Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle

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ARTICLE IN PRESS European Journal of Cell Biology xxx (2016) xxx–xxx

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Research paper

Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle Anita Helinska, Maciej Krupa, Karolina Archacka, Areta M. Czerwinska, Wladyslawa Streminska, Katarzyna Janczyk-Ilach, Maria A. Ciemerych, Iwona Grabowska ∗ Department of Cytology, Institute of Zoology, Faculty of Biology, University of Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 23 June 2016 Received in revised form 5 December 2016 Accepted 5 December 2016 Keywords: ES cells Myoblasts Skeletal muscle Differentiation Pax7 5-azacitidine

a b s t r a c t Regeneration of skeletal muscle relies on the presence of satellite cells. Satellite cells deficiency accompanying some degenerative diseases is the reason for the search for the “replacement cells” that can be used in the muscle therapies. Due to their unique properties embryonic stem cells (ESCs), as well as myogenic cells derived from them, are considered as a promising source of therapeutic cells. Among the factors crucial for the specification of myogenic precursor cells is Pax7 that sustains proper function of satellite cells. In our previous studies we showed that ESCs lacking functional Pax7 are able to form myoblasts in vitro when differentiated within embryoid bodies and their outgrowths. In the current study we showed that ESCs lacking functional Pax7, cultured in vitro in monolayer in the medium supplemented with horse serum and 5azaC, expressed higher levels of factors associated with myogenesis, such as Pdgfra, Pax3, Myf5, and MyoD. Importantly, skeletal myosin immunolocalization confirmed that myogenic differentiation of ESCs was more effective in case of cells lacking Pax7. Our in vivo studies showed that ESCs transplanted into regenerating skeletal muscles were detectable at day 7 of regeneration and the number of Pax7-/- ESCs detected was significantly higher than of control cells. Our results support the concept that lack of functional Pax7 promotes proliferation of differentiating ESCs and for this reason more of them can turn into myogenic lineage. © 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Skeletal muscle regeneration requires the presence of muscle specific stem cells, i.e. satellite cells. During vertebrate embryo development these cells originate from myogenic precursors characterized by the expression of two Pax genes, i.e. Pax3 and Pax7 (Messina and Cossu, 2009). In developing embryo these genes impact at MyoD1 expression inducing cell differentiation (Relaix et al., 2004; Tajbakhsh et al., 1997). Along the embryonic myogenesis differentiating muscle precursor cells downregulate Pax3 and Pax7. Satellite cells precursors, however, sustain Pax7 expression. In these cells Pax7 upregulate Id3, i.e. the inhibitor of differentiation (Kumar et al., 2009). It also regulates MyoD1 and Myf5 expression,

∗ Corresponding author at: Department of Cytology, Institute of Zoology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02−096, Warsaw, Poland. E-mail address: [email protected] (I. Grabowska).

as well as prevents apoptosis (Crist et al., 2012; McKinnell et al., 2008; Olguin and Olwin, 2004; Olguin et al., 2007). Satellite cells are also characterized by the expression of Myf5, as well as adhesion proteins such as M-cadherin and syndecan-4 (Cornelison and Wold, 1997). In adult skeletal muscles satellite cells remain quiescent occupying the niche between sarcolemma and basal lamina that surrounds muscle fiber (Relaix and Zammit, 2012). Skeletal muscle injury leads to the activation of satellite cells that resume the cell cycle, proliferate, differentiate into myoblasts that fuse with each other, and finally produce new fibers. Under physiological conditions satellite cells are the major source of cells securing correct muscle regeneration. However, also other endogenous stem cells were proven to be able to participate in this process (Brzoska et al., 2015). Various disorders, such as Duchenne’s muscular dystrophy or massive muscular injuries, might lead to the exhaustion of the satellite cells pool. As a result proper regeneration of skeletal muscles cannot be secured, regardless of the presence of other endogenous stem cells populations. Among the treatments aim-

http://dx.doi.org/10.1016/j.ejcb.2016.12.001 0171-9335/© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

Please cite this article in press as: Helinska, A., et al., Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.12.001

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ing to improve skeletal muscle regeneration are transplantations of potentially therapeutic cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Grabowska et al., 2012). Before the ESCs or iPSCs could be seriously considered in therapies aiming to improve skeletal muscle regeneration their myogenic differentiation has to be precisely analyzed at various levels – molecular (e.g. expression and the role of factors governing myogenesis), cellular (e.g. ability to form myotubes), and tissue level (e.g. ability to participate in muscle regeneration). In the current study we focused at all of these aspects. Previous studies documented the crucial role of Pax7 in the embryonic, postnatal, and adult myogenesis. Initial characterization of Pax7-null mice demonstrated the key function of this factor in neurogenesis (Mansouri et al., 1996) but involvement of Pax7 in myogenesis was soon documented in analyses proving its role in satellite cells function. Mutant mice were characterized by dramatic decrease or even absence of satellite cells in skeletal muscles (Oustanina et al., 2004; Seale et al., 2000) and tunica muscularis of esophagus (Worl et al., 2009). Directed deletion of Pax7 gene in satellite cells showed that in mice younger than 21 days skeletal muscle regeneration depends on this factor (Lepper et al., 2009). However, its role in older mice is disputable since some experiments documented that Pax7 is unessential for proper satellite cell function (Lepper et al., 2009) while other showed that it is crucial for myogenic differentiation even in mice older than 21 days (Gunther et al., 2013; von Maltzahn et al., 2013). Unquestionably, however, experimentally induced ablation of satellite cells resulted in the failure in proper skeletal muscle regeneration (Lepper et al., 2011; McCarthy et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011). In our previous study we showed that Pax7 function is dispensable for myogenic differentiation of pluripotent stem cells. We documented that in ESCs lacking functional Pax7 that were cultured in embryonic bodies (EBs) expression of other myogenic factors was not significantly affected (Czerwinska et al., 2016a). The lack of functional Pax7, however, seems to be advantageous for the proliferation of differentiating ESCs (Czerwinska et al., 2016b). In the current study, we took advantage of Pax7-/- ESCs and use them to follow their myogenic differentiation in vitro in the response to medium supplemented with horse serum and demethylating agent 5-azacitidine (5azaC), as well as in vivo, i.e. within the environment of regenerating skeletal muscles. 5-azacitidine was first characterized as a cytotoxic drug that could be potentially used in chemotherapy (Sorm et al., 1964). Further studies revealed that in the response to 5azaC treatment mouse fibroblasts are able to differentiate into myoblasts and other cells of mesodermal origins, e.g. chondroblasts or adipocytes (Constantinides et al., 1977; Constantinides et al., 1978; Taylor and Jones, 1979). The molecular basis of this phenomenon relied on the epigenetic changes caused by 5azaC, i.e. demethylation of cytosines leading to the derepression of certain gene expression (Konieczny and Emerson, 1984). Other studies showed that the differentiation of stem cells by 5azaC and other nucleoside drugs could be induced not only by changing the levels of cytosine methylation, but also by the induction of the Oct-4 and Nanog degradation mediated by caspases 3 and 7 (Musch et al., 2010). Importantly, Oct-4 was shown to prevent expression of MyoD1, thus, its degradation directly impacts at myogenic differentiation (Watanabe et al., 2011). 5azaC indeed induced MyoD expression in human ESCs (Zheng et al., 2006) as well as in mouse ESCs (Archacka et al., 2014). Archacka et al. suggested that 5azaC sensitize ESCs to factors present in horse serum which induce myogenic differentiation of these cells (Archacka et al., 2014). Many lines of evidence document that supplementation of culture media with horse serum is crucial for proper differentiation of such cells as primary myoblasts, C2C12 myoblasts and other cells, e.g. AC133+ stem cells or adipose derived mes-

enchymal stem cells (Di Rocco et al., 2006; Torrente et al., 2004). For this reason horse serum is consistently and commonly used for myoblast culture since 1960 (Capers, 1960). In the current study we followed myogenic differentiation of Pax7+/+ and Pax7-/- ESCs cultured in colonies eventually forming monolayer in the medium containing 5azaC and horse serum. We also tested myogenic potential of Pax7+/+ and Pax7-/- ESCs in vivo, i.e. after their transplantation into regenerating mouse muscle.

2. Materials and methods Animal studies were approved by Local Ethics Committee No. 1 in Warsaw, Poland (permit number 455/2013) according to European Union Directive on the approximation in laws, regulations and administrative provisions of the Member States regarding protection of animals used for experimental and scientific purpose (Close et al., 1996, 1997). All mice were raised on the premises and maintained in the 12 h light/12 h dark cycle.

2.1. Preparation of feeder cells and C2C12 myoblast culture Feeder cells, i.e. inactivated mouse embryonic fibroblasts (MEFs), were prepared according to Robertson (Robertson, 1987). Briefly, primary MEFs were derived from 13.5 days old embryos of F1(C57Bl6NxCBA/H) mice. MEFs were cultured in DMEM (with 4.500 mg/l glucose, Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), penicillin and streptomycin (5000 units/ml each, Gibco). Confluent MEFs were inactivated by treatment with mitomycin C (10 ␮g/ml, Sigma-Aldrich) for 2 h. Growth-arrested MEFs were then frozen and seeded as required. Murine C2C12 myoblasts (Sigma-Aldrich) were plated in culture plates (2.5 × 103 /cm2 ) and cultured in differentiation-inducing medium, that is DMEM (with 4.500 mg/l glucose, Gibco) supplemented with 10% of FBS, 10% of horse serum (HS, Gibco), penicillin and streptomycin (5000 units/ml each, Gibco) in 37 ◦ C, 5% CO2 . Once C2C12 myoblasts differentiated into myotubes, cells were collected and analyzed.

2.2. Generation and in vitro culture of ESCs C57Bl6N females carrying mutation in one allele of Pax7 gene were crossed with 129 Sv males and genotyped as described previously (Mansouri et al., 1996). ESCs were generated from blastocysts recovered from F1 (C57Bl6N x 129 Sv) Pax7+/− females induced to superovulate by injection of 10 IU of pregnant mare’s serum gonadotropin (PMSG, Folligon, Intervet) followed 48–52 h later by 10 IU of human chorionic gonadotropin (hCG, Folligon, Intervet) injection, allowed to mate with males of the same cross and genotype and sacrificed 96 h after hCG injection. ESC were derived as described previously (Hogan et al., 1994). Medium for ESC derivation was composed of KnockOut Dulbecco’s modified Eagle’s Medium (KnockOut DMEM, Gibco) supplemented with 10% serum replacement (SR, Gibco) with addition of nonessential amino acids (0.1 mM, Gibco), L-glutamine (2 mM, Gibco), ␤-mercaptoethanol (0.1 mM, Sigma-Aldrich), penicillin and streptomycin (5000 units/ml each, Gibco), murine leukemia inhibitory factor (LIF, 1000 IU/ml, ESGRO, Chemicon International), and 12.5 ␮M MEK1 inhibitor – PD98059 (Sigma-Aldrich). All experiments were carried out on three wild type Pax7+/+ ES cell lines (AE5B3S, CH7B5, AE5B8) and three knock-out Pax7-/- cell lines (AE5B4P, AI7.15, T2M4), as described previously (Czerwinska et al., 2016a).

Please cite this article in press as: Helinska, A., et al., Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.12.001

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2.3. Genotyping

2.7. Muscle injury and ESC transplantation to muscles

C57Bl6N females carrying mutation in one allele of Pax7 gene were crossed with 129 Sv males. Obtained progeny and ESCs lines were genotyped. Briefly, genomic DNA was isolated from tail tips of 2 weeks old mice or cells pellets that were placed in 100 ␮l of 10% Chelex 100 (Bio-Rad) solution in deionized water, in 98 ◦ C, for 15 min. Next, supernatant containing DNA was collected and 1 ␮l of obtained solution was used for PCR analysis using RedTaq Ready Mix (Sigma-Aldrich) and primers according to conditions described previously (Mansouri et al., 1996). PCR products were separated using 1.5% agarose gel (Bio-Rad) and visualized with ethidium bromide (1 mg/ml, Sigma-Aldrich). Agarose gels were analyzed with GelDoc 2000 (Bio-Rad) using Quantity One software (Bio-Rad). Wild type allele was represented by 200 bp and knock-out allele by 600 bp band (Mansouri et al., 1996).

The right gastrocnemius muscle was injured by cardiotoxin (CTX) injection. Before the procedure mice (three month-old male C57Bl6N x 129 Sv) were anesthetized by intraperitoneal injection of pentobarbital solution (30 ␮g/g of body mass). Muscles were injected with 50 ␮l of 10 ␮M cardiotoxin (Sigma-Aldrich) solution in 0.9% NaCl (Garry et al., 1997). Cell TrackerTM labeled wild type (Pax7+/ + ) or mutant (Pax7-/-) ESCs were transplanted three days after CTX-injection (1 × 106 cells in 10 ␮l of 0.9% NaCl). Muscles injured by CTX but injected with 10 ␮l of 0.9% NaCl only served as a control. At 7 and 14 day of regeneration mice were sacrificed and muscles were isolated. Immediately after isolation muscles were weighed and frozen in liquid nitrogen cooled with isopentane for further sectioning, or in liquid nitrogen for mRNA isolation, and preserved at −80 ◦ C. In each experiment muscles isolated from at least three animals were analyzed for each time point following CTX injury.

2.4. Karyotyping ESCs were incubated in medium containing 10 mg/ml of colchicine (Sigma-Aldrich) in 37 ◦ C for 1.5 h. Next, ESC colonies were disaggregated in 0.05% trypsin-EDTA for 3–5 min, washed in PBS, suspended and incubated in 1 ml of 0.56% KCl (SigmaAldrich) in room temperature for 20 min. Next, cells were fixed with methanol:acetic acid solution (3:1) in 4 ◦ C for 16 h. Finally, ESCs were dropped onto warm slides, allowed to dry and stained with Giemsa (Merck) according to the manufacturer’s protocol. Next, specimens were dehydrated in HistoChoice (Sigma-Aldrich), mounted with VectaMount Mounting Medium (Vector Laboratories) and analyzed using transmitted light microscopy (Axioskop, Zeiss). For each ESC line at least 30 metaphase plates were analyzed.

2.5. In vitro differentiation of ESCs Before 5-azacytidine (5azaC) incubation, ESCs were separated from MEFs by preplating, i.e. cells suspension was plated on dishes coated with 1% gelatin and incubated in 37 ◦ C, 5% CO2, for 20 min. Next, the medium containing unattached cells was transferred to next gelatin-coated dish; this procedure was repeated twice. Preplating enabled MEF attachment to the dish while ESCs remained suspended in the medium. After preplating ESCs were harvested and 2 × 105 ESCs were seeded onto 1% gelatin-coated cover slips (placed in 35 mm dishes) and cultured without MEFs in standard ESCs medium. After 24 h dishes were divided into 3 groups: one control and two treated for 24 h with 5 and 10 ␮M 5aza (Sigma Aldrich) in DMEM 4.500 mg/l glucose (Gibco), 10% FBS (Gibco), 10% HS (Invitrogen), penicillin and streptomycin (5000 units/ml each, Gibco). After 24 h incubation cells were washed twice with PBS and the medium was changed for medium without 5azaC (DMEM 4.500 mg/l glucose, Gibco; 10% FBS, Gibco; 10% HS, Invitrogen; penicillin and streptomycin 5000 units/ml each, Gibco). Medium was changed every 2 days until 10 day of the culture. Each experiment was performed in at least three independent replicates.

2.6. Fluorescent staining of ESCs ESCs were separated from MEFs by preplating (described above), then cells were incubated in Cell TrackerTM staining solution (25 ␮M, Life Technologies) at 37 ◦ C for 30 min. Cells were centrifuged and Cell TrackerTM solution was removed. Next, cells were washed twice in PBS and then 1 × 106 cells suspended in 10 ␮l of 0.9% NaCl were injected to injured muscles.

2.8. Immunolocalization The specimens, i.e. muscle cryo-sections or in vitro cultured ESCs and C2C12 myotubes, were fixed with 3% paraformaldehyde (Sigma-Aldrich) in PBS, at room temperature for 10 min, and permeabilized with 0.05% Triton-X 100 (Sigma-Aldrich) in PBS, at room temperature for 5 min. The nonspecific antibody binding sites were blocked by incubation in 3% bovine serum albumin (BSA) in PBS at room temperature for 30 min. Next, specimens were incubated in primary antibodies, i.e. anti: Oct-4 (sc 34918, Santa Cruz Biotechnology; 1:100), Nanog (RCAB0002P-F, Cosmo Bio USA; 1:200), Pax3 (DSHB, 1:20), Pax7 (DSHB, 1:20), MyoD (sc 760, Santa Cruz Biotechnology; 1:100), Myf5 (sc 302, Santa Cruz Biotechnology; 1:100), Myogenin (sc 576, Santa Cruz Biotechnology; 1:100), Mrf-4 (sc 301, Santa Cruz Biotechnology; 1:100), Laminin (L9393, SigmaAldrich; 1:200), skeletal Myosin Heavy Chains (skMyHC, M7523, Sigma-Aldrich; 1:100), or Ki67 (ab15580, Abcam; 1:200) antibodies diluted in 0.5% BSA in PBS, at 4 ◦ C overnight. Afterwards, they were incubated with appropriate secondary antibody conjugated with Alexa 488 (Molecular Probes) diluted 1:200 in 0.5% BSA in PBS, at room temperature for 2 h. To visualize the nuclei specimens were incubated in DRAQ5 (Biostatus Limited) diluted 1:500 in PBS for 5 min. Finally the specimens were mounted with Fluorescent Mounting Medium (DakoCytomation). Specificity of primary antibodies was confirmed by incubation of samples with secondary antibodies only. The specimens were analyzed using Axio Observer Z1 scanning confocal microscope (Zeiss) equipped with LSM 700 software. Figures were assembled using Adobe Photoshop CS6 Extended. Gimp2 software was used to evaluate the surface of skMyHC. Briefly, skMyHC visible at the picture was contoured and filled with color. Next, number of pixels within the colored area was counted automatically and the percentage of the “colored area” to the total surface, i.e. total number of pixels within the analyzed section was estimated. 2.9. Western blotting Protein extracts were produced by homogenization and lysis of in vitro differentiating ESCs. Briefly, cells were homogenized and lysed in RIPA buffer containing protease inhibitors (Complete, Roche) and obtained lysates were kept on ice for 30 min, microcentrifuged and stored at −80 ◦ C. Protein concentration was determined by using the Bradford’s assay (Sigma-Aldrich). 30 ␮g of protein was subjected to 10% SDS-PAGE and Western-blot analysis and probed with antibodies against Pdgfra (ab61219, Abcam; 1:1000), Myf5 (SAB 4501943, Sigma-Aldrich; 1:1000), Myogenin (sc 576, Santa Cruz Biotechnology; 1:1000), or Hsp90 (TA500494,

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OriGene Technologies). As secondary antibodies goat anti-rabbit HRP conjugate (170–6515, Bio-Rad) were used, followed by chemiluminescence detection. Films were photographed and optical density of resulting bands was measured using GelDocXR+ (BioRad) with ImageLab software.

incubation with primary antibody was omitted. Cells were analyzed with FACSCalibur instrument (Becton Dickinson) and CellQuest Pro software (BD Biosciences).

3. Results 2.10. Histological analyses Transverse, 10 ␮m thick muscle cross sections were obtained using a cryostat (Microm HM 505N, Microm Int.), air-dried, and stored at 4 ◦ C. Sections were stained with Harris hematoxylin for 40 min (Sigma-Aldrich), washed in water, stained with eosin Y for 3 min (Sigma-Aldrich), again washed in water, and mounted in aqueous permanent mounting agent (Dako Cytomation). Observations were made and pictures were taken using a Nikon TE200 microscope (Nikon Instruments, Tokyo, Japan) and NIS Elements software. The procedure was repeated three times and four sections obtained from one muscle were analyzed for each experiment. To compare the efficiency of regeneration the proportion of immature (with centrally located nucleus) and mature (with peripherally located nucleus) myofibres was established within the 1 mm2 surface of the muscles. Gimp2 software was used to evaluate the surface of connective tissue. Briefly, connective tissue visible at the section was contoured and filled with color. Next, number of pixels within the colored area was counted automatically and the percentage of the “colored area” to the total surface, i.e. total number of pixels within the analyzed section was estimated. 2.11. RNA isolation, RT-PCR and qPCR For mRNA analysis total RNA was isolated from undifferentiated ESCs, 5azaC-treated cells as well as control and regenerating muscles using mirVana Isolation Kit (Life Technologies). For RT-PCR 0.2 ␮g of RNA was used and reaction was carried with Titan One Tube RT-PCR System (Roche) and primers according to conditions described previously (Tamaki et al., 2002). PCR products were separated using 2% agarose gel and analyzed as indicated above. Reverse transcription was performed using 0.5 ␮g total RNA and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. qPCR was performed using specific ® TaqMan probes: Mm02019550 s1 (Nanog), Mm00435493 m1 (Pax3), Mm00435125 m1 (Myf5), Mm00440387 m1 (MyoD), Mm00446194 m1 (Myog), Mm00483191 m1 (Cdh15), Mm00488527 m1 (Sdc4), Mm01332463 m1 (Myh3), Mm01332564 m1 (Myh2), Mm01319006 g1 (Myh7), Mm01318252 m1 (T), Mm01222421 m1 (Flk1), Mm00440701 m1 (Pdgfra), Mm00492575 m1 (Id3), Mm01205647 g1 (Actb), the TaqMan Gene Expression Master Mix (Life Technologies) and LightCycler 96 instrument (Roche). Data were collected and analyzed with LightCycler 96 SW1.1 software (Roche). For each analysis three independent experiments were performed. 2.12. FACS ESCs were separated from MEFs by preplating and washed twice in PBS. Cells were fixed with 3% paraformaldehyde (Sigma-Aldrich) in PBS, at room temperature for 10 min, permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich) in PBS, at room temperature for 5 min. The nonspecific antibody binding was blocked by incubation in 3% bovine serum albumin in PBS at room temperature for 30 min. Next, cells were incubated at 4 ◦ C overnight with anti Nanog primary antibody (Cosmo Bio Co), diluted 1:100 in 0.5% BSA in PBS. Afterwards specimens were incubated with appropriate secondary antibody conjugated with Alexa 488 (Molecular Probes) diluted 1:200 in 0.5% BSA in PBS, at room temperature for 2 h. In control experiments,

3.1. 5azaC induced myogenic differentiation of control and Pax7 deficient ESCs The analysis of the role of Pax7 in mouse ESCs induced to differentiate in vitro by culture in the presence of horse serum and 5azaC was preceded by the characterization of the cell lines used in this study. We analyzed three control ESC lines (B3, B5, B8) and three ESC lines lacking functional Pax7 gene (B4, AI7.15, T2M4). All of them were derived from blastocysts obtained by crossing of mice carrying one mutated Pax7 allele that was created previously (Mansouri et al., 1996). Analyzed ESCs were characterized by normal karyotype, i.e. the number of diploid cells exceeded 80% in each of analyzed cell line, what suffice the requirements for the ESCs. ESCs cultured under pluripotency supporting conditions, i.e. in the LIF-containing medium, formed typical colonies and their morphology did not differ between cell lines analyzed (Fig. 1A). All analyzed cell lines expressed mRNA encoding pluripotency markers, such as Oct-4 and Nanog. FACS analysis revealed that on average 74.6% (± 9.5%) of cells of each line expressed Nanog (Fig. 1B), immunolocalization revealed Nanog and Oct-4 in all the ESCs colonies tested (Fig. 1C). These cells lacked, however, the mRNAs and proteins of factors characteristic for myogenic precursors and myoblasts, such as Pax3, Pax7, MyoD, Myf5, myogenin, and Mrf4 (Fig. 1C). Importantly, majority of “myogenic” factors, except Pax3 and Pax7, were expressed in mouse C2C12 cells differentiating into myotubes, that served as positive control, as shown by sqPCR (data not shown) and immunolocalization (Fig. 1C). Ability of ESC lines to differentiate into ecto-, endo-, and mesoderm derivatives was tested by in vitro differentiation in embryoid bodies (EBs) and by generating teratomas containing variety of tissues (Czerwinska et al., 2016a). Our previous analyses of ESCs lacking functional Pax7 involved their in vitro differentiation within EBs and their outgrowths. Under such conditions we did not reveal any dramatic differences between Pax7+/+ and Pax7-/- cells (Czerwinska et al., 2016a). Thus, we decided to induce in vitro differentiation of ESCs using more “aggressive” factors, i.e. DNA demethylating agent – 5azaC. We cultured them on gelatin coated dishes and supplemented culture medium with horse serum which was previously used as a factor promoting ESC differentiation into satellite cells and myoblasts (Chang et al., 2009; Zheng et al., 2006). We hoped that such treatment will allow us to uncover yet unknown phenotypes related to Pax7 deficiency. Thus, three wild type and three Pax7 deficient ESC lines were cultured in the presence of horse serum, as well as horse serum and 5 ␮M or 10 ␮M 5azaC. Such protocol was previously shown to allow the generation of skeletal myoblasts from human ESC (Zheng et al., 2006). After 10 days of culture in medium lacking LIF ESC colonies were round but flattened at their borders, suggesting that differentiation started (Fig. 2A). 5azaC treatment enhanced flattening and also induced the dispersal of colonies (Fig. 2A). In Pax7+/+ ESCs that effect was clearly pronounced when cells were cultured in the presence of 10 ␮M 5azaC (Fig. 2A). Interestingly, changes in the morphology of colonies were most evident in Pax7-/- ESCs suggesting that these cells are more prone to initiate differentiation in the response to the horse serum and demethylating agent (Fig. 2A). Immunolocalization of the skeletal isoform of myosin heavy chains (skMyHC) and calculation of the area of cells expressing this protein (Pax7+/+ ESCs 26.8% vs 72.8% in Pax7-/ESCs) confirmed that myogenic differentiation was more effective in case of ESC lacking functional Pax7 (Fig. 2A, B).

Please cite this article in press as: Helinska, A., et al., Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.12.001

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Fig. 1. Characteristics of Pax7+/+ (B3, B5, B8) and Pax7-/- (B4, AI7.15, T2M4) ESCs. (A) ESCs colonies cultured under pluripotency supporting conditions. Proportion of 2n metaphase plates (2n karyotype) in analyzed ESC lines is shown in the right bottom corner of each image; at least 30 metaphase plates were analyzed for each cell line; scale bar 100 ␮m. (B) FACS analysis of Nanog positive cells in all analyzed ESC lines; the mean values from 3 independent experiments are shown for each cell line; error bars indicate standard deviation. (C) Immunolocalization of pluripotency markers: Oct-4, Nanog, and myogenic markers: Pax3, Pax7, MyoD, Myf5, Myogenin, Mrf-4; differentiating C2C12 myoblasts served as positive control.

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Fig. 2. Morphology of differentiating Pax7+/+ and Pax7-/- ESCs. (A) Morphology of ESCs treated with 5 or 10 ␮M of 5azaC for 24 h and analyzed after 10 days of culture in HS containing differentiation medium. HS/Control − ESCs cultured in differentiation medium lacking 5azaC; 5azaC 5 ␮M − ESCs treated with 5 ␮m of 5azaC; 5azaC 10 ␮M − ESCs treated with 10 ␮m 5azaC. skMyHC − immunolocalization of skeletal myosin heavy chains (green) and localization of nuclei (blue) in Pax7+/+ and Pax7-/- ESCs cultured in the presence of 5 ␮m of 5azaC. Scale bar 100 ␮m. (B) Calculation of the area of cells expressing skMyHC. White bar − mean values for Pax7+/+ ESCs (B3, B5, B8), gray bar − mean values for Pax7-/- ESCs (B4, AI7.15, T2M4); error bars indicate standard deviation; statistically significant differences marked with stars, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

3.2. Expression of pluripotency, mesodermal, and myogenic markers in ESCs induced to differentiate by horse serum and 5azaC treatment We followed the expression of mRNAs encoding factors involved in the pluripotency, mesoderm formation, and myogenic differentiation, in ESCs induced for myogenic differentiation by culture in the presence of horse serum only as well as horse serum and

5azaC. Control ESCs were cultured in medium lacking LIF and supplemented with horse serum. Nanog expression levels were comparable between Pax7+/+ and Pax7-/- ESCs regardless of 5azaC treatment (Fig. 3A). However, ESCs lacking functional Pax7 presented lower levels of this transcript as compared to wild-type ones. Significant differences were observed between Pax7+/+ and Pax7-/- ESCs cultured in control medium as well as those ones treated with 5 ␮M 5azaC (Fig. 3A). Next, we followed the expression

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Fig. 3. Analysis of selected marker expression in differentiating Pax7+/+ and Pax7-/- ESCs. ESCs were treated with 5azaC for 24 h and analyzed after 10 days of culture in HS containing differentiation medium. (A) Data obtained by qPCR for each sample was normalized against actin and shown as Rq to the level of expression estimated for Pax7+/+ control cells, i.e. cultured in differentiation medium containing HS but lacking 5azaC. The results are shown as the mean values for three ESC lines of each genotype. White bars − mean of values for Pax7+/+ ESCs (B3, B5, B8), gray bars—mean values for Pax7-/- ESCs (B4, AI7.15, T2M4). (B) Western blotting analysis of Pdgfra, Myf5 and Myogenin in differentiating Pax7+/+ and Pax7-/- ESCs. Probing with anti-Hsp90 antibody was used as a loading control. Graph represents optical density of Pdgfra, Myf5 or Myogenin bands compared to density of Hsp90 bands (optical density of Hsp90 was taken as 100 arbitrary units). The results are shown as the mean values for three Western blotting analysis. Error bars indicate standard deviation; statistically significant differences marked with stars, *p < 0.05.

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of mesoderm markers T, Pdgfra, and Kdr (Fig. 3A). Expression of T, i.e. the gene encoding Brachyury which is a marker of primitive streak, was significantly higher only in Pax7-/- ESCs cultured in the control medium as compared to Pax7+/+. The level of Kdr, encoding Flk1 characteristic for lateral plate mesoderm which serves as a source of hematopoietic cells (Sakurai et al., 2008), was increased both in wild type and mutant ESCs, however, observed differences were not significant (Fig. 3A). In addition, the level of Pdgfra expression, i.e. the marker of paraxial mesoderm (Kataoka et al., 1997), was significantly higher in ESCs lacking functional Pax7 as compared to Pax7+/+ cells, again regardless of 5azaC treatment (Fig. 3A). Next, 5azaC did not significantly increase the levels of Pax3 during differentiation of ESCs, although, the expression of this factor was higher in Pax7-/- cells (Fig. 3A). Interestingly, Myf-5 and MyoD1 encoding mRNAs were induced by 5azaC and the levels of these transcripts were higher in differentiating Pax7-/- ESCs (Fig. 3A). As far as Myog expression is concerned in media containing horse serum the level of its mRNA was lower in Pax7-/- cells, however, 5azaC treatment increased it to the levels observed in wild type cells (Fig. 3A). Lack of functional Pax7 also resulted in the lower levels of Id3 transcripts encoding the inhibitor of differentiation (Kumar et al., 2009). The analyses of protein levels, i.e. Pdgfra and Myf5, showed that their amount was much higher in differentiating Pax7-/- cells, regardless of 5azaC treatment (Fig. 3B). Thus, in ESCs in that functional Pax7 was absent the levels of mRNAs and proteins (Pdfgra, Pax3, Myf5, Myogenin) were higher as compared to wild type cells. However, 5azaC treatment itself induced the changes at the mRNA levels only of few analyzed factors. Interestingly, the presence of horse serum was sufficient to uncover the differences between Pax7+/+ and Pax7-/- cells. 3.3. Regeneration of gastrocnemius muscles after ESC transplantation Lack of functional Pax7 facilitated upregulation of myogenic genes (Fig. 3) and also enhanced the cell differentiation (Fig. 2). Thus, we decided to test the behavior of Pax7-/- under in vivo conditions, i.e. after the transplantation of these cells into the injured and regenerating gastrocnemius mouse muscles. By doing that we also hoped to learn if the mutation is advantageous in terms of myogenic potential of ESCs in vivo. Wild type and Pax7 deficient ESCs of each cell line tested were transplanted into cardiotoxin injured gastrocnemius muscles. Control muscles were either left intact or injected with 0.9% NaCl. Next, control and experimental muscles were analyzed at day 7 and 14 of regeneration (Fig. 4A and B). First, we compared the weight of control muscle, i.e. injected with NaCl, with muscles that received Pax7+/+ and Pax7-/- ESCs. To standardize the results we related the muscle weight to the total body weight of each mice (Fig. 4C and D). At day 7 of regeneration we did not record any significant differences between control muscles and those ones that received ESCs, regardless of their genotype. At day 14 of regeneration we observed slight increase in the mass of muscles injected with ESCs, again regardless of cells genotype, however, this difference was not significant (Fig. 4D). The presence of ESCs did not impact at the level of connective tissue present in muscles at day 7 of regeneration (Fig. 4E). Histological analyses of such muscles showed that regeneration of all of them proceeded similarly (Fig. 4A and B). All analyzed muscles contained comparable proportion of muscle fibers with centrally positioned nuclei, i.e. the fibers that were newly formed (Fig. 4F). 3.4. Wild type and Pax7 deficient ESCs within the regenerating muscle To follow the fate of Pax7+/+ and Pax7-/- ESCs within the regenerating gastrocnemius muscle the cells were labeled with

fluorescent dye CellTrackerTM before transplantation. At day 7 of regeneration we analyzed the localization of ESCs within the muscles (Fig. 5A, control muscle). We were able to detect labeled cells in all analyzed samples. Majority of ESCs localized in-between muscle fibers (Fig. 5B). However, in some cases they were also visible within the satellite cells niche (Fig. 5C–E, white arrowheads). Further analyses revealed that the number of ESCs detectable within the muscle depended on their genotype. In case of Pax7+/+ ESCs 1% of nuclei visible in the muscle sections were of transplanted cell origin (Fig. 5B and C). In case of Pax7-/- ESCs 2% of nuclei originated from Pax7-/- cells (Fig. 5F). Thus, ESCs lacking Pax7 seemed to either have better potential to colonize or to survive within the regenerating muscle. The immunolocalization of Ki67, i.e. the marker of proliferating cells, revealed that 16.7% of Pax7+/+ expressed this factor, while as much as 45,3% of Pax7-/- cells was Ki67 positive (Fig. 5G). Thus, cells lacking functional Pax7 retained their proliferative capacity within the regenerating muscle. As a result more of them, as compared to wild type ESCs, were present within the muscle. Next, we analyzed expression of MyoD1, Myf5, Myog encoding Myogenin, Cdh15 encoding M-cadherin, Sdc4 encoding Syndecan4, and finally Myh3, Myh2, and Myh7 encoding embryonic, fast, and slow myosin heavy chains isoforms, respectively, in regenerating muscles (Fig. 6). As we already showed the transplantation of ESCs, regardless of their genotype, did not significantly affect the regenerating muscle fibers and connective tissue content, however, at the level of expression of all analyzed mRNAs we observed some differences (Fig. 6). Thus, the level of Myog expression was lower in muscles that received Pax7+/+ ESCs, as compared to control muscles (injected with NaCl). In case of the muscles that received Pax7-/- ESCs we observed lower level of MyoD1, Cdh15, Sdc4, and Myh2, as well as Myh7, as compared to the muscles injected with Pax7+/+ ESCs (Fig. 6). However, the levels of abovementioned mRNAs were comparable to the ones detected in the regenerating control muscles, suggesting that injection of wild type but not Pax7 deficient ESCs causes changes in the expression of genes crucial for myogenic differentiation. However, these changes do not translate into the differences in muscle histology (Fig. 4). Taken together, ESCs lacking functional Pax7 were characterized by higher proliferative ability when transplanted into regenerating muscle, as compared to wild type cells. However, the presence of these cells did not impact at the transcriptional profile of regenerating muscle. Most probably because they remain in their proliferative phase. The presence of Pax7+/+ cells within the muscle resulted in the higher expression of the myogenic factors, as compared to the control muscle. Thus, the levels of MyoD1, Cdh15, Sdc4, as well as Myh2 and Myh7 were higher in muscles receiving Pax7+/+ as compared to the ones into that Pax7-/- cells were transplanted.

4. Discussion Pluripotent stem cells, such as ESCs or iPSCs, are considered as cells suitable for regenerative medicine. Unfortunately, in vitro derivation of skeletal myoblasts from these cells was shown to be difficult. Majority of successful attempts based on the overexpression of crucial factors regulating myogenesis [e.g. (Darabi et al., 2008a; Dekel et al., 1992; Shani et al., 1992)] and only recently the methods allowing derivation of such cells by in vitro mimicking “myogenic interactions” occurring during embryonic development have been presented [(Borchin et al., 2013; Chal et al., 2015; Shelton et al., 2014), for a review see (Swierczek et al., 2015)]. Pluripotent stem cells beside being the possible source for regenerative medicine are also extensively used to study regulation of various developmental processes. Currently accessible methods permit to manipulate selected gene expression in ESCs or iPSCs or

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Fig. 4. Morphology of regenerating gastrocnemius muscles injected with Pax7+/+ or Pax7-/- ESCs. (A and B) Representative muscle cross sections stained with Harris hematoxylin and eosin Y; day 7 of regeneration. Inserts − magnification of muscle cross sections. Muscles injected with B8 Pax7+/+ ESCs (A) and T2M4 Pax7-/- ESCs (B). Muscles weight analyzed at days 7 (C) and 14 (D) of regeneration induced by CTX injection. Muscle mass is presented as a proportion of mouse weight. (E) Proportion of connective tissue in cross sections of muscles analyzed at day 7 of regeneration. (F) Proportion of regenerated (centrally positioned nuclei, pink color) and mature/undamaged (peripherally positioned nuclei, white and gray color) myofibers within the muscles analyzed at day 7 of regeneration. (C-F) Mean values of three cell lines of each genotype, white bars − control muscles (injected with NaCl) or injected with Pax7+/+ ESCs; gray bars − muscles injected with Pax7-/- ESCs. Scale bar − 100 ␮m. Error bars indicate standard deviation.

Please cite this article in press as: Helinska, A., et al., Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.12.001

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Fig. 5. Pax7+/+ or Pax7-/- ESCs localization in regenerating gastrocnemius muscles injected. Muscles were analyzed at day 7 of regeneration. (A) Control muscle (injected with NaCl). Pax7+/+ (B and C) and Pax7-/- ESCs (D and E) present within the muscle. Red arrows − myofibers with centrally located nuclei (blue), yellow arrows − ESCs present within in the intimate vicinity of muscle fibers. Inserts and arrowheads (white) − ESCs present within forming myofibers and within satellite cells niche. ESCs − red, nuclei − blue, laminin − green. (F) The proportion of ESCs in relation to all cell nuclei in muscle. Means values for three cell lines of each genotype, white bars − control muscles (injected with NaCl) and injected with Pax7+/+ ESCs; gray bar − muscles injected with Pax7-/- ESCs. (G) The proportion of proliferating (Ki67 positive) ESCs within regenerating muscle. Mean values for three cell lines of each genotype, white bar − Pax7+/+ ESCs; gray bar − Pax7-/- ESCs. Yellow arrowheads − Ki67 positive ESCs within the muscle. ESCs − red, nuclei − blue, Ki67 − green. Scale bar − 50 ␮m. Error bars indicate standard deviation, statistically significant differences are marked with stars, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Analysis of selected markers in regenerating gastrocnemius muscles injected with Pax7+/+ or Pax7-/- ESCs. Muscles were analyzed at day 7 of regeneration. Data presented in qPCR analysis was obtained for mRNAs as CT were normalized against CT of actin and represented as Rq of expression observed in Pax7+/+ control muscles (injected with saline solution). Control (white bars) − muscles injected with NaCl (n = 3), Pax7+/+ (white bars) − muscles injected with B3, B5 or B8 ESCs (n = 9), Pax7-/- (gray bars) − muscles injected with B4, AI7.15 or T2M4 ESCs (n = 9); error bars indicate standard deviation; statistically significant differences are marked with stars, *p < 0.05.

to derive iPSCs from patients carrying certain mutations, allowing to study the role of certain factors in cell function. Among the genes which role has been studied using pluripotent stem cells are also those ones encoding factors regulating myogenesis, including Pax7 which plays crucial role in both embryonic and postnatal myogenesis [e.g. (Armour et al., 1999; Craft et al., 2008; Darabi et al., 2011b; Dekel et al., 1992; Gianakopoulos et al., 2011; Shani et al., 1992)]. In developing embryo both Pax3 and Pax7 regulate MyoD expression, and thus, impact myoblasts differentiation (Relaix et al., 2004; Tajbakhsh et al., 1997). In adult muscles Pax7 is present in quiescent satellite cells in that it induces expression of the Id3, i.e. inhibitor of differentiation (Kumar et al., 2009), regulates MyoD (Olguin and Olwin, 2004; Olguin et al., 2007) and Myf5 expression (Crist et al., 2012; McKinnell et al., 2008). In consequence, it prevents differentiation of these cells into myoblasts. The role of Pax7 in mouse development was studied using Pax7-null mice [e.g. (Kuang et al., 2006; Oustanina et al., 2004; Seale et al., 2000)] or mice in that Pax7 gene was conditionally ablated in satellite cells [e.g. (Lepper et al., 2009; Lepper et al., 2011; von Maltzahn et al., 2013)]. Importantly, it was shown that Pax7 is crucial for the proper function of these stem cells. The role of Pax7 in pluripotent stem

cells differentiation was mostly studied in “overexpression experiments” proving that by increasing its level one can force stem cells to differentiate into skeletal myoblasts by triggering embryonic myogenesis pathway (Darabi et al., 2009; Darabi et al., 2008a; Darabi et al., 2011a; Darabi and Perlingeiro, 2008; Darabi et al., 2011b; Darabi et al., 2008b). However, it has to be noted that to allow myogenesis to complete Pax7 expression has to be eventually silenced. The outcome of abolishing Pax7 expression in pluripotent stem cells was studied indirectly by using siRNA technique (Bhagavati et al., 2007) and directly using Pax7-null ESCs [current work, (Czerwinska et al., 2016a; Czerwinska et al., 2016b). To induce differentiation we cultured ESCs in the presence of 5azacytidine (5azaC) and horse serum. 5azaC has been previously used to provoke phenotype conversion of fibroblasts and other embryonic cells into elongated myoblasts-like cells (Constantinides et al., 1977; Constantinides et al., 1978; Taylor and Jones, 1979, 1982) and treatment with this drug resulted in the de-repression of subset of genes and led to the induction of muscle protein synthesis, such as ␣-actin, ␣ and ␤ tropomyosin, embryonic myosin light chains, and troponins (Konieczny and Emerson, 1984). Such myogenic conversion was also observed when rat bone marrow

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mesenchymal stem cells were subjected to azacytidine analogues and multinucleated myotubes expressing muscle-specific myosin were formed (Wakitani et al., 1995). Similar reaction was observed when pluripotent stem cells were treated with 5azaC or other azacytidine analogues. Under such culture conditions mouse (Archacka et al., 2014) and human ESCs (Zheng et al., 2006) were shown to upregulate MyoD1 expression. Interestingly, in the current study we showed that the absence of the functional Pax7 but not the applied treatments was the major factor impacting at the levels of myogenic factors in differentiating ESCs. In vitro differentiating Pax7-/- ESCs were characterized by the higher levels of Pdgrfa, Pax3, and Myf5. At the protein levels we also observed the increase in Myogenin despite that the levels of Myog transcript were comparable between Pax7+/+ and Pax7-/- cells. 5azaC further increased the expression of MyoD1 and Myf5. After 10 days of 5azaC treatment MyoD1, as well as Myf5, were upregulated in cells lacking functional Pax7, in contrast to wild-type, i.e. Pax7 expressing cells. This reaction can be linked with the fact that in satellite cells Pax7 restricts MyoD expression (Kumar et al., 2009; Olguin and Olwin, 2004; Olguin et al., 2007) and also controls the levels of Myf5 mRNA (McKinnell et al., 2008), which in satellite cells remains localized in mRNPs what prevents its translation (Crist et al., 2012). In activated satellite cells expression of Pax7 is downregulated allowing synthesis of MyoD and Myf5. Thus, in the absence of Pax7 function expression of these two myogenic transcription factors is more readily de-repressed. It is also possible that upregulation of Pax3, shown by us at the mRNA level, which also activates Myf5 and MyoD1 expression (Relaix et al., 2006; Tajbakhsh et al., 1997), led to the increase in the levels of these factors. Myogenic differentiation of Pax7-/- ESCs cultured in the presence of 5azaC and horse serum might be also facilitated due to the fact that these cells are more prone to induce expression of Pdgfr˛ which directs the stem cell fate towards paraxial mesoderm cells, i.e. the cells which eventually give rise to myogenic precursor cells (Kataoka et al., 1997). Again, this might be achieved by the upregulation of Pax3, which as previously shown, also impacts at the Pdgfra levels (Magli et al., 2014; Darabi et al., 2008a). Interestingly, 5azaC was shown to act at differentiating myogenic cells not only by upregulation of MRFs but also via preventing proliferation of these cells. C2C12 myoblasts in the reaction to 5azaC upregulated not only Myf5 and MyoD but also p21cip1, i.e. the inhibitor of cyclin dependent kinases (CDK) crucial for the proper cell cycle progression (Montesano et al., 2013). We did not, however, test this issue in our study. In our experiments we used the medium supplemented with horse serum and showed that under such conditions Pax7-/- ESCs more readily expressed T, Pdgfr˛, Pax3, and Myf5, regardless of 5azaC treatment. Thus, it proves that these cells are more prone to initiate myogenic differentiation than the wild-type ones, which do not elevate the expression of these genes in the response to the presence of horse serum in the culture medium. Such characteristic of Pax7-/- ESC was suspected by us in our previous study, in which we analyzed ESC differentiation within EBs (Czerwinska et al., 2016a). We observed that Pax7-/- cells generated more myoblasts in in vitro cultured EB outgrowths, however, we were not able to detect any dramatic differences in the expression of abovementioned genes. The higher number of myoblasts observed most probably reflected the fact that Pax7-/- ESCs are characterized by increased proliferative potential in vitro (Czerwinska et al., 2016b) and also in vivo (current study). Archacka et al. postulated that treatment of ESCs with 5azaC induces epigenetic modification that make cells “sensitive” to environmental signals, such as factors present in horse serum (Archacka et al., 2014). In fact, horse serum is a common supplement of myoblast differentiation media previously used to induce myogenic differentiation of other stem cells [e.g. cells derived from the stromal vascular fraction of adi-

pose tissue (Di Rocco et al., 2006) or AC133 + cells (Torrente et al., 2004)]. However, its mode of action remains obscure, despite it is used to induce myogenic differentiation of proliferating myoblasts for more than 70 years (Capers, 1960). Having shown that lack of functional Pax7 facilitates expression of MRFs in horse serum and 5azaC treated cells we asked whether ESC subjected to in vivo myogenic environment would undergo myogenic differentiation more readily. To this point we transplanted wild type and Pax7 deficient ESCs into cardiotoxin injured gastrocnemius mouse muscles and followed their fate within the regenerating tissue. Up to now only the transplantation of pluripotent cells that were induced to differentiate either by overexpression of Pax3 and/or Pax7 [e.g. (Darabi et al., 2009; Darabi et al., 2008a; Darabi et al., 2011a; Darabi et al., 2011b)] or were subjected to precisely selected extracellular signals before the transplantation (Chal et al., 2015) resulted in the successful engraftment. Thus, we assumed that Pax7-/- ESCs, that are more prone to upregulate MRFs in vitro, will be characterized by enhanced ability to participate in the muscle regeneration. Unfortunately, we did not notice any differences between both types of cells – we were not able to detect muscle fibers formed by transplanted ESCs. Importantly, we observed that the number of Pax7-/- ESCs, present within regenerating muscles after 7 days of regeneration, was significantly higher than in the case of muscles that received wild type ESCs. Thus, ESCs lacking functional Pax7 were characterized either by higher survival rate or by increased ability to proliferate after transplantation. Since the higher proportion of these cells expressed Ki67, i.e. the marker of proliferating cells, we postulate that they retained their ability to proliferate. This is in agreement with the results of our recent study documenting the involvement of Pax7 in the regulation of cell cycle and apoptosis (Czerwinska et al., 2016b). However, other available data shows that Pax7 rather inhibits apoptosis. Downregulation of Pax7 in muscle progenitors leads to their death (Kassar-Duchossoy et al., 2005). Next, in Pax7/- mice satellite cells are progressively lost as result of increased apoptosis (Relaix et al., 2006) and downregulation of Pax7 in rhabdomyosarcoma cells also results in their elimination (Bernasconi et al., 1996). In addition, MyoD-/- myoblasts exhibit remarkable resistance to apoptosis (Hirai et al., 2010). Thus, if the molecular machinery operating in satellite cells and ESCs is similar, lack of functional Pax7 should not lead to the increased cell survival. Our results, however, do not support this notion. In satellite cells Pax7 plays anti-proliferative role (Olguin and Olwin, 2004) and its expression was shown to correlate with the presence of cell cycle inhibitors such as p21cip1 (Tanaka et al., 2008). Thus, proliferation of Pax7 null ESCs might be enhanced, as compared to the wild type ones. Indeed, we showed that ESCs lacking functional Pax7 do proliferate more vigorously at the early stages of differentiation induced in vitro, within the embryoid bodies (Czerwinska et al., 2016b) and also in vivo within the regenerating muscle (current study). Unfortunately, the lack of functional Pax7 does not provide sufficient “impulse” to drive their myogenic differentiation in vivo, within the regenerating muscle. Summarizing, lack of functional Pax7 seems to have two-step impact at differentiating ESCs. First, in the absence of Pax7 activity proliferative capacity of differentiating ESCs is improved both in vitro (Czerwinska et al., 2016b) and in vivo (current study). Second, such Pax7-/- ESCs phenotype allows the formation of increased number of myogenic cells, what is also manifested by the higher expression of myogenic markers, in vitro. Unfortunately, this latter phenotype of Pax7-/- ESCs was not observed during our in vivo studies. We were only able to pinpoint that Pax7-/- ESCs proliferate more vigorously within the regenerating muscle. Moreover, we noticed that for unknown reason the presence of Pax7-/- cells did not have impact at the expression of myogenic regulators, as it was observed in regenerating muscles which received Pax7+/+

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cells. The mechanisms of this action remains, however, currently obscure. Nevertheless, as we shown here, Pax7 deficiency does not prevent neither myogenic differentiation in vitro nor colonizing of the regenerating muscle in vivo. Importantly, in vitro differentiating Pax7-/- ESCs are more prone to initiate myogenic differentiation, showing that this process might be facilitated not only by the overexpression of myogenic factors but also by the lack of some of them (Abujarour et al., 2014; Darabi et al., 2009; Darabi et al., 2008a; Darabi et al., 2011a; Darabi and Perlingeiro, 2016; Darabi et al., 2011b; Dekel et al., 1992; Goudenege et al., 2012; Maffioletti et al., 2015; Shoji et al., 2016; Tanaka et al., 2013). Authors’ contribution AH carried out the experimental work, analyzed data, edited manuscript; KM carried out the experimental work; KA carried out the experimental work, analyzed data, edited manuscript, AMC derived ESC lines, performed ESC initial characteristic; WS carried out the experimental work; KJI carried out the experimental work; MAC discussed the concept, analyzed data, wrote and edited the manuscript; IG developed concept, designed the research, carried out the experimental work, wrote and edited the manuscript. Conflict of interest None of the authors has any conflicts of interest to disclose and all authors support submission to this journal. Acknowledgements We would like to thank all members of the Department of Cytology for their constant support and helpful discussions. This work was supported by grant provided by budget funds from National Science Centre (Poland) for Iwona Grabowska—N N303 548139 for 2010-2013. References Abujarour, R., Bennett, M., Valamehr, B., Lee, T.T., Robinson, M., Robbins, D., Le, T., Lai, K., Flynn, P., 2014. Myogenic differentiation of muscular dystrophy-specific induced pluripotent stem cells for use in drug discovery. Stem Cells Transl. Med. 3, 149–160. Archacka, K., Denkis, A., Brzoska, E., Swierczek, B., Tarczyluk, M., Janczyk-Ilach, K., Ciemerych, M.A., Moraczewski, J., 2014. Competence of In vitro cultured mouse embryonic stem cells for myogenic differentiation and fusion with myoblasts. Stem Cells Dev. 23, 2455–2468. Armour, C., Garson, K., McBurney, M.W., 1999. Cell-cell interaction modulates myoD-induced skeletal myogenesis of pluripotent P19 cells in vitro. Exp. Cell Res. 251, 79–91. Bernasconi, M., Remppis, A., Fredericks, W.J., Rauscher, F.J., Schafer, B.W., 1996. Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins. Proc. Natl. Acad. Sci. U. S. A. 93, 13164–13169. Bhagavati, S., Song, X., Siddiqui, M.A., 2007. RNAi inhibition of Pax3/7 expression leads to markedly decreased expression of muscle determination genes. Mol. Cell. Biochem. 302, 257–262. Borchin, B., Chen, J., Barberi, T., 2013. Derivation and FACS-Mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells. Stem Cell Rep. 1, 620–631. Brzoska, E., Kowalski, K., Markowska-Zagrajek, A., Kowalewska, M., Archacki, R., Plaskota, I., Streminska, W., Janczyk-Ilach, K., Ciemerych, M.A., 2015. Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration. Stem Cell Res. Ther. 6, 46. Capers, C.R., 1960. Multinucleation of skeletal muscle in vitro. J. Biophys. Biochem. Cytol. 7, 559–566. Chal, J., Oginuma, M., Al Tanoury, Z., Gobert, B., Sumara, O., Hick, A., Bousson, F., Zidouni, Y., Mursch, C., Moncuquet, P., Tassy, O., Vincent, S., Miyanari, A., Bera, A., Garnier, J.M., Guevara, G., Hestin, M., Kennedy, L., Hayashi, S., Drayton, B., Cherrier, T., Gayraud-Morel, B., Gussoni, E., Relaix, F., Tajbakhsh, S., Pourquie, O., 2015. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat. Biotechnol. Chang, H., Yoshimoto, M., Umeda, K., Iwasa, T., Mizuno, Y., Fukada, S., Yamamoto, H., Motohashi, N., Miyagoe-Suzuki, Y., Takeda, S., Heike, T., Nakahata, T., 2009. Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells. FASEB J. 23, 1907–1919.

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Please cite this article in press as: Helinska, A., et al., Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.12.001