Pax3-induced expansion enables the genetic correction of dystrophic satellite cells
- Antonio Filareto†1Email author,
- Fabrizio Rinaldi†1,
- Robert W. Arpke2,
- Radbod Darabi1,
- Joseph J. Belanto3,
- Erik A. Toso2,
- Auston Z. Miller1,
- James M. Ervasti3,
- R. Scott McIvor4,
- Michael Kyba2 and
- Rita CR Perlingeiro1Email author
© Filareto et al. 2015
Received: 26 June 2015
Accepted: 8 October 2015
Published: 26 October 2015
Satellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as self-renewal and long-term regenerative potential.
We develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice.
Here, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human μDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury.
These results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties.
KeywordsSatellite cells Muscular dystrophy Gene correction Sleeping Beauty Dystrophin Pax3 Regeneration
Duchenne muscular dystrophy is a fatal neuromuscular disease affecting about 1 in 5000 boys , which is caused by mutations in the gene coding for the dystrophin protein . Its absence results in continual damage and regeneration, which becomes impaired over time, leading to displacement of muscle fibers with fat and connective tissue, resulting in diminished muscle function . Loss of regeneration is thought to be due to exhaustion or impairment of satellite cells. These are resident adult muscle stem cells located underneath the basal lamina  that actively contribute to skeletal muscle growth and regeneration throughout life . Under normal physiological conditions, satellite cells are quiescent  and express Pax7 . In response to injury, satellite cells become activated and proliferate as myogenic progenitor cells (MPCs), migrate to the damaged area, and fuse to form new multinucleated myofibers [8, 9]. This period of extensive proliferation terminates when homeostasis is achieved and injury is repaired . During this process, a small proportion of satellite cells re-enter quiescence [11, 12] and reconstitute the satellite cell pool. These characteristics make satellite cells an attractive cell population to be utilized in cell-based therapies to treat muscular dystrophies.
Numerous studies have investigated the regenerative potential of satellite cells and their progeny derivatives [10, 13–20]. However, ex vivo expanded satellite cells have diminished regenerative potential in vivo . Tremblay and colleagues have conducted a phase I clinical trial for MPC cultures using a high-density injection approach in DMD patients, which has been reported encouraging in the sense dystrophin expression could be detected [22–24]. Nevertheless, MPCs are not the most desirable population to be transplanted since these cells are characterized by limited survival and migration upon injection [25–28] and, more importantly, are devoid of self-renewal, which limits their long-term regeneration potential. Thus, an essential requirement for muscle cell-based therapies is the development of an approach that enables the ex vivo expansion of satellite cells while maintaining their “stemness” and regeneration potential.
Here, we show that this can be accomplished in mouse satellite cells by transient expression of Pax3, the master regulator of the embryonic myogenic program, and that ex vivo expanded satellite cell progeny have a high capacity for engraftment and regeneration. Most importantly, we demonstrate that this system can be combined with genetic correction of cells from dystrophic animals and utilized to promote muscle regeneration and improve functional properties in vivo when transplanted back into dystrophic mice.
Mice and satellite cell isolation
Animal maintenance and experimental use were performed according to protocols approved by the University of Minnesota Institutional Animal Care and Use Committee. The transgenic Pax7-ZsGreen reporter mouse line was generated by injection of the purified Pax7-BAC (RP23–218H13) containing a ZsGreen fluorescence protein into the first coding exon, as described . Pax7-ZsGreen/mdx mice were generated by breeding mdx mice (C57BL/10ScSn), purchased from Jackson Laboratories (Bar Harbor, ME, http://www.jax.org), to WT-Pax7-ZsGreen mice . Female progeny containing both genes were crossed to hemizygous mdx male mice. R26-M2rtTA/M2rtTA mice  were also bred to Pax7-ZsGreen mice. Resulting mice from this breeding were intercrossed, and mice homozygous for at the R26-M2rtTA were identified. NSG-mdx 4Cv mice  were used as transplantation recipients. Pax7-ZsGreen satellite cells were isolated from soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), and gastrocnemius (GAS) muscles of 6–8-week-old Pax7-ZsGreen/mdx or R26-M2rtTA/M2rtTA;Pax7-ZsGreen mice, as described previously . Analysis and cell sorting were performed on a Cytomation MoFlo cytometer (Dako, Carpinteria, CA, http://www.dako.com).
Generation of Pax3-induced cells
Freshly isolated satellite cells were immediately transduced with the inducible Pax3-IRES-mCherry-expressing lentivector  to generate the Pax3-induced satellite cells and mdx-Pax3-induced satellite cells; control satellite cells were transduced with mCherry lentiviral vector.
Sleeping Beauty system and generation of corrected μDYS-Pax3-induced cells
We developed a bicistronic T2-inverted terminal repeat transposon (Tn) vector (pKt2-GFP-Neo/μDYSTROPHIN) carrying an 11.3-kb engineered transgene containing the skeletal α-actin promoter (pHSA) (generously provided by Jeffrey Chamberlain, Department of Neurology, University of Washington School of Medicine) that drives μDYS and a ubiquitin promoter (hEF1a-eIF4g) that drives a GFP-2A-Neo reporter gene, which allows for the selection of μDYS-corrected mdx-Pax3-induced cells. The whole transgene is flanked by the terminal inverted repeats (IR/DR), each of which contains two binding sites for the transposase. For the transposase, we used pCMV-SB100X (generously provided by Zoltan Ivics from Max Delbruck Center for Molecular Medicine, Berlin), which yields high levels of Tn integration. SB transposase-mediated gene delivery was done using an Amaxa Nucleofector (Lonza) according to the manufacturer’s protocol (fibroblast Nucleofector kit solution, Nucleofector program U-023). 5 × 105 mdx-Pax3-induced cells were nucleofected with 1 μg of pCMV-SB100X and 4 μg of Tn:pKt2-GFP-Neo/μH2-μDYS. Corrected μDYS-Pax3-induced satellite cell (SC) Pax3 cells were purified based on sorting for GFP+ cells. The μDYS was generated using the full-length human dystrophin cDNA in the Gateway entry vector pENTR223.1 (NM_004006) that was obtained from the ORFeome Collaboration. The entry vector was N-terminally FLAG-tagged via PCR using primers with overhangs encoding the tag. The μ -dystrophinΔR4–23/ΔCT (μDYS) was built by deletion using previously described methods . Briefly, PCR primers were designed such that they amplified the entire plasmid except the region being deleted, namely spectrin-like repeats 4–23. These linear PCR products were then circularized via the addition of T4 polynucleotide kinase and T4 DNA ligase (New England Biolabs) and sequence verified. A second round of PCR and circularization was performed to delete the C-terminus. All PCRs were performed using PfuUltra II HS polymerase (Stratagene).
RNA extraction and real-time PCR analysis
RNA was isolated from cultured cells using TRIzol reagent (Life Technology). One microgram of total RNA was reverse-transcribed using the ThermoScript™ Reverse Transcriptase kit (Life Technology). In control uninduced and Pax3-induced cells, real-time PCR was performed for muscle-specific genes with probe sets from Applied Biosystems . To confirm μDYS expression in corrected Pax3-induced cells, specific primers were designed for the μDYS gene (F: 5′-TTCTAAGTTTGGGAAGCAGCA-3′ and R: GGTCTGGCCTATGACTATGGA. Primers for GAPDH were F: AGGCCGGTGCTGAGTATGTC and R: TGCCCTGCTTCACCACCTTCT).
Muscle injury and transplantation studies
Four-month-old NSG-mdx 4Cv mice were used as recipients for all transplantation studies described here. Muscle injury was performed as described previously . Briefly, both hind limbs were subjected to 1200 cGy of irradiation at day 2; muscle injury was induced 24 hours later (day 1) using 15 μl of cardiotoxin (10 μM, SIGMA) in both right and left TA muscle; on day 0, cells were injected into the left TA of each mouse using a Hamilton syringe. For each set of transplantation, cells were collected using cell dissociation buffer, enzyme-free (GIBCO) (10 min at 37 °C), resuspended in PBS, and then injected directly into the left TA muscle (350,000 cells per 10 μl PBS). Control TA muscles were injected with the same volume of PBS.
Immunofluorescence of cultured cells and tissue sections
TA muscles were embedded in Tissue-Tek OCT compound and immediately frozen in liquid nitrogen-cooled isopentane. Cut tissues (10–12 μm) were permeabilized with 0.3 % Triton X-100 in PBS for 10 min, then blocked for 1 h in 20 % goat serum, and incubated overnight with specific primary antibody in antibody diluent (Dako). Primary antibodies used were rabbit anti-dystrophin polyclonal antibody (1:250, ab 15277; Abcam), mouse anti-dystrophin polyclonal antibody specific for human μDys (1:50, MAB1690; Chemicon, Millipore), mouse anti-Pax7 (1:250; MAB 1675; R&D System), rabbit anti-laminin (1:400; Sigma), anti-rabbit ZsGreen (1:100; Clontech), and anti-embryonic MHC (1:20; F1.652; Developmental Studies Hybridoma Bank). For ZsGreen staining, tissues were collected and immediately fixed in 4 % PFA for 1 h. Next slides were incubated in a solution of 30 % sucrose in 0.01 M PBS for 2 h and left over night in a solution of 20 % sucrose in 0.01 M PBS. The next day, TA muscles were embedded in OCT compound (Leica). A MOM kit (Vector Laboratory) was used following the manufacturer’s instruction. After three PBS washes, sections were incubated for 45 min with secondary antibody. For secondary staining, goat Alexa-555 anti-rabbit or mouse, Alexa-488 anti-rabbit or mouse, Alexa-647 anti-rabbit, and Alexa-488 anti-chicken (1:1000) were used (Molecular Probes). Control tissues were processed simultaneously in the same manner.
For in vitro cultures, cells were maintained on gelatin-coated plates and processed as described above. Cells were first fixed for 10 min at RT in 4 % PFA, washed twice in PBS, and incubated for 10 min with 0.3 % Triton X-100 in PBS. The following primary antibodies were used: anti-Pax3 (1:100; R&D Systems) and anti-MHC (1:50; MF20; Developmental Studies Hybridoma Bank). Alexa Fluor 555 goat anti-rabbit and anti-mouse (Molecular Probes) was used for secondary staining. 4, 6-Diamidino-2-phenylindole (DAPI) was used to counter-stain nuclei (Sigma).
Muscle preparation for mechanical studies
For the measurement of contractile properties, mice were anesthetized with avertin (250 mg kg−1 intraperitoneal) and analyzed as described previously [28, 30]. Intact TA muscles were analyzed ex vivo in an experimental organ bath filled with mammalian Ringer buffer, containing platinum electrodes placed longitudinally on either side of the muscle. Muscles were stimulated by electric field (square wave pulses 25 V, 0.2 ms in duration, 150 Hz) using an optimal muscle length (L 0) for the development of maximum isometric tetanic force (F 0). Specific force (sF 0) was determined by normalizing maximum isometric tetanic force (F 0) to cross-sectional area (CSA). Total muscle CSA was calculated by dividing muscle mass (mg) by the product of muscle length (mm), and 1.06 mg/mm3 is the density of mammalian skeletal muscle.
Results and discussion
Derivation and ex vivo expansion of satellite cells using Pax3
In vivo regenerative potential of ex vivo expanded satellite cells
To assess whether Pax3-induced cells have the capacity to engraft the host SC compartment, and therefore contribute to ongoing regeneration, engrafted TA muscles were stained for ZsGreen and Pax7 to identify donor-derived SC contribution. Histological analysis of transverse sections of TA muscles 1 month after transplantation clearly identified the presence of Pax7+ZsGreen+ cells beneath the basal lamina, suggesting that Pax3-induced cells can engraft the SC pool (Fig. 3d). To investigate whether donor-derived iPax3 SCs would be able to contribute to ongoing muscle regeneration, a cohort of mice transplanted with unlabelled Pax3-induced cells were reinjured with CTX 1 month after cell transplantation. Ten days after reinjury, we detected donor-derived newly regenerated myofibers, as indicated by the presence of DYS+/embryonic MHC+ myofibers (Fig. 3e, white arrows). Since we have used half of the usual dose of CTX (5ul/5uM, instead of 10ul/10uM) for these reinjury studies, CTX injection did not result in degeneration of the whole tissue, and accordingly the presence of DYS+/eMHC− fibers was detected. These results suggest that at least some of engrafted Pax3-induced cells remain less differentiated and are able to respond to a second round of muscle injury.
Genetic repair of dystrophic Pax3-induced cells
SCs were isolated by flow cytometry from Pax7-ZsGreen/ mdx mice (Fig. 4a), immediately transduced with the doxycycline-inducible Pax3 vector, and grown in doxycycline to induce Pax3 expression. It should be noted that almost immediately upon placing the Pax7-ZsGreen SCs into culture, the ZsGreen fluorescence is lost. We now then transduced these non-fluorescent cells with the μ-dystrophin correction vector, which contained a GFP reporter, and sorted on this signal; therefore, the culture was now constitutively green. Dystrophin-deficient Pax3-induced cells were subsequently nucleofected with Tn vector and transposase (engineered hyperactive variant SB100X ; Fig. 4b, upper panel), using a plasmid ratio of 4:1, respectively, which we have previously found to provide optimal in vitro gene transfer for a large transgene . Five days after nucleofection, flow cytometry analysis revealed a cell sub-population positive for GFP/μDYS (~1.2 %) (Fig. 4b, lower panel). Following two rounds of sorting, a highly enriched μDYS + (GFP+) population was obtained (>96 %) (Fig. 4b, lower panel). Expression of the transgene in corrected cells was confirmed by RT-PCR analysis using specific primers for the human μDYS transgene (Fig. 4c). These results demonstrate the capacity for the Sleeping Beauty system to deliver a large transgene (11.3 Kb) into dystrophic activated SCs.
Regenerative potential of μDYS-Pax3-induced cells
To determine whether engrafted μDYS-corrected Pax3-induced cells would have the same ability to respond to injury as shown above for WT cells and would therefore be capable of providing μDYSTROPHIN continuously, we reinjured muscles that had been previously transplanted with μDYS-Pax3-induced cells. Ten days following CTX injection, we stained muscle sections with embryonic MHC and human DYS antibodies. This clearly showed the presence of donor-derived newly regenerated muscle fibers that were double-positive for μDYS and embryonic MHC (Fig. 6d, white arrows and Additional file 3). Altogether, these results show that transplantation of corrected μDYS-Pax3-induced cells provides functional improvement of dystrophic muscles, both in terms of muscle force generation and in terms of their ability to respond to ongoing muscle injury and stably express μDYS protein.
SCs isolated by flow cytometry have been demonstrated to possess a tremendous capacity to improve muscle function in mdx mice ; however, the impracticality of isolating large numbers of SCs from living donors as well as the requirement for gene correction, if considering an autologous transplantation setting, necessitates ex vivo expansion. To date, only one study has reported a combined cell/gene therapy approach using SCs in the context of muscular dystrophy . In this study, the authors isolated SCs from a dystrophic mouse, transduced them with a lentiviral vector encoding the mouse μDYS transgene, and immediately transplanted them into the dystrophic muscle and found that they were able to differentiate into DYS+ fibers.
Several studies have investigated the transplantation of cultures derived from prospectively isolated SCs. Blau and colleagues demonstrated that culturing mouse SCs on a substrate that mimics muscle tissue elasticity, and in the presence of an inhibitor for p38MAPK, helped maintain “stemness” features [10, 39]. Following a different approach, Tapscott and colleagues expanded freshly isolated canine SCs by activating the Notch signaling pathway, which bestowed superior in vivo regenerative ability upon SC-initiated cultures compared to controls . In a recent study, Rudnicki and colleagues reported that short treatment of SCs with Wnt7a resulted in enhanced engraftment that was accompanied by improved muscle function .
Herein, we demonstrate that upon conditional expression of Pax3, freshly isolated SCs can be successfully expanded when compared to their cultured empty vector control counterparts (Fig. 1d). Following their intramuscular transplantation into dystrophic mice, Pax3-induced cells display greater regenerative potential than control SCs, and engraftment levels correlated with a significant improvement in muscle strength (Fig. 3a–c). Importantly, we also show that engrafted Pax3-induced cells are capable of seeding the SC pool and responding to a second round of CTX-induced damage by generating newly formed DYS+ fibers (Fig. 4d, e). In addition, we show that Pax3-induced dystrophic SCs are amenable to genetic correction. Using a non-viral Sleeping Beauty system carrying a human μDYS transgene, we corrected SCs from dystrophin-deficient mice and found that these were capable of differentiating into functional muscle fibers in vivo (Fig. 5), increasing force generation capacity of dystrophic muscles (Fig. 6a–c), and producing new myofibers upon CTX reinjury that remain positive for the μDYS transgene.
We describe here a method for the ex vivo expansion of SCs that facilitates genetic correction and importantly allows for the retention of a population with the capacity to regenerate muscle function. Unlike viral approaches that target the myofiber, this approach provides a genetic correction that is persistent and not transient as corrected fibers are eventually lost due to normal muscle turnover. If future studies demonstrate that this approach is similarly efficient to expand human SCs, it may be useful to provide human adult stem cell populations endowed with muscle regeneration potential in vivo.
basic fibroblast growth factor
Duchenne muscular dystrophy
embryonic myosin heavy chain
green fluorescent protein
hybrid promoter of human elongation factor 1 and alpha-human eukaryotic initiation factor 4G
myosin heavy chain
muscle progenitors cells
- NSG mice:
NOD scid gamma mice
skeletal α-actin promoter
reverse transcription polymerase chain reaction
The project was supported by NIH grants: RC1AR058118 and AR055299 (RCRP), AR055685 and AG034370 (MK), the Muscular Dystrophy Center Core Laboratories P30-AR0507220, and the Minnesota Muscle Training Grant AR007612 (RWA), as well as funding from the MDA (#238127 to RCRP). We thank the Dr. Bob and Jean Smith Foundation (RCRP) and the Greg Marzolf Jr. Foundation (AF) for their generous support. Monoclonal antibodies to MHC were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We are thankful to Dr. Jeffrey Chamberlain and Dr. Zoltan Ivics for providing, respectively, the human skeletal α-actin promoter (pHSA) and the pCMV-SB100X plasmid.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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.
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