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Pluripotent stem cell-derived skeletal muscle fibers preferentially express myosin heavy-chain isoforms associated with slow and oxidative muscles

Pluripotent stem cell-derived skeletal muscle fibers preferentially express myosin heavy-chain... Background: Skeletal muscle function is essential for health, and it depends on the proper activity of myofibers and their innervating motor neurons. Each adult muscle is composed of different types of myofibers with distinct contractile and metabolic characteristics. The proper balance of myofiber types is disrupted in most muscle degenerative disorders, representing another factor compromising muscle function. One promising therapeutic approach for the treatment of these diseases is cell replacement based on the targeted differentiation of pluripotent stem cells (PSCs) towards the myogenic lineage. We have previously shown that transient induction of Pax3 or Pax7 in PSCs allows for the generation of skeletal myogenic progenitors endowed with myogenic regenerative potential, but whether they contribute to different fiber types remains unknown. Results: Here, we investigate the fiber type composition of mouse PSC-derived myofibers upon their transplantation into dystrophic and non-dystrophic mice. Our data reveal that PSC-derived myofibers express slow and oxidative myosin heavy-chain isoforms, along with developmental myosins, regardless of the recipient background. Furthermore, transplantation of the mononuclear cell fraction re-isolated from primary grafts into secondary recipients results in myofibers that maintain preferential expression of slow and oxidative myosin heavy- chain isoforms but no longer express developmental myosins, thus indicating postnatal composition. Conclusions: Considering oxidative fibers are commonly spared in the context of dystrophic pathogenesis, this feature of PSC-derived myofibers could be advantageous for therapeutic applications. Keywords: Pluripotent stem cells (PSC), Skeletal myogenesis, Myofibers, Fiber types, Fast, Slow, Pax3, Pax7, Engraftment, Muscle stem cell Background cells [1]. Upon injury or disease, muscle stem cells are acti- Adult skeletal muscles are characterized by several major vated to regenerate new muscle tissue [2]. Each adult components: myofibers, their innervating motor neurons muscle is a heterogeneous combination of different types of and muscle resident stem cells, along with fibroblasts, myofibers characterized by specific contractile and meta- fibro-adipogenic progenitors, endothelial cells, and immune bolic properties, which are classified into three main cat- egories: (i) oxidative slow-twitch, expressing type I myosin heavy chain (MyHC); (ii) oxidative fast-twitch, expressing * Correspondence: perli032@umn.edu type IIA MyHC; and (iii) glycolytic fast-twitch, expressing Lillehei Heart Institute, Department of Medicine, University of Minnesota, type IIX and IIB MyHC isoforms [3]. In mammals, prenatal 4-128 CCRB, 2231 6th St. SE, Minneapolis, MN 55455, USA Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Incitti et al. Skeletal Muscle (2020) 10:17 Page 2 of 11 fibers initially express the embryonic MyHC isoform, oxidative slow myofibers compared to freshly isolated followed by fetal/neonatal, type I, and, later on, type II, primary muscle stem cells. These results suggest that which is further classified into IIA, IIX, and/or IIB, with the PSC-derived myofibers could be beneficial in the context latter not expressed in humans [4]. Developmental MyHC of muscular dystrophies, not only for their regenerative isoforms disappear during the first weeks of postnatal life capacity, but also for their ability to provide slow-twitch, but the embryonic type becomes re-expressed for a short dystrophy-resistant tissue. time window during regeneration in adults, a process that partially recapitulates developmental myogenesis [3]. Even Methods though each muscle acquires a unique pattern of MyHC Cell culture and differentiation expression and consequently, fiber type characteristics, their Inducible iPax3 and iPax7 mouse embryonic stem (ES) composition is highly plastic and can be influenced by cell lines were generated as previously described [16]. several factors, such as muscle loading, exercise, hormone- mES cells were maintained in knock-out DMEM mediated signaling, and disease [3]. Moreover, myofibers (Invitrogen) supplemented with 15% FBS (Embryomax undergo fiber type transitioning, which generate hybrid ES-qualified FBS—Millipore), 1% penicillin/strepto- myofibers expressing two or more different MyHC iso- mycin (Invitrogen), 2 mM glutamax (Invitrogen), 0.1 forms at the same time [5]. mM non-essential aminoacids (Invitrogen), 0.1 mM β- Many muscle degenerative disorders, including Du- mercaptoethanol (Invitrogen), and 1000 U/ml LIF chenne muscular dystrophy (DMD), facioscapulohumeral (Millipore). Skeletal myogenic differentiation was muscular dystrophy (FSHD), myotonic dystrophies 1 and achieved as described [17]. Briefly, cells were detached 2 (DM1-2), and Pompe disease, exhibit disrupted regula- and the supernatant was then incubated in an orbital tion of fiber type composition, thus further compromising shaker at 80 rpm at the concentration of 40000 cells/ muscle function [6]. For instance, in DMD, the fast fibers ml in embryoid body (EB) differentiation medium, are preferentially affected while the slow fibers degenerate composed of IMDM (Invitrogen) supplemented with more slowly, suggesting that dystrophin may have a major 15% FBS (Embryomax ES-qualified FBS), 1% penicil- role in fibers with the ability to respond to the highest lin/streptomycin (Invitrogen), 2 mM glutamax (Invi- frequency stimulation [7]. One promising therapeutic trogen), 50 μg/ml ascorbic acid (Sigma-Aldrich), and approach for muscle wasting diseases is cell replacement 4.5 mM monothioglycerol (MP Biomedicals). Pax3 or based on the myogenic differentiation of pluripotent stem Pax7 induction is induced starting from day 3 by ad- cells (PSCs). We developed a doxycycline-inducible sys- ministering 1 μM doxycycline (dox). Day 5 EBs were tem that enables the generation of large numbers of prolif- disaggregated, incubated with Fc block (1 μl/million erating early skeletal myogenic progenitors from PSCs cells, BD Biosciences) for 5 min and then with Flk1- through the transient expression of the myogenic tran- APC and Vcam1-biotin-conjugated antibodies (1 μl/ scription factors Pax3 or Pax7. We have documented that million cells, e-Bioscience) for 20 min on ice followed upon transplantation, these myogenic progenitors give rise by 5-min incubation with streptavidin-PeCy7. Cells to new myofibers, seed the muscle stem cell compartment, were washed twice with PBS and then resuspended in provide functional muscle improvement, and remodel staining buffer (SB) composed of PBS containing 10% their molecular signature to acquire a more mature FBS and propidium iodide (PI) to exclude dead cells. phenotype [8–10]. Vcam1+FLK1- cells were sorted using a FACSAria II A few studies have investigated the fiber type compos- (BD Biosciences), replated on gelatin-coated dishes, ition of PSC-derived skeletal muscle in vitro [11, 12], but and further expanded for 2–3 passages before collec- to date, only one report has documented in vivo fiber tion for RNA extraction or transduced with H B-GFP- type characteristics of engrafted tissue, which was gener- encoding lentiviral vector fortransplantation studies ated from PSC-derived teratomas [13]. Since fiber type into NSG mice. For terminal differentiation, 50,000 composition plays an important role in skeletal muscle cells were deposited onto a well of 24-well plates, adaptation to pathological stimuli [7, 14, 15], determining allowed to reach confluence, and then cultured in EB how in vitro-generated PSC-derived myogenic progenitors media without dox for 5-7 days, after which cells were participate in this process will provide important informa- collected for RNA extraction or immunofluorescence tion for the development of effective cell-based skeletal staining. muscle replacement therapies. Here, we sought to investigate the fiber type compos- Labeling of myogenic progenitors ition of engrafted muscles following the transplantation H B-GFP-encoding lentiviral vectors were prepared as of PSC-derived myogenic progenitors in a mouse model described [9]. Briefly, vectors were co-transfected with of DMD. These in vivo studies revealed that PSC- packaging plasmids Δ8.91 and pVSVG into 293T cells derived skeletal myofibers display a higher proportion of using the LTX transfection reagent (Thermofisher Incitti et al. Skeletal Muscle (2020) 10:17 Page 3 of 11 Scientific). Lentiviral-containing supernatant was col- 4 °C. The following day, samples were rinsed with PBS lected 48 h after transfection, filtered, and used for trans- and then incubated with the secondary antibodies goat duction of myogenic progenitors upon centrifugation for anti-rabbit Alexa Fluor-488 or 647, and with goat anti- 1.5 h at 2500 rpm and 37 °C. mouse Alexa Fluor-555 and DAPI for 1 h at RT. After washing three times with PBS, sections were briefly dried Primary cell isolation and transplantation studies and mounted using Prolong Gold with DAPI (Invitrogen). Animal experiments were carried out in strict accordance Pictures were acquired with Axioimager M1 fluorescence to protocols approved by the University of Minnesota Insti- microscope (Zeiss) and analyzed with ZEN Blue software. tutional Animal Care and Use Committee. Primary embry- Quantification of positive myofibers and calculation of onic and fetal myoblasts were isolated and dissected from cross-sectional area (CSA) were performed using Fiji Ima- E10.5 and E14.5 Myf5Cre-Rosa26YFP [5, 6]mouse em- geJ software. bryos and fetuses, respectively, while neonatal and adult satellite cells were isolated from hindlimbs of Pax7- RNA isolation and gene expression analyses ZsGreen mice [7] as previously described [10]. For RNA Samples for RNA were resuspended in Qiazol (Qiagen) extraction and transplantation studies, we used YFP+ cells and RNA extracted with the Qiagen MiRNeasy Mini Kit, for prenatal myoblasts, ZsGreen+ cells for postnatal satellite according to manufacturer’s instructions. After RNA ex- cells, and CD31-/CD45-/Itga7+/Vcam1+/GFP+ for PSC traction, in column DNase digestion was performed and donor-derived satellite cells. Freshly isolated ZsGreen+ cells samples were retro-transcribed using Superscript Vilo were also seeded on gelatin-coated multiwell plates in the (Invitrogen). Gene expression analyses were performed presence of EB medium and cultured for 5 to 7 days to using an amount of cDNA corresponding to 5 ng of induce terminal differentiation, which was followed by im- starting RNA for each reaction. RT-qPCR was per- munofluorescence analyses. formed using Premix Ex Taq (Probe qPCR) Master Mix For transplantation studies, hindlimbs of 6–8-week-old (Takara) and TaqMan probes (Applied Biosystems). mdx4cv male NOD-scid IL2Rgnull (NSG) and NSG [14] mice were irradiated with a 12-Gy single dose at 24 h prior RNA sequencing and data availability to injury of both tibialis anterior (TA) muscles with 15 μl Sequencing data and corresponding analyses have been of cardiotoxin 10 μM (Latoxan). One day later, iPax myo- conducted previously [10]. Raw and processed data have genic progenitors and freshly isolated embryonic and fetal been deposited to the NCBI Gene Expression Omnibus myoblasts were resuspended in PBS at the concentration (GEO) database and are accessible under the GEO ac- of 3 × 10 cells/μl, while freshly isolated neonatal and sat- cession number GSE121639. ellite cells were resuspended between 3 and 5 × 10 cells/ μl. Donor-derived iPax mononuclear cells (iPax MNCs) Functional annotation were re-isolated 4 weeks after the transplantation of PSC- Gene listsweresubmittedtoGeneOntologyConsortium derived myogenic progenitors into primary recipients, as (geneontology.org) and annotated for complete cellular previously described [10]. For secondary transplantation, components (GO CC) analyses. Top enriched categories iPax MNCs were resuspended in PBS at the concentration were selected based on p valuecorrected formultiplehy- of 1.5 × 10 cells/μl. Ten microliters of a given cell suspen- pothesis testing (B+H FDR). Logarithm of p value was then sion were injected in each TA while the contralateral TA plotted using Microsoft Office Excel or Prism (Graphpad). received the same volume of PBS as internal control. Four to 6 weeks after transplantation, mice were euthanized, Statistical analysis and TAs collected for immunostaining analysis as previ- Differences between samples were assessed by using the ously described [15]. Briefly, muscles were frozen in iso- unpaired two-tailed Student’s t test for single compari- pentane cooled in liquid nitrogen, and serial 10-μm-thick sons, or one-way ANOVA followed by post hoc Tukey cryosections were collected and analyzed. test among multiple groups. p values < 0.05 were consid- ered statistically significant. Immunofluorescence Immunofluorescence staining was performed on fixed cul- Results tured cells and on unfixed TA cryosections as described Predominance of slow and oxidative fibers in primary [18]. Briefly, unfixed TA cryosections were permeabilized transplants by PSC-derived myogenic progenitors with 0.3% Triton/PBS, followed by blocking with 3% BSA/ To assess the in vivo fiber type composition produced by PBS before incubating with the primary antibodies for PSC-derived myogenic progenitors, we generated myo- dystrophin (rabbit polyclonal 1:250, Abcam), MyHC type I genic progenitors from dox-inducible (i)Pax3 and iPax7 (BA-D5), type IIA (sc-71), type IIB (BF-F3), type I+IIA+IIB mouse embryonic stem cells [8, 9]. These myogenic pro- (BF-35), all mouse monoclonal from DSHB, 1:100, ON at genitors were then transplanted into pre-injured tibialis Incitti et al. Skeletal Muscle (2020) 10:17 Page 4 of 11 anterior (TA) muscles of immunocompromised dys- and iPax7-injected muscles. Consequently, the ratio of 4cv trophic NSGmdx mice [19]. As reference control, we total oxidative (I+IIA) myofibers over the total glyco- performed a similar set of transplantations using satellite lytic (IIX+IIB) myofibers is higher in PSC-derived cells freshly isolated from TAs of 3-month-old Pax7- myofibers (Fig. S2a). Of note, engraftment from adult ZsGreen mice [20]. Our immunofluorescence results show satellite cells exhibits the same fiber type composition that both iPax3 and iPax7 myogenic progenitors give rise observed in PBS-injected controls, which is what is to a high proportion of slow-twitch type I, as indicated by expected in TA muscles, mainly composed of type II the expression of MyHC type I when compared to fast-twitch fibers [21]. We observed the same pheno- 4cv muscles injected with adult satellite cells or PBS alone type (Fig. S2b), when NSGmdx mice were injected with (Fig. 1a, b). Similar results were observed using the satellite cells isolated from the slow-twitch soleus muscle in vitro differentiation system, demonstrating that the [3], corroborating published data suggesting that satellite type I MyHC isoform is expressed in PSC-derived cells do not retain a heritable fiber type phenotype [22]. myotubes, unlike samples derived from adult satellite These data suggest that, unlike primary satellite cells, cells freshly isolated from TAs (Fig. S1). As shown in iPax3 and iPax7 PSC-derived myogenic progenitors pref- Fig. 1a and b, fast-twitch glycolytic fibers IIX and IIB erentially express type I MyHC and that they do not were detected at a much lower frequency in iPax3- acquire the fiber type composition of recipient muscles. Fig. 1 PSC-derived myogenic progenitors preferentially give rise to myofibers expressing oxidative MyHC isoforms. a Representative images show 4cv staining for MyHC isoforms (red) and dystrophin (green) in NSGmdx . Nuclei were counterstained with DAPI (blue). Magnification bar, 100 μm. b Bar graphs show the percentage of DYS+MyHC+ double positive with respect to total donor-derived DYS+ myofibers. Data are shown as mean ± 4cv SEM (n =7–8 per group). ***p < 0.001 and ****p < 0.0001. Adult, satellite cells from TAs of 3-month-old NSGmdx mice Incitti et al. Skeletal Muscle (2020) 10:17 Page 5 of 11 Due to the embryonic nature of PSC-derived myogenic fiber type composition similar to the one observed in progenitors [10], a high number of iPax3- and iPax7- dystrophic muscles (Fig. S4c). Of note, transplantation of derived myofibers express embryonic MyHC (Fig. S2c- PSC-derived myofibers into non-dystrophic mice resulted d), in contrast to adult satellite cell-derived myofibers. in a higher frequency of type IIA MyHC fibers with Consistently, cross-sectional area (CSA) analysis shows respect to the host environment (Fig. 2b), and this is prob- that myofibers from PSC-derived myogenic progenitor ably due to the low numbers of IIA myofibers found in transplants display smaller size than those produced by NSG TA muscles. adult satellite cells, as a high frequency of myofibers is Altogether, these data suggest that iPax3/iPax7 PSC- smaller than 100 μm (Fig. S2e). derived skeletal myogenic progenitors give rise to specific Since in vitro-generated iPax3 and iPax7 PSC-derived myofiber types independently from the host environment. myogenic progenitors display a prenatal molecular signa- ture profile [10], we also determined myofiber type engraft- PSC-derived myofibers express genes associated with ment of primary cells isolated from E10.5 embryos mitochondrial function (embryonic), E14.5 fetuses (fetal), and 3-day-old pups (neo- As slow and oxidative fibers are characterized by oxidative natal) samples (Fig. S3a). Embryonic myoblasts give rise to metabolism [3], we reasoned that genes related to mito- a significantly lower proportion of type IIB myofibers chondria function would be highly expressed in PSC- compared to fetal and neonatal satellite cells, in agreement derived myogenic progenitors and myofibers. Before asses- with published data [23]. Nevertheless, they do not show a sing the in vivo gene expression of engrafted PSC-derived preferential expression of slow and oxidative MyHC iso- myofibers, we took advantage of our transcriptome study forms, as observed with myofibers generated by iPax3/ on in vitro-generated cells [10] to investigate whether dif- iPax7 myogenic progenitors, thus suggesting that this is a ferences in the expression of metabolic genes are already unique feature of PSC-derived myofibers rather than an visible at the myogenic progenitor stage. Gene ontology earlier developmental characteristic. analysis of the genes upregulated in iPax3/7 myogenic pro- Altogether, these data suggest that PSC-derived pro- genitors with respect to adult satellite cells demonstrated genitors preferentially give rise to a high proportion of that mitochondrial complexes represent half of the top ten slow-twitch type I and fast-twitch type IIA oxidative categories based on cellular component classification (red myofibers. boxes, Fig. 3a). Accordingly, genes encoding for known components of the mitochondrial respiratory machinery, Fiber type composition of PSC-derived skeletal muscle is such as the proton-transporting two-sector ATPase com- cell-autonomous in primary transplants plex (Atp5c1), mitochondrial chain complex III (Cyc1), and It has been suggested that dystrophic muscles show a IV (Cox5a), and the mitochondrial fatty acid beta-oxidation higher proportion of oxidative, type I myofibers with re- multienzyme complex (Hadha), are highly expressed in spect to wild-type (WT) controls, and this phenomenon iPax3 and iPax7 PSC-derived myogenic progenitors when has been ascribed to a slower degeneration rate of the compared to adult satellite cells (SC, Fig. 3b). To verify slow-twitch fibers in pathologies such as DMD and whether this transcriptional profile is maintained in PSC- FSHD [7, 14, 15]. In agreement, we observed a similar derived myofibers, we performed gene expression analyses 4cv phenotype in TA muscles of NSGmdx mice, which in engrafted TA muscles that had been injected with iPax3 show type I MyHC-positive myofibers, unlike age- and myogenic progenitors. As shown in Fig. 3c, the expression gender-matched muscles from NSG mice (Fig. S4a-b). levels of genes associated with mitochondrial function, Of note, the relative proportion of all analyzed myofibers including Atp5c1, Cox5a, and Cyc1, are similar between is significantly different between the two mouse strains, PSC-derived in vitro-differentiated myotubes (iPax3) and with dystrophic mice showing more oxidative myofibers in vivo-generated myofibers (iPax3 TA). However, the than NSG mice, while the number of glycolytic myofi- expression levels of these genes are significantly lower when bers is significantly lower, as shown in Fig. S4b. Based compared to control non-injected TA muscles from 4cv on these observations, we sought to determine whether NSGmdx (ctrl TA). the observed high proportion of PSC-derived type I Thesedatasuggest that in vitro-generated myogenicpro- myofibers is due to the dystrophic environment. To test genitors are endowed with oxidative properties, which are this hypothesis, we performed transplantation studies in maintained upon in vivo differentiation into myofibers. non-dystrophic recipients. As shown in Fig. 2a,PSC- derived myogenic progenitors injected in a non- Exposure to the muscle environment does not influence dystrophic environment generated preferentially slow and the oxidative phenotype of PSC-derived myofibers in oxidative myofibers. Quantification of the engrafted tissue, secondary transplants as indicated by PSC-derived GFP expression with respect Along with developmental MyHCs, type I MyHC is to the MyHC expression area (Fig. 2b), revealed a trend of abundant in primary prenatal myofibers [24]. Since PSC- Incitti et al. Skeletal Muscle (2020) 10:17 Page 6 of 11 Fig. 2 Fiber type composition of PSC-derived skeletal muscle is not dependent on the background of recipient muscles. a Representative images show staining for MyHC isoforms (red) and dystrophin (white) in TA muscles from NSG mice that had been injected with iPax3 or iPax7 myogenic progenitors. PBS served as control. Green nuclei represent epifluorescence of H B-GFP. Nuclei were counterstained with DAPI (blue). Yellow boxes indicate position of the higher magnification insets below. Magnification bar, 100 μm. b Bar graphs show respective quantification (panel a). Percentages represent the ratio between the engraftment area (GFP) and MyHC expression, in comparison to PBS, represented as the percentage of total MyHC positive. Data are shown as mean ± SEM (n = 4 per group). **p < 0.05, ***p < 0.001, and ****p < 0.0001 derived myogenic progenitors show a prenatal molecular secondary dystrophic recipients. Our results show that signature, and exposure to the endogenous muscle en- both type IIA and IIB MyHC isoforms are expressed at vironment induce their maturation towards a postnatal similar levels in primary and secondary engrafted PSC- phenotype [10], we assessed whether the high expression derived myofibers. The frequency of type I isoform is of MyHC type I in PSC-derived myofibers is due to the lower in secondary recipients, but this is not significantly prenatal developmental stage of these cells. To test this, different from primary grafts (p = 0.064, Fig. 4a). Of we re-isolated iPax3/iPax7 donor-derived MNCs from note, we observed reduced frequency of embryonic primary recipient muscles and injected these into MyHC-positive fibers (Fig. S5a-b). These data suggest Incitti et al. Skeletal Muscle (2020) 10:17 Page 7 of 11 Fig. 3 PSC-derived skeletal muscle fibers express genes associated with mitochondrial function. a Plot shows gene ontology cellular components terms, ranked based on hierarchy, of the genes upregulated in PSC-derived iPax3 and iPax7 myogenic progenitors. Black bars indicate p value in logarithmic scale representing B+H FDR. Red boxes highlight mitochondrial specific categories. b Graphs show the average count per million (CPM) values for representative differentially expressed genes from the categories highlighted in panel a. Data are shown as mean ± SEM (n =4 per group). ***p < 0.001 and ****p < 0.0001. c Graph shows RT-qPCR results of mRNA expression levels of the genes for ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 (Atp5c1), cytochrome c oxidase subunit 5A (Cox5a), and cytochrome c1 (Cyc1). 4cv Data are shown as mean ± SEM (n = 4 per group). **p < 0.01. iPax3 TA, NSGmdx muscles injected with iPax3 myogenic progenitors; ctrl TA, 4cv non-injected muscles of 3-month-old NSGmdx mice Incitti et al. Skeletal Muscle (2020) 10:17 Page 8 of 11 Fig. 4 Exposure to the adult endogenous environment does not influence the oxidative phenotype of PSC-derived myofibers. a Representative 4cv images show staining for MyHC isoforms (red) and dystrophin (green) in TA muscles from NSGmdx mice that had been injected with iPax3 or iPax7-derived re-isolated mononuclear cells (MNCs). PBS served as control. Nuclei were counterstained with DAPI (blue). Magnification bar, 100 μm. b Bar graphs show respective quantification (panel a), as indicated by the percentage of DYS+MyHC+ double positive with respect to total DYS+ myofibers for iPax3-injected MNCs (gray bar, secondary recipients) in comparison to data from iPax3-injected myogenic progenitors (black bars, primary recipients). Data are shown as mean ± SEM (n = 4 per group). **p < 0.01 that the preferred composition of oxidative myofibers is Discussion not due to the immature phenotype of the primary grafts Several studies have suggested that disease pathogenesis but rather due to an intrinsic property of iPax3 and due to neuromuscular disorders can affect fiber type iPax7-derived skeletal muscle. Importantly, muscles composition and therefore muscle properties, such as injected with re-isolated MNCs display higher frequency endurance and resistance to fatigue [6, 25]. Within this of glycolytic fast type IIX myofibers compared to their heterogeneous group of disorders, DMD is characterized primary engrafted counterparts (Fig. 4). Type IIX MyHC by highly fibrotic skeletal muscle [26] and increased is only expressed in myofibers after birth, arising from number of slow myofibers, which display higher resist- the secondary myogenesis wave [24]. Therefore, the ance to pathogenesis, while glycolytic fast fibers progres- maturation of PSC-derived myogenic progenitors, sively vanish with disease progression [7]. Therefore, occurring upon exposure to the endogenous muscle when developing a cell replacement strategy for skeletal environment, is also reflected in the generation of post- muscle disorders, it is important to determine the fiber natal myofibers. type composition of engrafted cells. Altogether, these results demonstrate that PSC-derived In this study, we investigated the content of myofiber myogenic progenitors have an intrinsic capacity to pref- types generated following the transplantation of PSC- erentially give rise to oxidative myofibers. derived myogenic progenitors. We focused on mouse- Incitti et al. Skeletal Muscle (2020) 10:17 Page 9 of 11 to-mouse engraftment to prevent species-specific differ- metabolic properties of human PSC-derived myogenic ences that could introduce bias to our data, as human progenitors. muscles lack the type IIB MyHC isoform [4]. Our results To further assess whether the level of maturation of showed that PSC-derived engrafted muscle preferentially transplanted cells impacts the fiber type composition of expresses types I and IIA MyHC isoforms, which are fea- engrafted skeletal muscle, we re-isolated the donor- tures of slow and/or oxidative myofibers [3]. Our data derived MNC fraction from primary grafts and trans- also show that a high number of PSC-derived myofibers planted these into secondary recipient mice. Our results express the embryonic MyHC isoform, indicating that show that engrafted muscle in secondary recipients dis- the prenatal molecular signature of iPax3 and iPax7 plays an increased proportion of glycolytic fast-twitch PSC-derived myogenic progenitors is likely maintained fiber IIX with respect to primary counterparts. Of note, in their respective regenerating muscle in primary grafts they also show lower number of fibers expressing the [10]. Based on this observation, we hypothesized that the embryonic MyHC isoform. These results reinforce and high proportion of oxidative myofibers could be ascribed complement our recent data demonstrating that the to two different reasons: (1) PSC-derived myofibers maturation of iPax3 and iPax7 myogenic progenitors generate oxidative fibers as an indirect result of the occurs upon exposure to the endogenous adult muscle pressure from the dystrophic environment, which de- environment [10]. However, when looking at the num- lays degeneration of diseased slow-twitch fibers, (2) bers of slow and oxidative donor-derived myofibers, we PSC-derived myofibers are immature because they de- found that these are comparable between primary and rive from myogenic progenitors with a prenatal mo- secondary engrafted muscle. This observation suggests lecular signature, hence they express more type I and that the prenatal molecular signature of in vitro-gener- IIA MyHC isoforms, which arise earlier during devel- ated PSC-derived myogenic progenitors might be only opmental myogenesis [24]. marginally responsible for the high number of slow-twitch To rule out the influence of the recipient environment, and oxidative myofibers found upon engraftment, al- we transplanted non-dystrophic mice and compared the though we cannot exclude that the donor-derived MNC pattern of fiber type composition to engrafted muscles fraction did not sufficiently mature after the 4-week- from dystrophic mice. Our data show that PSC-derived exposure to the endogenous adult muscle environment. myofibers express more type I MyHC, regardless of the When generating cells suitable for replacement therap- recipient genetic background, suggesting this is a cell- ies in the context of skeletal muscle, several characteris- autonomous feature of the donor cells. Accordingly, we tics should be evaluated, including engraftment and interrogated the transcriptome of PSC-derived iPax3 and regenerative capacity, ability to repopulate the satellite iPax7 myogenic progenitors [10], and we found that cell compartment, and the long-term contribution to the mitochondrial complexes are the most represented cellu- recipient tissue’s functionality. One of the key functions lar components in both cell lines, which is in accordance of skeletal muscle is the ability to respond to innervation with oxidative metabolic needs. Moreover, we found that stimuli and to contract according to specific endurance representative genes encoding for proteins of mitochon- and speed abilities, which are unique to different groups drial complexes are also expressed at similar levels in of muscles, but highly plastic. To achieve this level of PSC-derived in vitro myotubes and in vivo myofibers, complexity, it is fundamental to assess the ability of suggesting that PSC-derived myogenic progenitors main- in vitro-generated myogenic progenitors to contribute to tain oxidative properties upon in vivo differentiation to the tissue physiology, thus ensuring proper integration myofibers. However, we also observed that non-injected into the host environment. Up to date, several studies dystrophic muscles express higher levels of the selected have assessed the therapeutic properties of muscle cells mitochondrial genes. These data indicate that the myofi- from different sources [30]. However, very few studies bers generated from PSC-derived myogenic progenitor, have analyzed the fiber type specification of cells gener- although expressing genes related to mitochondrial ac- ated in vitro [11, 12, 31], and only one assessed the con- tivity, may have a metabolic profile proper of immature tribution of transplanted cells to fiber type specification organelles, as recently shown for fetal and neonatal satel- in vivo [13], which showed that myofibers generated by lite cells [27]. In the same paper, Pala et al. suggest that PSC-derived teratomas preferentially express type I, IIA, mouse myogenic progenitors at different developmental and IIB MyHC isoforms, while IIX myofibers were not and functional stages have different metabolic require- detected. ments, and metabolic reprogramming may influence the The present study documents for the first time the progression throughout these stages. Of note, human in vivo myofiber composition resulting from the trans- PSC-derived myogenic progenitors have been described plantation of in vitro-generated PSC-derived myogenic to display fetal characteristics [18, 28, 29]. Therefore, fu- progenitors. Our findings show that donor-derived myo- ture studies should focus on fiber type composition and fiber contribution by PSC-derived myogenic progenitors Incitti et al. Skeletal Muscle (2020) 10:17 Page 10 of 11 is characterized by the expression of slow and oxidative DYS+MyHC+ double positive with respect to total donor-derived DYS+ myosin heavy-chain isoforms, in addition to develop- myofibers. Data are shown as mean ± SEM (n = 4-5 per group) **p < 0.01. Figure S4. Characterization of fiber type composition based on re- mental myosins, and that transplantation of the donor- cipient genetic background. a) Representative images show staining for derived MNC fraction into secondary recipients results type I MyHC (red) and Laminin (green) in TA muscles from dystrophic 4cv in myofibers still predominantly expressing slow and NSGmdx and NSG mice. b) Graph shows respective quantification (panel a), as indicated by the number of MyHC+ myofibers in TA muscles oxidative myosin heavy-chain isoforms, whereas expres- 4cv from dystrophic NSGmdx and NSG mice. Data are shown as mean ± sion of developmental myosins is reduced and type IIX SEM (n = 6 per group). *p < 0.05, **p < 0.01, and ***p < 0.001. c) Graph is increased. Taken together, our data suggest that ex- bar shows percentage of MyHC+ donor-derived myofibers from Fig. 2a in comparison to iPax3 and iPax7 samples from Fig. 1b. Data are shown as posure to the adult muscle environment favors postnatal mean ± SEM (n = 6-8 per group), and no statistically significant differ- myofiber composition switch and that elevated oxidative ences were observed among groups. Figure S5. Characterization of fiber fiber type composition is a cell-autonomous characteris- types from secondary grafts. a) Representative image shows staining for embryonic MyHC (red) and dystrophin (green) in TA muscles of second- tic of iPax3 and iPax7 PSC-derived myofibers, which ary recipients that had been injected with iPax3-derived MNCs. Magnifica- could be relevant for the development of cell replace- tion bar: 100 μm. b) Graph shows respective quantification (panel a), as ment therapy for DMD. indicated by the number of DYS+MyHC+ double positive with respect to total DYS+ myofibers of secondary grafts (iPax3 MNCs) in comparison to primary-injected TAs (iPax3). Data are shown as mean ± SEM (n = 4 per Conclusions group). **p < 0.01. We have shown that iPax3 and iPax7 PSC-derived myo- genic progenitors cell-autonomously generate slow and Abbreviations oxidative myofibers upon transplantation into dystrophic CSA: Cross-sectional area; DAPI: 4,6-diamidino-2-phenylindole; DM1- 2: Myotonic dystrophy 1 and 2; DMD: Duchenne muscular dystrophy; mice. This capacity is maintained upon re-isolation of FSHD: Facioscapulohumeral muscular dystrophy; H2B-GFP: Histone 2B-green donor-derived MNCs and their injection into secondary fluorescent protein; iPax3/7: Inducible Pax3/7; MyHC: Myosin heavy chain; recipients. If these findings are recapitulated with human MNC: Mononuclear cell; NSG: NOD-scid IL2Rgnull; PBS: Phosphate-buffer saline; PSC: Pluripotent stem cells; SB: Staining buffer; TA: Tibialis anterior PSC-derived myofibers, these results would suggest that in vitro-generated myogenic progenitors could have a Acknowledgements double benefit in the therapy of DMD: they may (i) re- The authors are grateful to Magally Ramirez for histology assistance and to all the members of the Perlingeiro laboratory for helpful discussion. place diseased skeletal muscle and (ii) provide myofibers more resistant to disease progression. Authors’ contributions T.I. designed and performed the research, analyzed the data, and wrote the Supplementary information manuscript. A.M. contributed with experimental design, research, Supplementary information accompanies this paper at https://doi.org/10. interpretation of the data, and writing of the manuscript. J.J., K.L., and A.Y. 1186/s13395-020-00234-5. performed the research. R.C.R.P. contributed with experimental design, interpretation of the data, and writing of the manuscript. The authors read and approved the final manuscript. Additional file 1: Figure S1. Fiber type composition of myotubes resulting from the in vitro differentiation of PSC-derived iPax3/iPax7 myo- Funding genic progenitors and adult satellite cells. Representative images show This project was supported by grants from the National Institute of Arthritis immunofluorescence staining for pan, embryonic, type I and IIA MyHC and Musculoskeletal and Skin Diseases, R01 AR055299 and R01 AR071439 isoforms (red) in myotubes resulting from the in vitro differentiation of (R.C.R.P.). iPax3 and iPax7 PSC-derived myogenic progenitors, as well as adult satel- lite cells (adult). Nuclei were counterstained with DAPI (blue). Magnifica- Availability of data and materials tion bar: 100 μm. Figure S2. Characterization of fiber type composition. The datasets used during the current study are deposited and publicly a) Graph shows the ratio of oxidative DYS+MyHC+ type I and type IIA available. Materials used in this study are commercially available. over glycolytic type IIX and IIB myofibers following the transplantation with iPax3 and iPax7 myogenic progenitors, and adult satellite cells. Data are shown as mean ± SEM (n = 7-8 per group). **p < 0.01. b) Graph bars Ethics approval and consent to participate show percentage of DYS+MyHC+ double positive with respect to total All procedures were approved by the University of Minnesota Institutional DYS+ in TA muscles transplanted with satellite cells isolated from TA or Animal Care and Use Committee and were performed in accordance with soleus muscles. Data are shown as mean ± SEM (n = 4 per group) and the National Research Council’s Guide for the Care and Use of Laboratory no statistically significant differences were observed among groups. c) Animals (2011). Representative images show staining for embryonic MyHC (red) and dys- 4cv trophin (green) in TA muscles from NSGmdx mice that had been Consent for publication injected with iPax3 or iPax7 myogenic progenitors. Adult satellite cells Not applicable. served as control. Magnification bar: 100 μm. d) Graph shows respective quantification (panel c), as indicated by the number of DYS+MyHC+ Competing interests double positive with respect to total DYS+. Data are shown as mean ± The authors declare no competing financial interests. SEM (n = 6-8 per group). *p < 0.05 and ***p < 0.001. d) Quantification of CSA (μm ) of grafts from indicated groups. Data are shown as mean ± Received: 12 January 2020 Accepted: 17 May 2020 SEM (n = 4-5 per group). *p < 0.05. Figure S3. Characterization of fiber type composition following the transplantation of embryonic, fetal, and neonatal primary cells. a) Representative images show staining for MyHC References isoforms (red) and dystrophin (green). Nuclei were counterstained with 1. Yin H, Price F, Rudnicki MA. Satellite cells and the muscle stem cell niche. DAPI (blue). Magnification bar: 100 μm. b) Percentage quantification of Physiol Rev. 2013;93(1):23–67. Incitti et al. Skeletal Muscle (2020) 10:17 Page 11 of 11 2. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 26. Kharraz Y, Guerra J, Pessina P, Serrano AL, Munoz-Canoves P. Understanding 1961;9:493–5. the process of fibrosis in Duchenne muscular dystrophy. Biomed Res Int. 3. Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol 2014;2014:965631. Rev. 2011;91(4):1447–531. 27. Pala F, Di Girolamo D, Mella S, Yennek S, Chatre L, Ricchetti M, et al. 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Pluripotent stem cell-derived skeletal muscle fibers preferentially express myosin heavy-chain isoforms associated with slow and oxidative muscles

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Abstract

Background: Skeletal muscle function is essential for health, and it depends on the proper activity of myofibers and their innervating motor neurons. Each adult muscle is composed of different types of myofibers with distinct contractile and metabolic characteristics. The proper balance of myofiber types is disrupted in most muscle degenerative disorders, representing another factor compromising muscle function. One promising therapeutic approach for the treatment of these diseases is cell replacement based on the targeted differentiation of pluripotent stem cells (PSCs) towards the myogenic lineage. We have previously shown that transient induction of Pax3 or Pax7 in PSCs allows for the generation of skeletal myogenic progenitors endowed with myogenic regenerative potential, but whether they contribute to different fiber types remains unknown. Results: Here, we investigate the fiber type composition of mouse PSC-derived myofibers upon their transplantation into dystrophic and non-dystrophic mice. Our data reveal that PSC-derived myofibers express slow and oxidative myosin heavy-chain isoforms, along with developmental myosins, regardless of the recipient background. Furthermore, transplantation of the mononuclear cell fraction re-isolated from primary grafts into secondary recipients results in myofibers that maintain preferential expression of slow and oxidative myosin heavy- chain isoforms but no longer express developmental myosins, thus indicating postnatal composition. Conclusions: Considering oxidative fibers are commonly spared in the context of dystrophic pathogenesis, this feature of PSC-derived myofibers could be advantageous for therapeutic applications. Keywords: Pluripotent stem cells (PSC), Skeletal myogenesis, Myofibers, Fiber types, Fast, Slow, Pax3, Pax7, Engraftment, Muscle stem cell Background cells [1]. Upon injury or disease, muscle stem cells are acti- Adult skeletal muscles are characterized by several major vated to regenerate new muscle tissue [2]. Each adult components: myofibers, their innervating motor neurons muscle is a heterogeneous combination of different types of and muscle resident stem cells, along with fibroblasts, myofibers characterized by specific contractile and meta- fibro-adipogenic progenitors, endothelial cells, and immune bolic properties, which are classified into three main cat- egories: (i) oxidative slow-twitch, expressing type I myosin heavy chain (MyHC); (ii) oxidative fast-twitch, expressing * Correspondence: perli032@umn.edu type IIA MyHC; and (iii) glycolytic fast-twitch, expressing Lillehei Heart Institute, Department of Medicine, University of Minnesota, type IIX and IIB MyHC isoforms [3]. In mammals, prenatal 4-128 CCRB, 2231 6th St. SE, Minneapolis, MN 55455, USA Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Incitti et al. Skeletal Muscle (2020) 10:17 Page 2 of 11 fibers initially express the embryonic MyHC isoform, oxidative slow myofibers compared to freshly isolated followed by fetal/neonatal, type I, and, later on, type II, primary muscle stem cells. These results suggest that which is further classified into IIA, IIX, and/or IIB, with the PSC-derived myofibers could be beneficial in the context latter not expressed in humans [4]. Developmental MyHC of muscular dystrophies, not only for their regenerative isoforms disappear during the first weeks of postnatal life capacity, but also for their ability to provide slow-twitch, but the embryonic type becomes re-expressed for a short dystrophy-resistant tissue. time window during regeneration in adults, a process that partially recapitulates developmental myogenesis [3]. Even Methods though each muscle acquires a unique pattern of MyHC Cell culture and differentiation expression and consequently, fiber type characteristics, their Inducible iPax3 and iPax7 mouse embryonic stem (ES) composition is highly plastic and can be influenced by cell lines were generated as previously described [16]. several factors, such as muscle loading, exercise, hormone- mES cells were maintained in knock-out DMEM mediated signaling, and disease [3]. Moreover, myofibers (Invitrogen) supplemented with 15% FBS (Embryomax undergo fiber type transitioning, which generate hybrid ES-qualified FBS—Millipore), 1% penicillin/strepto- myofibers expressing two or more different MyHC iso- mycin (Invitrogen), 2 mM glutamax (Invitrogen), 0.1 forms at the same time [5]. mM non-essential aminoacids (Invitrogen), 0.1 mM β- Many muscle degenerative disorders, including Du- mercaptoethanol (Invitrogen), and 1000 U/ml LIF chenne muscular dystrophy (DMD), facioscapulohumeral (Millipore). Skeletal myogenic differentiation was muscular dystrophy (FSHD), myotonic dystrophies 1 and achieved as described [17]. Briefly, cells were detached 2 (DM1-2), and Pompe disease, exhibit disrupted regula- and the supernatant was then incubated in an orbital tion of fiber type composition, thus further compromising shaker at 80 rpm at the concentration of 40000 cells/ muscle function [6]. For instance, in DMD, the fast fibers ml in embryoid body (EB) differentiation medium, are preferentially affected while the slow fibers degenerate composed of IMDM (Invitrogen) supplemented with more slowly, suggesting that dystrophin may have a major 15% FBS (Embryomax ES-qualified FBS), 1% penicil- role in fibers with the ability to respond to the highest lin/streptomycin (Invitrogen), 2 mM glutamax (Invi- frequency stimulation [7]. One promising therapeutic trogen), 50 μg/ml ascorbic acid (Sigma-Aldrich), and approach for muscle wasting diseases is cell replacement 4.5 mM monothioglycerol (MP Biomedicals). Pax3 or based on the myogenic differentiation of pluripotent stem Pax7 induction is induced starting from day 3 by ad- cells (PSCs). We developed a doxycycline-inducible sys- ministering 1 μM doxycycline (dox). Day 5 EBs were tem that enables the generation of large numbers of prolif- disaggregated, incubated with Fc block (1 μl/million erating early skeletal myogenic progenitors from PSCs cells, BD Biosciences) for 5 min and then with Flk1- through the transient expression of the myogenic tran- APC and Vcam1-biotin-conjugated antibodies (1 μl/ scription factors Pax3 or Pax7. We have documented that million cells, e-Bioscience) for 20 min on ice followed upon transplantation, these myogenic progenitors give rise by 5-min incubation with streptavidin-PeCy7. Cells to new myofibers, seed the muscle stem cell compartment, were washed twice with PBS and then resuspended in provide functional muscle improvement, and remodel staining buffer (SB) composed of PBS containing 10% their molecular signature to acquire a more mature FBS and propidium iodide (PI) to exclude dead cells. phenotype [8–10]. Vcam1+FLK1- cells were sorted using a FACSAria II A few studies have investigated the fiber type compos- (BD Biosciences), replated on gelatin-coated dishes, ition of PSC-derived skeletal muscle in vitro [11, 12], but and further expanded for 2–3 passages before collec- to date, only one report has documented in vivo fiber tion for RNA extraction or transduced with H B-GFP- type characteristics of engrafted tissue, which was gener- encoding lentiviral vector fortransplantation studies ated from PSC-derived teratomas [13]. Since fiber type into NSG mice. For terminal differentiation, 50,000 composition plays an important role in skeletal muscle cells were deposited onto a well of 24-well plates, adaptation to pathological stimuli [7, 14, 15], determining allowed to reach confluence, and then cultured in EB how in vitro-generated PSC-derived myogenic progenitors media without dox for 5-7 days, after which cells were participate in this process will provide important informa- collected for RNA extraction or immunofluorescence tion for the development of effective cell-based skeletal staining. muscle replacement therapies. Here, we sought to investigate the fiber type compos- Labeling of myogenic progenitors ition of engrafted muscles following the transplantation H B-GFP-encoding lentiviral vectors were prepared as of PSC-derived myogenic progenitors in a mouse model described [9]. Briefly, vectors were co-transfected with of DMD. These in vivo studies revealed that PSC- packaging plasmids Δ8.91 and pVSVG into 293T cells derived skeletal myofibers display a higher proportion of using the LTX transfection reagent (Thermofisher Incitti et al. Skeletal Muscle (2020) 10:17 Page 3 of 11 Scientific). Lentiviral-containing supernatant was col- 4 °C. The following day, samples were rinsed with PBS lected 48 h after transfection, filtered, and used for trans- and then incubated with the secondary antibodies goat duction of myogenic progenitors upon centrifugation for anti-rabbit Alexa Fluor-488 or 647, and with goat anti- 1.5 h at 2500 rpm and 37 °C. mouse Alexa Fluor-555 and DAPI for 1 h at RT. After washing three times with PBS, sections were briefly dried Primary cell isolation and transplantation studies and mounted using Prolong Gold with DAPI (Invitrogen). Animal experiments were carried out in strict accordance Pictures were acquired with Axioimager M1 fluorescence to protocols approved by the University of Minnesota Insti- microscope (Zeiss) and analyzed with ZEN Blue software. tutional Animal Care and Use Committee. Primary embry- Quantification of positive myofibers and calculation of onic and fetal myoblasts were isolated and dissected from cross-sectional area (CSA) were performed using Fiji Ima- E10.5 and E14.5 Myf5Cre-Rosa26YFP [5, 6]mouse em- geJ software. bryos and fetuses, respectively, while neonatal and adult satellite cells were isolated from hindlimbs of Pax7- RNA isolation and gene expression analyses ZsGreen mice [7] as previously described [10]. For RNA Samples for RNA were resuspended in Qiazol (Qiagen) extraction and transplantation studies, we used YFP+ cells and RNA extracted with the Qiagen MiRNeasy Mini Kit, for prenatal myoblasts, ZsGreen+ cells for postnatal satellite according to manufacturer’s instructions. After RNA ex- cells, and CD31-/CD45-/Itga7+/Vcam1+/GFP+ for PSC traction, in column DNase digestion was performed and donor-derived satellite cells. Freshly isolated ZsGreen+ cells samples were retro-transcribed using Superscript Vilo were also seeded on gelatin-coated multiwell plates in the (Invitrogen). Gene expression analyses were performed presence of EB medium and cultured for 5 to 7 days to using an amount of cDNA corresponding to 5 ng of induce terminal differentiation, which was followed by im- starting RNA for each reaction. RT-qPCR was per- munofluorescence analyses. formed using Premix Ex Taq (Probe qPCR) Master Mix For transplantation studies, hindlimbs of 6–8-week-old (Takara) and TaqMan probes (Applied Biosystems). mdx4cv male NOD-scid IL2Rgnull (NSG) and NSG [14] mice were irradiated with a 12-Gy single dose at 24 h prior RNA sequencing and data availability to injury of both tibialis anterior (TA) muscles with 15 μl Sequencing data and corresponding analyses have been of cardiotoxin 10 μM (Latoxan). One day later, iPax myo- conducted previously [10]. Raw and processed data have genic progenitors and freshly isolated embryonic and fetal been deposited to the NCBI Gene Expression Omnibus myoblasts were resuspended in PBS at the concentration (GEO) database and are accessible under the GEO ac- of 3 × 10 cells/μl, while freshly isolated neonatal and sat- cession number GSE121639. ellite cells were resuspended between 3 and 5 × 10 cells/ μl. Donor-derived iPax mononuclear cells (iPax MNCs) Functional annotation were re-isolated 4 weeks after the transplantation of PSC- Gene listsweresubmittedtoGeneOntologyConsortium derived myogenic progenitors into primary recipients, as (geneontology.org) and annotated for complete cellular previously described [10]. For secondary transplantation, components (GO CC) analyses. Top enriched categories iPax MNCs were resuspended in PBS at the concentration were selected based on p valuecorrected formultiplehy- of 1.5 × 10 cells/μl. Ten microliters of a given cell suspen- pothesis testing (B+H FDR). Logarithm of p value was then sion were injected in each TA while the contralateral TA plotted using Microsoft Office Excel or Prism (Graphpad). received the same volume of PBS as internal control. Four to 6 weeks after transplantation, mice were euthanized, Statistical analysis and TAs collected for immunostaining analysis as previ- Differences between samples were assessed by using the ously described [15]. Briefly, muscles were frozen in iso- unpaired two-tailed Student’s t test for single compari- pentane cooled in liquid nitrogen, and serial 10-μm-thick sons, or one-way ANOVA followed by post hoc Tukey cryosections were collected and analyzed. test among multiple groups. p values < 0.05 were consid- ered statistically significant. Immunofluorescence Immunofluorescence staining was performed on fixed cul- Results tured cells and on unfixed TA cryosections as described Predominance of slow and oxidative fibers in primary [18]. Briefly, unfixed TA cryosections were permeabilized transplants by PSC-derived myogenic progenitors with 0.3% Triton/PBS, followed by blocking with 3% BSA/ To assess the in vivo fiber type composition produced by PBS before incubating with the primary antibodies for PSC-derived myogenic progenitors, we generated myo- dystrophin (rabbit polyclonal 1:250, Abcam), MyHC type I genic progenitors from dox-inducible (i)Pax3 and iPax7 (BA-D5), type IIA (sc-71), type IIB (BF-F3), type I+IIA+IIB mouse embryonic stem cells [8, 9]. These myogenic pro- (BF-35), all mouse monoclonal from DSHB, 1:100, ON at genitors were then transplanted into pre-injured tibialis Incitti et al. Skeletal Muscle (2020) 10:17 Page 4 of 11 anterior (TA) muscles of immunocompromised dys- and iPax7-injected muscles. Consequently, the ratio of 4cv trophic NSGmdx mice [19]. As reference control, we total oxidative (I+IIA) myofibers over the total glyco- performed a similar set of transplantations using satellite lytic (IIX+IIB) myofibers is higher in PSC-derived cells freshly isolated from TAs of 3-month-old Pax7- myofibers (Fig. S2a). Of note, engraftment from adult ZsGreen mice [20]. Our immunofluorescence results show satellite cells exhibits the same fiber type composition that both iPax3 and iPax7 myogenic progenitors give rise observed in PBS-injected controls, which is what is to a high proportion of slow-twitch type I, as indicated by expected in TA muscles, mainly composed of type II the expression of MyHC type I when compared to fast-twitch fibers [21]. We observed the same pheno- 4cv muscles injected with adult satellite cells or PBS alone type (Fig. S2b), when NSGmdx mice were injected with (Fig. 1a, b). Similar results were observed using the satellite cells isolated from the slow-twitch soleus muscle in vitro differentiation system, demonstrating that the [3], corroborating published data suggesting that satellite type I MyHC isoform is expressed in PSC-derived cells do not retain a heritable fiber type phenotype [22]. myotubes, unlike samples derived from adult satellite These data suggest that, unlike primary satellite cells, cells freshly isolated from TAs (Fig. S1). As shown in iPax3 and iPax7 PSC-derived myogenic progenitors pref- Fig. 1a and b, fast-twitch glycolytic fibers IIX and IIB erentially express type I MyHC and that they do not were detected at a much lower frequency in iPax3- acquire the fiber type composition of recipient muscles. Fig. 1 PSC-derived myogenic progenitors preferentially give rise to myofibers expressing oxidative MyHC isoforms. a Representative images show 4cv staining for MyHC isoforms (red) and dystrophin (green) in NSGmdx . Nuclei were counterstained with DAPI (blue). Magnification bar, 100 μm. b Bar graphs show the percentage of DYS+MyHC+ double positive with respect to total donor-derived DYS+ myofibers. Data are shown as mean ± 4cv SEM (n =7–8 per group). ***p < 0.001 and ****p < 0.0001. Adult, satellite cells from TAs of 3-month-old NSGmdx mice Incitti et al. Skeletal Muscle (2020) 10:17 Page 5 of 11 Due to the embryonic nature of PSC-derived myogenic fiber type composition similar to the one observed in progenitors [10], a high number of iPax3- and iPax7- dystrophic muscles (Fig. S4c). Of note, transplantation of derived myofibers express embryonic MyHC (Fig. S2c- PSC-derived myofibers into non-dystrophic mice resulted d), in contrast to adult satellite cell-derived myofibers. in a higher frequency of type IIA MyHC fibers with Consistently, cross-sectional area (CSA) analysis shows respect to the host environment (Fig. 2b), and this is prob- that myofibers from PSC-derived myogenic progenitor ably due to the low numbers of IIA myofibers found in transplants display smaller size than those produced by NSG TA muscles. adult satellite cells, as a high frequency of myofibers is Altogether, these data suggest that iPax3/iPax7 PSC- smaller than 100 μm (Fig. S2e). derived skeletal myogenic progenitors give rise to specific Since in vitro-generated iPax3 and iPax7 PSC-derived myofiber types independently from the host environment. myogenic progenitors display a prenatal molecular signa- ture profile [10], we also determined myofiber type engraft- PSC-derived myofibers express genes associated with ment of primary cells isolated from E10.5 embryos mitochondrial function (embryonic), E14.5 fetuses (fetal), and 3-day-old pups (neo- As slow and oxidative fibers are characterized by oxidative natal) samples (Fig. S3a). Embryonic myoblasts give rise to metabolism [3], we reasoned that genes related to mito- a significantly lower proportion of type IIB myofibers chondria function would be highly expressed in PSC- compared to fetal and neonatal satellite cells, in agreement derived myogenic progenitors and myofibers. Before asses- with published data [23]. Nevertheless, they do not show a sing the in vivo gene expression of engrafted PSC-derived preferential expression of slow and oxidative MyHC iso- myofibers, we took advantage of our transcriptome study forms, as observed with myofibers generated by iPax3/ on in vitro-generated cells [10] to investigate whether dif- iPax7 myogenic progenitors, thus suggesting that this is a ferences in the expression of metabolic genes are already unique feature of PSC-derived myofibers rather than an visible at the myogenic progenitor stage. Gene ontology earlier developmental characteristic. analysis of the genes upregulated in iPax3/7 myogenic pro- Altogether, these data suggest that PSC-derived pro- genitors with respect to adult satellite cells demonstrated genitors preferentially give rise to a high proportion of that mitochondrial complexes represent half of the top ten slow-twitch type I and fast-twitch type IIA oxidative categories based on cellular component classification (red myofibers. boxes, Fig. 3a). Accordingly, genes encoding for known components of the mitochondrial respiratory machinery, Fiber type composition of PSC-derived skeletal muscle is such as the proton-transporting two-sector ATPase com- cell-autonomous in primary transplants plex (Atp5c1), mitochondrial chain complex III (Cyc1), and It has been suggested that dystrophic muscles show a IV (Cox5a), and the mitochondrial fatty acid beta-oxidation higher proportion of oxidative, type I myofibers with re- multienzyme complex (Hadha), are highly expressed in spect to wild-type (WT) controls, and this phenomenon iPax3 and iPax7 PSC-derived myogenic progenitors when has been ascribed to a slower degeneration rate of the compared to adult satellite cells (SC, Fig. 3b). To verify slow-twitch fibers in pathologies such as DMD and whether this transcriptional profile is maintained in PSC- FSHD [7, 14, 15]. In agreement, we observed a similar derived myofibers, we performed gene expression analyses 4cv phenotype in TA muscles of NSGmdx mice, which in engrafted TA muscles that had been injected with iPax3 show type I MyHC-positive myofibers, unlike age- and myogenic progenitors. As shown in Fig. 3c, the expression gender-matched muscles from NSG mice (Fig. S4a-b). levels of genes associated with mitochondrial function, Of note, the relative proportion of all analyzed myofibers including Atp5c1, Cox5a, and Cyc1, are similar between is significantly different between the two mouse strains, PSC-derived in vitro-differentiated myotubes (iPax3) and with dystrophic mice showing more oxidative myofibers in vivo-generated myofibers (iPax3 TA). However, the than NSG mice, while the number of glycolytic myofi- expression levels of these genes are significantly lower when bers is significantly lower, as shown in Fig. S4b. Based compared to control non-injected TA muscles from 4cv on these observations, we sought to determine whether NSGmdx (ctrl TA). the observed high proportion of PSC-derived type I Thesedatasuggest that in vitro-generated myogenicpro- myofibers is due to the dystrophic environment. To test genitors are endowed with oxidative properties, which are this hypothesis, we performed transplantation studies in maintained upon in vivo differentiation into myofibers. non-dystrophic recipients. As shown in Fig. 2a,PSC- derived myogenic progenitors injected in a non- Exposure to the muscle environment does not influence dystrophic environment generated preferentially slow and the oxidative phenotype of PSC-derived myofibers in oxidative myofibers. Quantification of the engrafted tissue, secondary transplants as indicated by PSC-derived GFP expression with respect Along with developmental MyHCs, type I MyHC is to the MyHC expression area (Fig. 2b), revealed a trend of abundant in primary prenatal myofibers [24]. Since PSC- Incitti et al. Skeletal Muscle (2020) 10:17 Page 6 of 11 Fig. 2 Fiber type composition of PSC-derived skeletal muscle is not dependent on the background of recipient muscles. a Representative images show staining for MyHC isoforms (red) and dystrophin (white) in TA muscles from NSG mice that had been injected with iPax3 or iPax7 myogenic progenitors. PBS served as control. Green nuclei represent epifluorescence of H B-GFP. Nuclei were counterstained with DAPI (blue). Yellow boxes indicate position of the higher magnification insets below. Magnification bar, 100 μm. b Bar graphs show respective quantification (panel a). Percentages represent the ratio between the engraftment area (GFP) and MyHC expression, in comparison to PBS, represented as the percentage of total MyHC positive. Data are shown as mean ± SEM (n = 4 per group). **p < 0.05, ***p < 0.001, and ****p < 0.0001 derived myogenic progenitors show a prenatal molecular secondary dystrophic recipients. Our results show that signature, and exposure to the endogenous muscle en- both type IIA and IIB MyHC isoforms are expressed at vironment induce their maturation towards a postnatal similar levels in primary and secondary engrafted PSC- phenotype [10], we assessed whether the high expression derived myofibers. The frequency of type I isoform is of MyHC type I in PSC-derived myofibers is due to the lower in secondary recipients, but this is not significantly prenatal developmental stage of these cells. To test this, different from primary grafts (p = 0.064, Fig. 4a). Of we re-isolated iPax3/iPax7 donor-derived MNCs from note, we observed reduced frequency of embryonic primary recipient muscles and injected these into MyHC-positive fibers (Fig. S5a-b). These data suggest Incitti et al. Skeletal Muscle (2020) 10:17 Page 7 of 11 Fig. 3 PSC-derived skeletal muscle fibers express genes associated with mitochondrial function. a Plot shows gene ontology cellular components terms, ranked based on hierarchy, of the genes upregulated in PSC-derived iPax3 and iPax7 myogenic progenitors. Black bars indicate p value in logarithmic scale representing B+H FDR. Red boxes highlight mitochondrial specific categories. b Graphs show the average count per million (CPM) values for representative differentially expressed genes from the categories highlighted in panel a. Data are shown as mean ± SEM (n =4 per group). ***p < 0.001 and ****p < 0.0001. c Graph shows RT-qPCR results of mRNA expression levels of the genes for ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 (Atp5c1), cytochrome c oxidase subunit 5A (Cox5a), and cytochrome c1 (Cyc1). 4cv Data are shown as mean ± SEM (n = 4 per group). **p < 0.01. iPax3 TA, NSGmdx muscles injected with iPax3 myogenic progenitors; ctrl TA, 4cv non-injected muscles of 3-month-old NSGmdx mice Incitti et al. Skeletal Muscle (2020) 10:17 Page 8 of 11 Fig. 4 Exposure to the adult endogenous environment does not influence the oxidative phenotype of PSC-derived myofibers. a Representative 4cv images show staining for MyHC isoforms (red) and dystrophin (green) in TA muscles from NSGmdx mice that had been injected with iPax3 or iPax7-derived re-isolated mononuclear cells (MNCs). PBS served as control. Nuclei were counterstained with DAPI (blue). Magnification bar, 100 μm. b Bar graphs show respective quantification (panel a), as indicated by the percentage of DYS+MyHC+ double positive with respect to total DYS+ myofibers for iPax3-injected MNCs (gray bar, secondary recipients) in comparison to data from iPax3-injected myogenic progenitors (black bars, primary recipients). Data are shown as mean ± SEM (n = 4 per group). **p < 0.01 that the preferred composition of oxidative myofibers is Discussion not due to the immature phenotype of the primary grafts Several studies have suggested that disease pathogenesis but rather due to an intrinsic property of iPax3 and due to neuromuscular disorders can affect fiber type iPax7-derived skeletal muscle. Importantly, muscles composition and therefore muscle properties, such as injected with re-isolated MNCs display higher frequency endurance and resistance to fatigue [6, 25]. Within this of glycolytic fast type IIX myofibers compared to their heterogeneous group of disorders, DMD is characterized primary engrafted counterparts (Fig. 4). Type IIX MyHC by highly fibrotic skeletal muscle [26] and increased is only expressed in myofibers after birth, arising from number of slow myofibers, which display higher resist- the secondary myogenesis wave [24]. Therefore, the ance to pathogenesis, while glycolytic fast fibers progres- maturation of PSC-derived myogenic progenitors, sively vanish with disease progression [7]. Therefore, occurring upon exposure to the endogenous muscle when developing a cell replacement strategy for skeletal environment, is also reflected in the generation of post- muscle disorders, it is important to determine the fiber natal myofibers. type composition of engrafted cells. Altogether, these results demonstrate that PSC-derived In this study, we investigated the content of myofiber myogenic progenitors have an intrinsic capacity to pref- types generated following the transplantation of PSC- erentially give rise to oxidative myofibers. derived myogenic progenitors. We focused on mouse- Incitti et al. Skeletal Muscle (2020) 10:17 Page 9 of 11 to-mouse engraftment to prevent species-specific differ- metabolic properties of human PSC-derived myogenic ences that could introduce bias to our data, as human progenitors. muscles lack the type IIB MyHC isoform [4]. Our results To further assess whether the level of maturation of showed that PSC-derived engrafted muscle preferentially transplanted cells impacts the fiber type composition of expresses types I and IIA MyHC isoforms, which are fea- engrafted skeletal muscle, we re-isolated the donor- tures of slow and/or oxidative myofibers [3]. Our data derived MNC fraction from primary grafts and trans- also show that a high number of PSC-derived myofibers planted these into secondary recipient mice. Our results express the embryonic MyHC isoform, indicating that show that engrafted muscle in secondary recipients dis- the prenatal molecular signature of iPax3 and iPax7 plays an increased proportion of glycolytic fast-twitch PSC-derived myogenic progenitors is likely maintained fiber IIX with respect to primary counterparts. Of note, in their respective regenerating muscle in primary grafts they also show lower number of fibers expressing the [10]. Based on this observation, we hypothesized that the embryonic MyHC isoform. These results reinforce and high proportion of oxidative myofibers could be ascribed complement our recent data demonstrating that the to two different reasons: (1) PSC-derived myofibers maturation of iPax3 and iPax7 myogenic progenitors generate oxidative fibers as an indirect result of the occurs upon exposure to the endogenous adult muscle pressure from the dystrophic environment, which de- environment [10]. However, when looking at the num- lays degeneration of diseased slow-twitch fibers, (2) bers of slow and oxidative donor-derived myofibers, we PSC-derived myofibers are immature because they de- found that these are comparable between primary and rive from myogenic progenitors with a prenatal mo- secondary engrafted muscle. This observation suggests lecular signature, hence they express more type I and that the prenatal molecular signature of in vitro-gener- IIA MyHC isoforms, which arise earlier during devel- ated PSC-derived myogenic progenitors might be only opmental myogenesis [24]. marginally responsible for the high number of slow-twitch To rule out the influence of the recipient environment, and oxidative myofibers found upon engraftment, al- we transplanted non-dystrophic mice and compared the though we cannot exclude that the donor-derived MNC pattern of fiber type composition to engrafted muscles fraction did not sufficiently mature after the 4-week- from dystrophic mice. Our data show that PSC-derived exposure to the endogenous adult muscle environment. myofibers express more type I MyHC, regardless of the When generating cells suitable for replacement therap- recipient genetic background, suggesting this is a cell- ies in the context of skeletal muscle, several characteris- autonomous feature of the donor cells. Accordingly, we tics should be evaluated, including engraftment and interrogated the transcriptome of PSC-derived iPax3 and regenerative capacity, ability to repopulate the satellite iPax7 myogenic progenitors [10], and we found that cell compartment, and the long-term contribution to the mitochondrial complexes are the most represented cellu- recipient tissue’s functionality. One of the key functions lar components in both cell lines, which is in accordance of skeletal muscle is the ability to respond to innervation with oxidative metabolic needs. Moreover, we found that stimuli and to contract according to specific endurance representative genes encoding for proteins of mitochon- and speed abilities, which are unique to different groups drial complexes are also expressed at similar levels in of muscles, but highly plastic. To achieve this level of PSC-derived in vitro myotubes and in vivo myofibers, complexity, it is fundamental to assess the ability of suggesting that PSC-derived myogenic progenitors main- in vitro-generated myogenic progenitors to contribute to tain oxidative properties upon in vivo differentiation to the tissue physiology, thus ensuring proper integration myofibers. However, we also observed that non-injected into the host environment. Up to date, several studies dystrophic muscles express higher levels of the selected have assessed the therapeutic properties of muscle cells mitochondrial genes. These data indicate that the myofi- from different sources [30]. However, very few studies bers generated from PSC-derived myogenic progenitor, have analyzed the fiber type specification of cells gener- although expressing genes related to mitochondrial ac- ated in vitro [11, 12, 31], and only one assessed the con- tivity, may have a metabolic profile proper of immature tribution of transplanted cells to fiber type specification organelles, as recently shown for fetal and neonatal satel- in vivo [13], which showed that myofibers generated by lite cells [27]. In the same paper, Pala et al. suggest that PSC-derived teratomas preferentially express type I, IIA, mouse myogenic progenitors at different developmental and IIB MyHC isoforms, while IIX myofibers were not and functional stages have different metabolic require- detected. ments, and metabolic reprogramming may influence the The present study documents for the first time the progression throughout these stages. Of note, human in vivo myofiber composition resulting from the trans- PSC-derived myogenic progenitors have been described plantation of in vitro-generated PSC-derived myogenic to display fetal characteristics [18, 28, 29]. Therefore, fu- progenitors. Our findings show that donor-derived myo- ture studies should focus on fiber type composition and fiber contribution by PSC-derived myogenic progenitors Incitti et al. Skeletal Muscle (2020) 10:17 Page 10 of 11 is characterized by the expression of slow and oxidative DYS+MyHC+ double positive with respect to total donor-derived DYS+ myosin heavy-chain isoforms, in addition to develop- myofibers. Data are shown as mean ± SEM (n = 4-5 per group) **p < 0.01. Figure S4. Characterization of fiber type composition based on re- mental myosins, and that transplantation of the donor- cipient genetic background. a) Representative images show staining for derived MNC fraction into secondary recipients results type I MyHC (red) and Laminin (green) in TA muscles from dystrophic 4cv in myofibers still predominantly expressing slow and NSGmdx and NSG mice. b) Graph shows respective quantification (panel a), as indicated by the number of MyHC+ myofibers in TA muscles oxidative myosin heavy-chain isoforms, whereas expres- 4cv from dystrophic NSGmdx and NSG mice. Data are shown as mean ± sion of developmental myosins is reduced and type IIX SEM (n = 6 per group). *p < 0.05, **p < 0.01, and ***p < 0.001. c) Graph is increased. Taken together, our data suggest that ex- bar shows percentage of MyHC+ donor-derived myofibers from Fig. 2a in comparison to iPax3 and iPax7 samples from Fig. 1b. Data are shown as posure to the adult muscle environment favors postnatal mean ± SEM (n = 6-8 per group), and no statistically significant differ- myofiber composition switch and that elevated oxidative ences were observed among groups. Figure S5. Characterization of fiber fiber type composition is a cell-autonomous characteris- types from secondary grafts. a) Representative image shows staining for embryonic MyHC (red) and dystrophin (green) in TA muscles of second- tic of iPax3 and iPax7 PSC-derived myofibers, which ary recipients that had been injected with iPax3-derived MNCs. Magnifica- could be relevant for the development of cell replace- tion bar: 100 μm. b) Graph shows respective quantification (panel a), as ment therapy for DMD. indicated by the number of DYS+MyHC+ double positive with respect to total DYS+ myofibers of secondary grafts (iPax3 MNCs) in comparison to primary-injected TAs (iPax3). Data are shown as mean ± SEM (n = 4 per Conclusions group). **p < 0.01. We have shown that iPax3 and iPax7 PSC-derived myo- genic progenitors cell-autonomously generate slow and Abbreviations oxidative myofibers upon transplantation into dystrophic CSA: Cross-sectional area; DAPI: 4,6-diamidino-2-phenylindole; DM1- 2: Myotonic dystrophy 1 and 2; DMD: Duchenne muscular dystrophy; mice. This capacity is maintained upon re-isolation of FSHD: Facioscapulohumeral muscular dystrophy; H2B-GFP: Histone 2B-green donor-derived MNCs and their injection into secondary fluorescent protein; iPax3/7: Inducible Pax3/7; MyHC: Myosin heavy chain; recipients. If these findings are recapitulated with human MNC: Mononuclear cell; NSG: NOD-scid IL2Rgnull; PBS: Phosphate-buffer saline; PSC: Pluripotent stem cells; SB: Staining buffer; TA: Tibialis anterior PSC-derived myofibers, these results would suggest that in vitro-generated myogenic progenitors could have a Acknowledgements double benefit in the therapy of DMD: they may (i) re- The authors are grateful to Magally Ramirez for histology assistance and to all the members of the Perlingeiro laboratory for helpful discussion. place diseased skeletal muscle and (ii) provide myofibers more resistant to disease progression. Authors’ contributions T.I. designed and performed the research, analyzed the data, and wrote the Supplementary information manuscript. A.M. contributed with experimental design, research, Supplementary information accompanies this paper at https://doi.org/10. interpretation of the data, and writing of the manuscript. J.J., K.L., and A.Y. 1186/s13395-020-00234-5. performed the research. R.C.R.P. contributed with experimental design, interpretation of the data, and writing of the manuscript. The authors read and approved the final manuscript. Additional file 1: Figure S1. Fiber type composition of myotubes resulting from the in vitro differentiation of PSC-derived iPax3/iPax7 myo- Funding genic progenitors and adult satellite cells. Representative images show This project was supported by grants from the National Institute of Arthritis immunofluorescence staining for pan, embryonic, type I and IIA MyHC and Musculoskeletal and Skin Diseases, R01 AR055299 and R01 AR071439 isoforms (red) in myotubes resulting from the in vitro differentiation of (R.C.R.P.). iPax3 and iPax7 PSC-derived myogenic progenitors, as well as adult satel- lite cells (adult). Nuclei were counterstained with DAPI (blue). Magnifica- Availability of data and materials tion bar: 100 μm. Figure S2. Characterization of fiber type composition. The datasets used during the current study are deposited and publicly a) Graph shows the ratio of oxidative DYS+MyHC+ type I and type IIA available. Materials used in this study are commercially available. over glycolytic type IIX and IIB myofibers following the transplantation with iPax3 and iPax7 myogenic progenitors, and adult satellite cells. Data are shown as mean ± SEM (n = 7-8 per group). **p < 0.01. b) Graph bars Ethics approval and consent to participate show percentage of DYS+MyHC+ double positive with respect to total All procedures were approved by the University of Minnesota Institutional DYS+ in TA muscles transplanted with satellite cells isolated from TA or Animal Care and Use Committee and were performed in accordance with soleus muscles. Data are shown as mean ± SEM (n = 4 per group) and the National Research Council’s Guide for the Care and Use of Laboratory no statistically significant differences were observed among groups. c) Animals (2011). Representative images show staining for embryonic MyHC (red) and dys- 4cv trophin (green) in TA muscles from NSGmdx mice that had been Consent for publication injected with iPax3 or iPax7 myogenic progenitors. Adult satellite cells Not applicable. served as control. Magnification bar: 100 μm. d) Graph shows respective quantification (panel c), as indicated by the number of DYS+MyHC+ Competing interests double positive with respect to total DYS+. Data are shown as mean ± The authors declare no competing financial interests. SEM (n = 6-8 per group). *p < 0.05 and ***p < 0.001. d) Quantification of CSA (μm ) of grafts from indicated groups. Data are shown as mean ± Received: 12 January 2020 Accepted: 17 May 2020 SEM (n = 4-5 per group). *p < 0.05. Figure S3. 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Published: Jun 3, 2020

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