To determine whether SCs maintained engraftment potential when expanded ex vivo using Fig. 1.<\/strong> Derivation and characterization of Pax3-induced satellite cells. a<\/strong> (I<\/em>) FACS purification of satellite cells based on ZsGreen expression (Pax7), (II<\/em>) transduction of Pax7 To evaluate in vivo repopulation potential after 1\u00c2\u00a0week of ex vivo expansion, Pax3-induced Fig. 2.<\/strong> Regenerative potential of Pax3-induced satellite cells following their transplantation Next, we determined whether myofiber engraftment was accompanied by improvement in Fig. 3.<\/strong> Contractile properties of transplanted muscles and satellite cell homing. a<\/strong> Representative examples of maximum isometric tetanic force in TA muscles that had To assess whether Pax3-induced cells have the capacity to engraft the host SC compartment, We next applied genetic correction to ex vivo expanded dystrophic SCs following the Fig. 4.<\/strong> Correction of dystrophin-deficient Pax3-induced satellite cells using a human ?DYS<\/em> transgene. a<\/strong> FACS plot shows gate for the purification of ZsGreen SCs were isolated by flow cytometry from Pax7-ZsGreen\/<\/strong>mdx<\/em> mice (Fig.\u00c2\u00a04a), immediately transduced with the doxycycline-inducible Pax3 vector, and grown in To assess the regenerative potential of corrected ?Dys-Pax3-induced cells in vivo, Fig. 5.<\/strong> Engraftment of ?DYS<\/em>-Pax3-induced cells into NSG-mdx 4Cv<\/sup><\/em> We next investigated whether engraftment of corrected ?DYS<\/em>-Pax3-induced cells was accompanied by functional improvement. Engrafted muscles showed Fig. 6.<\/strong> Contractile function and response to reinjury by muscles engrafted with ?DYS<\/em>-Pax3-induced cells. a<\/strong> Representative examples of maximum isometric tetanic force in TA muscles that had To determine whether engrafted ?DYS<\/em>-corrected Pax3-induced cells would have the same ability to respond to injury as SCs isolated by flow cytometry have been demonstrated to possess a tremendous capacity Several studies have investigated the transplantation of cultures derived from prospectively Herein, we demonstrate that upon conditional expression of Pax3, freshly isolated Derivation and ex vivo expansion of satellite cells using Pax3 To determine whether SCs maintained engraftment potential when expanded ex vivo using conditional expression of Pax3, we followed the strategy summarized in Fig.\u00c2\u00a01a, in which SCs from the transgenic Pax7-ZsGreen reporter mouse 27] were (I) purified by flow cytometry, (II) genetically modified with a lentiviral […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-26990","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/26990","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/comments?post=26990"}],"version-history":[{"count":0,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/26990\/revisions"}],"wp:attachment":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/media?parent=26990"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/categories?post=26990"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/tags?post=26990"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n conditional expression of Pax3, we followed the strategy summarized in Fig.\u00c2\u00a01a, in which SCs from the transgenic Pax7-ZsGreen reporter mouse 27] were (I) purified by flow cytometry, (II) genetically modified with a lentiviral
\n vector encoding a doxycycline-inducible Pax3 transgene, (III) expanded ex vivo in
\n the presence of doxycycline, and then (IV) transplanted into immune-deficient, dystrophin-deficient
NSG-mdx 4Cv<\/sup><\/em>31] mice. After enzymatic digestion, the muscle mononuclear fraction of Pax7-ZsGreen
\n was FACS-purified based on ZsGreen expression, which reflects Pax7
+<\/sup>
\n cells (Fig.\u00c2\u00a01b), and accordingly gave rise to a homogeneous SC population (Fig.\u00c2\u00a01c). These cells were immediately transduced with a doxycycline-regulated conditional
\n Pax3-IRES-mCherry-expressing lentivector (Pax3 induced) 32]. As a control, SCs were transduced with empty vector (mCherry only). Pax3
+<\/sup>
\n (mCherry
+<\/sup>
\n ) cells were detected only when doxycycline (dox) was added to the culture medium
\n (Additional file 1). To determine the effect of Pax3 on the expansion of transduced SCs, we evaluated
\n the proliferation rate of Pax3-induced cells side-by-side with control cell preparations
\n (empty vector) grown under identical culture conditions: proliferation medium with
\n basic fibroblast growth factor (bFGF) and dox. Notable expansion advantage was observed
\n in Pax3-induced cultures when compared to control counterparts (Fig.\u00c2\u00a01d). Although under these proliferation conditions, both control and Pax3-induced cells
\n displayed similar morphology (Fig.\u00c2\u00a01e, f, panel I<\/em>), only Pax3-induced cells showed abundant Pax3 expression, as evidenced by immunofluorescence
\n staining (Fig.\u00c2\u00a01e, f, panel II<\/em>) and gene expression analyses (Additional file 2). As expected, Pax3 overexpression in SCs was accompanied by upregulation of its
\n target gene Myf5 35] (Additional file 2). Under proliferation conditions, Pax3-induced cells showed no signs of myotube formation,
\n as indicated by the absence of signal for myosin heavy chain (MHC) (Fig.\u00c2\u00a01f, panel III<\/em>, and Additional file 2), whereas the control uninduced population spontaneously differentiated into MHC-positive
\n myotubes (Fig.\u00c2\u00a01e, panel III<\/em>, and Additional file 2). Nevertheless, when Pax3-induced and control cells were subjected to differentiation
\n conditions (5\u00c2\u00a0% horse serum and withdrawal of dox and bFGF), both cultures gave rise
\n to multinucleated myotubes displaying abundant expression of MHC (Fig.\u00c2\u00a01e, f, panels IV<\/em> and V<\/em>, and Additional file 2). Control cultures expressed significant levels of MHC under proliferation conditions,
\n suggesting the propensity of these cells to begin differentiation as soon as they
\n have reached confluence. We next quantified the fusion index of control and Pax3-induced
\n cultures. Upon in vitro differentiation, Pax3-induced SCs exhibited elevated fusion
\n index (67\u00c2\u00a0%) relative to control cultures (47\u00c2\u00a0%). Thus, under the conditions tested
\n here, Pax3 induction allows the in vitro expansion of less differentiated SCs, without
\n affecting their ability to terminally differentiate into fusing myotubes.<\/p>\n
+<\/sup>
\n cells with an inducible expression system encoding Pax3, (III<\/em>) in vitro expansion of Pax3-induced cells and control empty vector counterparts,
\n and (IV<\/em>) transplantation of iPax3 and control cells into NSG-mdx 4Cv<\/sup><\/em>
\n . b<\/strong> Representative FACS profile for ZsGreen (Pax7) expression in digested muscles isolated
\n from Pax7-ZsGreen reporter mice. Sorting gate for ZsGreen
+<\/sup>
\n (Pax7
+<\/sup>
\n ) satellite cells is shown. c<\/strong> Phase-contrast image of sorted ZsGreen
+<\/sup>
\n (Pax7
+<\/sup>
\n ) satellite cells. d<\/strong> Cell growth curve of Pax3-induced cells and control counterparts at several passages
\n (P1\u00e2\u20ac\u201cP4) (n<\/em>?=?2, mean?\u00c2\u00b1?SD). e<\/strong>, f<\/strong> In vitro characterization of ex vivo expanded satellite cells grown under proliferation
\n and differentiation culture conditions. Phase-contrast images of control empty vector
\n (e<\/strong>) and Pax3-induced (f<\/strong>) monolayers. Representative immunofluorescence staining for Pax3 (red<\/em>, upper panels<\/em>) and MHC (red<\/em>, lower panels<\/em>) in control empty vector SCs (e<\/strong>) and Pax3-induced SCs (f<\/strong>). Cells are co-stained with DAPI (blue<\/em>). Scale bar<\/em> 200\u00c2\u00a0?m. g<\/strong> Fusion index calculation. Error bars<\/em> represent s.e.m. (n<\/em>?=?3). **P<\/em>??0.01\n <\/p>\nIn vivo regenerative potential of ex vivo expanded satellite cells<\/h4>\n
\n and respective control cell preparations were transplanted into the TA muscles of
NSG-mdx 4Cv<\/sup><\/em>
\n mice. Prior to cell transplantation, both hind limbs were subjected to irradiation
\n (12\u00c2\u00a0Gy\/leg) to deplete endogenous SCs 31] and injury with cardiotoxin (CTX). While the contra-lateral TA was injected with
\n PBS, 350,000 Pax3-induced or control cells were injected into the right TA. Five weeks
\n after transplantation, TA muscles were harvested and evaluated for engraftment by
\n immunofluorescence staining for dystrophin. Whereas DYS
+<\/sup>
\n myofibers were virtually undetectable in PBS-injected muscles (Fig.\u00c2\u00a02a, c), dystrophin expression was observed in TA muscles that had been transplanted with
\n control (Fig.\u00c2\u00a02b) or Pax3-induced (Fig.\u00c2\u00a02d) cell preparations, with the latter showing higher engraftment levels (Fig.\u00c2\u00a02e, 14?\u00c2\u00b1?7.4 vs. 37?\u00c2\u00b1?5.7\u00c2\u00a0%, respectively).<\/p>\n
\n into NSG-mdx 4Cv<\/sup><\/em>
\n mice. Engraftment analysis of control empty vector (a<\/strong>, b<\/strong>) and Pax3-induced cells (iPax3) (c<\/strong>, d<\/strong>). Cross sections of TA muscles harvested from NSG-mdx 4Cv<\/sup><\/em>
\n mice that had been injected with PBS (a<\/strong>, c<\/strong>) or satellite cells (b<\/strong>, d<\/strong>) were stained with antibody to dystrophin (red<\/em>). Engrafted tissues from control and Pax3-induced cells are represented by mice #03<\/em> and #05<\/em> and #07<\/em> and #09<\/em>, respectively. DAPI is shown in blue. Scale bar<\/em>, 50\u00c2\u00a0?m. e<\/strong> Quantification of DYS
+<\/sup>
\n myofibers in treated muscles. Error bars<\/em> represent s.e.m. (n<\/em>?=?6). *P<\/em>??0.03\n <\/p>\n
\n muscle strength. As expected, the maximum isometric force for PBS-injected TA muscles
\n (contra-lateral legs) was low (Fig.\u00c2\u00a03a, gray lines). In contrast, engrafted TA muscles showed enhanced isometric force (Fig.\u00c2\u00a03a, red lines). Cell transplantation of both control and Pax3-induced preparations resulted
\n in improved absolute (Fig.\u00c2\u00a03b) and specific (Fig.\u00c2\u00a03c) force of engrafted muscles when compared with their respective PBS-injected contra-lateral
\n muscles. However, muscles that had been transplanted with Pax3-induced cells displayed
\n significantly superior functional improvement (Fig.\u00c2\u00a03b, c) when compared to control cells (1.52-fold). No statistical difference was observed
\n in forces between the contra-lateral legs (PBS) of the two groups of mice. These results
\n demonstrate that 7-day cultured SCs expanded with Pax3 have a superior ability to
\n improve muscle function, compared to control empty vector transduced counterparts.<\/p>\n
\n been injected with PBS (contra-lateral leg, gray line<\/em>) and control or Pax3-induced cells (red lines<\/em>). Wild-type Bl6 mice were used for reference control (dashed line<\/em>). b<\/strong>, c<\/strong> Cell transplantation produces an improvement in absolute (F<\/em>0<\/sub>
\n , b<\/strong>) and specific (sF<\/em>0<\/sub>
\n = F<\/em>0<\/sub>
\n normalized to CSA, c<\/strong>) force. Error bars<\/em> represent s.e.m. from a total of six mice. *P<\/em>??0.05, **P<\/em>??0.01, ***P<\/em>??0.001. d<\/strong> In situ analysis reveals the presence of donor-derived satellite cells (ZsGreen\/Pax3-induced
\n cells) in the host stem cell pool, as shown by the presence of cells co-stained for
\n both Pax7 (red<\/em>) and ZsGreen (green<\/em>) (white arrow<\/em>) beneath the basal lamina (gray<\/em>). e<\/strong> Upon reinjury, engrafted donor-derived satellite cells give rise to newly formed
\n myofibers, as indicated by the co-expression of DYS (red<\/em>) and embryonic MHC (green<\/em>) (white arrow<\/em>). Arrowheads<\/em> denote DYS
?<\/sup>
\n \/eMHC
+<\/sup>
\n host-derived new formed myofibers. DAPI is shown in blue. Scale bar<\/em>, 50\u00c2\u00a0?m\n <\/p>\n
\n and therefore contribute to ongoing regeneration, engrafted TA muscles were stained
\n for ZsGreen and Pax7 to identify donor-derived SC contribution. Histological analysis
\n of transverse sections of TA muscles 1\u00c2\u00a0month after transplantation clearly identified
\n the presence of Pax7
+<\/sup>
\n ZsGreen
+<\/sup>
\n cells beneath the basal lamina, suggesting that Pax3-induced cells can engraft the
\n SC pool (Fig.\u00c2\u00a03d). To investigate whether donor-derived iPax3 SCs would be able to contribute to ongoing
\n muscle regeneration, a cohort of mice transplanted with unlabelled Pax3-induced cells
\n were reinjured with CTX 1\u00c2\u00a0month after cell transplantation. Ten days after reinjury,
\n we detected donor-derived newly regenerated myofibers, as indicated by the presence
\n of DYS
+<\/sup>
\n \/embryonic MHC
+<\/sup>
\n myofibers (Fig.\u00c2\u00a03e, white arrows). Since we have used half of the usual dose of CTX (5ul\/5uM, instead
\n of 10ul\/10uM) for these reinjury studies, CTX injection did not result in degeneration
\n of the whole tissue, and accordingly the presence of DYS
+<\/sup>
\n \/eMHC
?<\/sup>
\n fibers was detected. These results suggest that at least some of engrafted Pax3-induced
\n cells remain less differentiated and are able to respond to a second round of muscle
\n injury.\n <\/p>\nGenetic repair of dystrophic Pax3-induced cells<\/h4>\n
\n protocol outlined in Fig.\u00c2\u00a01a, but using SCs harvested from mdx<\/em> mice bred to carry the Pax7-ZsGreen reporter (Fig.\u00c2\u00a04a). For genetic repair, we used the human micro-dystrophin
?R4\u00e2\u20ac\u201c23\/?CT<\/sup>
\n (?DYS<\/em>) transgene lacking the spectrin-like repeats 4\u00e2\u20ac\u201c23 and the C-terminus 36] and the non-viral Sleeping Beauty system<\/em> for transduction. First, we generated a Tn vector (pKT2-Neo selection marker driven
\n by the-Neo\/hH2 ?DYS<\/em>; Fig.\u00c2\u00a04b) containing two divergent genes: a GFP\/Neo selection marker driven by the hEF1a-eIF4g
\n promoter and the human ?Dystrophin (?DYS<\/em>) gene under the control of a pHSA 37].<\/p>\n
+<\/sup>
\n (Pax7
+<\/sup>
\n ) satellite cells from Pax7-ZsGreen\/<\/strong>mdx<\/em> mice. b<\/strong> The Sleeping Beauty transposon<\/em> system consists of transposon (Tn) and transposase (SB100X) vectors. The Tn is a
\n bicistronic promoter vector of 11.3\u00c2\u00a0Kb containing the ubiquitin hEF1a-eIF4g (Pr, in
gray<\/em>) and the skeletal muscle-specific skeletal ?-actin promoter (pHSA, in black<\/em>). The ubiquitin promoter drives a GFP-2A-Neo. This selection marker cassette is flanked
\n by lox P sequences (red<\/em>). The human ?DYS<\/em> gene is under control of the pHSA. SB100X transposase proteins (red spheres<\/em>) bind the DR sequences (yellow arrows<\/em>) within the two inverted repeats (IR\/DR, arrowheads<\/em>) and catalyze integration of the whole transposon transgene into the genome with
\n high efficiency. c<\/strong> Representative FACS profiles for enrichment steps used to isolate a pure and stable
\n population of corrected GFP
+<\/sup>
\n cells (?DYS<\/em>-Pax3-induced cells) following transfection with pKT2\/?DYS<\/em> and SB100X. Control consisted of dystrophin-deficient Pax3-induced cells (CTL<\/em>) nucleofected with pKT2 transposon vector only (no transposase). d<\/strong> RT-PCR analysis for uncorrected (UNC<\/em>, dystrophin-deficient Pax3-induced cells) and corrected (Corr<\/em>, ?DYS<\/em>-Pax3-induced cells) cells grown under proliferation (P<\/em>) and differentiation (D<\/em>) culture conditions shows the expression of human ?DYS<\/em> solely in corrected cells. GAPDH was used as housekeeping gene\n <\/p>\n
\n doxycycline to induce Pax3 expression. It should be noted that almost immediately
\n upon placing the Pax7-ZsGreen SCs into culture, the ZsGreen fluorescence is lost.
\n We now then transduced these non-fluorescent cells with the ?-dystrophin correction
\n vector, which contained a GFP reporter, and sorted on this signal; therefore, the
\n culture was now constitutively green. Dystrophin-deficient Pax3-induced cells were
\n subsequently nucleofected with Tn vector and transposase (engineered hyperactive variant
\n SB100X 38]; Fig.\u00c2\u00a04b, upper panel), using a plasmid ratio of 4:1, respectively, which we have previously
\n found to provide optimal in vitro gene transfer for a large transgene 32]. Five days after nucleofection, flow cytometry analysis revealed a cell sub-population
\n positive for GFP\/?DYS<\/em> (~1.2\u00c2\u00a0%) (Fig.\u00c2\u00a04b, lower panel). Following two rounds of sorting, a highly enriched ?DYS<\/em>+<\/sup>
\n (GFP
+<\/sup>
\n ) population was obtained (96\u00c2\u00a0%) (Fig.\u00c2\u00a04b, lower panel). Expression of the transgene in corrected cells was confirmed by RT-PCR
\n analysis using specific primers for the human ?DYS<\/em> transgene (Fig.\u00c2\u00a04c). These results demonstrate the capacity for the Sleeping Beauty<\/em> system to deliver a large transgene (11.3\u00c2\u00a0Kb) into dystrophic activated SCs.\n <\/p>\nRegenerative potential of ?DYS<\/em>-Pax3-induced cells
<\/h4>\n
\n these cells were transplanted into CTX-injured TA muscles of NSG-mdx 4Cv<\/sup><\/em>
\n mice. We did not irradiate these mice as irradiation would be discouraged in the clinical
\n setting. One month following transplantation, TA muscles were harvested and sections
\n were evaluated for engraftment by immunostaining using a human DYSTROPHIN antibody
\n that recognizes the N-terminal epitope, which is preserved in the human ?DYS<\/em> transgene. While no DYS expression was detected in PBS-injected muscles (Fig.\u00c2\u00a05a), muscles that had been transplanted with ?DYS<\/em>-Pax3-induced cells generated large engrafted areas with DYS
+<\/sup>
\n myofibers (Fig.\u00c2\u00a05b). Quantification of engraftment revealed that approximately 20\u00c2\u00a0% of fibers in transplanted
\n muscles were ?DYSTROPHIN
+<\/sup>
\n , confirming the regeneration potential of ex vivo corrected activated SCs.<\/p>\n
\n mice. TA muscles harvested from NSG-mdx 4Cv<\/sup><\/em>
\n mice that had been injected with PBS (a<\/strong>) or corrected ex vivo expanded satellite cells (?DYS<\/em>-Pax3-induced cells) (b<\/strong>) were stained using an antibody specific for human DYSTROPHIN (red<\/em>). The DYS protein was detected only in the transplanted muscles. Two representative
\n transplanted mice (b<\/strong>) are shown. DAPI is shown in blue. Scale bar<\/em>, 50\u00c2\u00a0?m. c<\/strong> Quantification of human ?DYSTROPHIN
+<\/sup>
\n myofibers in these transplanted muscles. Error bars<\/em> represent s.e.m (n<\/em>?=?6)\n <\/p>\n
\n superior isometric (Fig.\u00c2\u00a06a), absolute (Fig.\u00c2\u00a06b), and specific (Fig.\u00c2\u00a06c) force when compared to PBS-injected TA muscles.<\/p>\n
\n been injected with PBS (contra-lateral leg, gray line<\/em>) or Pax3 induced (red line<\/em>). b<\/strong>, c<\/strong>?DYS<\/em>-Pax3-induced cell transplantation produced a significant improvement in absolute
\n (F<\/em>0<\/sub>
\n , b<\/strong>) and specific (sF<\/em>0<\/sub>
\n = F<\/em>0<\/sub>
\n normalized to CSA, c<\/strong>) forces. Error bars<\/em> represent s.e.m. from a total of six mice. **P<\/em>??0.01. d<\/strong> Immunofluorescence staining for embryonic MHC and ?DYS in engrafted TA muscles analyzed
\n 10\u00c2\u00a0days after CTX reinjury indicates the presence of newly formed donor myofibers
\n as denoted by co-expression of human ?DYS (red<\/em>) and eMHC (green<\/em>) (arrows<\/em>). Arrowheads<\/em> show ?DYS
?<\/sup>
\n \/eMHC
+<\/sup>
\n host-derived newly formed myofibers. Alexa-647 was used to detect eMHC. DAPI is shown
\n in blue. Scale bar<\/em>, 50\u00c2\u00a0?m\n <\/p>\n
\n shown above for WT cells and would therefore be capable of providing ?DYSTROPHIN continuously,
\n we reinjured muscles that had been previously transplanted with ?DYS<\/em>-Pax3-induced cells. Ten days following CTX injection, we stained muscle sections
\n with embryonic MHC and human DYS antibodies. This clearly showed the presence of donor-derived
\n newly regenerated muscle fibers that were double-positive for ?DYS and embryonic MHC
\n (Fig.\u00c2\u00a06d, white arrows and Additional file 3). Altogether, these results show that transplantation of corrected ?DYS<\/em>-Pax3-induced cells provides functional improvement of dystrophic muscles, both in
\n terms of muscle force generation and in terms of their ability to respond to ongoing
\n muscle injury and stably express ?DYS protein.\n <\/p>\n
\n to improve muscle function in mdx<\/em> mice 31]; however, the impracticality of isolating large numbers of SCs from living donors
\n as well as the requirement for gene correction, if considering an autologous transplantation
\n setting, necessitates ex vivo expansion. To date, only one study has reported a combined
\n cell\/gene therapy approach using SCs in the context of muscular dystrophy 18]. In this study, the authors isolated SCs from a dystrophic mouse, transduced them
\n with a lentiviral vector encoding the mouse ?DYS<\/em> transgene, and immediately transplanted them into the dystrophic muscle and found
\n that they were able to differentiate into DYS+ fibers.\n <\/p>\n
\n isolated SCs. Blau and colleagues demonstrated that culturing mouse SCs on a substrate
\n that mimics muscle tissue elasticity, and in the presence of an inhibitor for p38MAPK,
\n helped maintain \u00e2\u20ac\u0153stemness\u00e2\u20ac\u009d features 10], 39]. Following a different approach, Tapscott and colleagues expanded freshly isolated
\n canine SCs by activating the Notch signaling pathway, which bestowed superior in vivo
\n regenerative ability upon SC-initiated cultures compared to controls 20]. In a recent study, Rudnicki and colleagues reported that short treatment of SCs
\n with Wnt7a resulted in enhanced engraftment that was accompanied by improved muscle
\n function 40].\n <\/p>\n
\n SCs can be successfully expanded when compared to their cultured empty vector control
\n counterparts (Fig.\u00c2\u00a01d). Following their intramuscular transplantation into dystrophic mice, Pax3-induced
\n cells display greater regenerative potential than control SCs, and engraftment levels
\n correlated with a significant improvement in muscle strength (Fig.\u00c2\u00a03a\u00e2\u20ac\u201cc). Importantly, we also show that engrafted Pax3-induced cells are capable of seeding
\n the SC pool and responding to a second round of CTX-induced damage by generating newly
\n formed DYS
+<\/sup>
\n fibers (Fig.\u00c2\u00a04d, e). In addition, we show that Pax3-induced dystrophic SCs are amenable to genetic correction.
\n Using a non-viral Sleeping Beauty<\/em> system carrying a human ?DYS<\/em> transgene, we corrected SCs from dystrophin-deficient mice and found that these were
\n capable of differentiating into functional muscle fibers in vivo (Fig.\u00c2\u00a05), increasing force generation capacity of dystrophic muscles (Fig.\u00c2\u00a06a\u00e2\u20ac\u201cc), and producing new myofibers upon CTX reinjury that remain positive for the ?DYS<\/em> transgene.\n <\/p>\n","protected":false},"excerpt":{"rendered":"