mg/ml), an intercalating agent, and fluorescence microscopy. In vitro cells were treated with 2 ␮M STS (Fig. 3A), a broad range kinase inhibitor and a powerful inducer of apoptosis. A peak in the DEVDase activity was observed at 12 h and as shown in Fig. 3, no cell death was observed in 3 days old mpc cultures grown in conditioned media from mpc only in contrast to cells grown in presence of STS for 12 h as chromatin condensation visualized using Hoechst 33342 staining was only observed when the mpc were grown in presence of the kinase inhibitor (STS) (Fig. 3B). Spontaneous cell death was analyzed using a

quantitative assay with substrates of the different caspases. As illustrated in Fig. 3C, spontaneous apoptosis was mainly due to the activities of executioner caspase-3 and caspase-6 and to that of the initiator caspase-8. Quantification of the STS-induced apoptosis by measurement of the caspase-3 activity at its peak revealed that mpc grown in the presence of ventral neuronconditioned media were less sensitive to apoptosis than cells cultured in conditioned media from dorsal neurons or mpc alone (Fig. 3D). Taken together, our results indicate that soluble elements released by neurons from the ventral region of the neural tube could control the proliferation and the resistance to apoptosis of muscle precursor cells. Muscle stem cells are a self-renewing

Fig. 3. In (A), 2 ␮M of staurosporine (STS) was added to mpc grown in the presence of conditioned media from ventral or dorsal neuron or from mpc alone and DEVDase activity was assayed every 2 h for 16 h. In (B), investigation of chromatin condensation in the absence or in the presence of STS for 0, 5 or 16 h, using Hoechst 33342 staining of mpc on day 3. In (C) and (D), caspase activities in mpc cultured in the presence or in the absence of STS in presence of conditioned media from mpc alone (S alone), plus dorsal neurons (S + DN) or plus ventral neurons (S + VN); * P < 0.05, Scheffe test.

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pool of cells that give rise to daughter mpc in adult skeletal muscle [11]. The mpc are also believed to represent a committed stem cell population [2] and play an essential role in postnatal tissue growth and regeneration (for review see [25]). Numerous studies have established that in vivo and in vitro developmental myogenesis is regulated by the neural tube and notochord factors [28], which might have different roles in early myogenesis [20]. Of note, Shh and the neurotrophin 3 which are known to be in a high concentration in the ventral part of the neural tube could play a role on the effects we observed in the presence of ventral tube-conditioned medium (for review see [24]). Additional data from our group also showed that the ventral part as compared to the dorsal one of the neural tube modified the regulation of the Na+ /Ca2+ exchanger in mpc [24]. This exchanger plays an important role in regulating intracellular free calcium levels [3] which might be involved in some apoptotic triggering pathways [14]. On the other hand, as opposed to the dorsal region of the neural tube, the ventral region does not express the paired-box homeodomain transcription factor Pax3, an essential upstream regulator for the muscle determination factor MyoD [1]. This could explain, in part, the delay in mpc differentiation observed. Motor innervation plays also an important role which was investigated by different methods (denervation, crossreinnervation, nerve or muscle electrical stimulation) and electrical activity has been long thought to be the primary neural stimulus for the regulation of skeletal muscle differentiation and activity [15]. Therefore, the relative contribution of neural influences has mainly focused on the effect of loss of innervation on muscle physiology [22]. However, it has been recently demonstrated that some neural effects independent of electrical activity are not only involved in the preservation of muscle mass and the regulation of muscle-specific genes but also in the protection of the mpc pool in inactive muscles [12]. This observation is obviously of particular interest in designing of replacement strategies of damaged skeletal muscles by cell therapy involving adult muscle precursor cells. The differential influence of soluble factors observed in conditioned-media conditions from either the dorsal or the ventral region of the neural tube on the proliferation and/or the resistance to apoptosis of mpc reported here is thus a novelty. As we did not notice any cell death during the 3 days of conditioned-media culture (i.e. no floating cells in the culture media), it is likely that cell proliferation was specifically enhanced in the presence of ventral cells soluble factors and rule out the hypothesis of similar growth rates between the three culture conditions at the start of the experiment but having a mpc death increased in cultures other than the one with ventral neuron-conditioned media. In addition, during development, post-mitotic neurons (including motoneurons) produce signals that affect the fates of adjacent cells [8] thus, we can postulate a direct effect of soluble factors released from ventral neurons on mpc. It is, however, not known what factors released by ventral neurons (mainly motoneurons as visualized with an anti-islet-I antibody) [27] are able to prevent mpc from entering the differentiation program and instead to continue to self-renew. Few factors have been identified to have an effect on the chemotactic control of mpc proliferation and differentiation both under

normal and pathological conditions such as aging or muscular dystrophy [21]. Experiments using macrophage-conditioned media or macrophage-stimulating factors have shown that mpc growth is mediated by soluble factors, which exert their effects not only on mpc in vitro [23] but also in vivo during muscle regeneration [18]. We can also rule out any effect of macrophages present in the E-11-day-old neural tube as macrophage progenitors (the microglia) enter the central nervous system in mammals via the surrounding mesenchyma or through blood vessels only shortly before birth and postnatally [5]. On one hand, the absence of astrocytes in our 3-day-old culture is in agreement with other data [19] showing that GFAP positive astrocytes are not present in the rodent neural tube before E-16. At the light of these results, we could suggest that the effects we observed using E-11 old neural tube were certainly induced by factors released by neurons and not by astrocytes. However, we cannot totally exclude that factors released by neurons did not then induce the release by the mpc of some molecules able to trigger in an apocrine fashion the resulting effects on proliferation and resistance to apoptosis. However, to our knowledge, no data are available concerning the proliferative and the anti-apoptotic effects of mpc on any cell types. Our results suggest that neurons can influence the regeneration of skeletal muscle through the specific control of proliferation and survival of mpc. The nature of the specificity of these soluble factors as well as the proliferation and anti-apoptotic mechanisms involved in this process are still to be characterized. Acknowledgements MP was a recipient of a fellowship from the Association pour la Recherche contre le Cancer (ARC). This work was supported by INSERM and the University of Nantes (F.M.V., L.L.), by CNRS and Association Franc¸aise contre les Myopathies grants to J.F.-P. References [1] H.H. Arnold, B. Winter, Muscle differentiation: more complexity to the network of myogenic regulators, Curr. Opin. Genet. Dev. 8 (1998) 539–544. [2] A. Asakura, M. Komaki, M. Rudnicki, Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic and adipogenic differentiation, Differentiation 68 (2001) 245–253. [3] M.P. Blaustein, W.J. Lederer, Sodium/calcium exchange: Ist physiological implications, Physiol. Rev. 79 (1999) 763–854. [4] B. Chazaud, C. Sonnet, P. Lafuste, G. Bassez, A.C. Rimaniol, F. Poron, F.J. Authier, P.A. Dreyfus, R.K. Gherardi, Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth, J. Cell. Biol. 163 (2003) 1133–1143. [5] V. Corbisiero, G. Hagger, S. Topps, S. Kohsaka, Y. Imai, D. Male, P. Rezaie, Colonization of developing mouse brain by microglial progenitors. Third international symposium on normal and abnormal development of human fetal brain, Neuroendocrinology 2 (2003) 180–188. [6] S. Creuzet, L. Lescaudron, Z. Li, J. Fontaine-P´erus, MyoD, myogenin, and desmin-nls-lacZ transgene emphasize the distinct patterns of satellite cell activation in growth and regeneration, Exp. Cell Res. 243 (1998) 241–253. [7] P.A. Dreyfus, F. Chretien, B. Chazaud, Y. Kirova, P. Caramelle, L. Garcia, G. Butler-Browne, R.K. Gherardi, Adult bone marrow-derived stem cells in muscle connective tissue and satellite cell niches, Am. J. Pathol. 164 (2004) 773–779.

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