Valproic acid stimulates myogenesis in pluripotent stem cell-derived mesodermal progenitors in a NOTCH-dependent manner.

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

Minimally Invasive Muscle Embedding Generates Donor-Cell-Derived Muscle Fibers that Express Desmin and Dystrophin.

INTRODUCTION: The aim of this study was to quantify the extent of donor-cell-derived myogenesis achieved by a novel surgical technique known as Minimally Invasive Muscle Embedding (MIME). MATERIALS AND METHODS: Through MIME, we implanted a single extensor digitorum longus muscle from donor mice (N = 2) that expressed a red fluorescent protein (RFP), into the left tibialis anterior (TA) muscle of immunodeficient host mice (N = 4) that expressed a green fluorescent protein (GFP). Soon after MIME, we injected a myotoxin (barium chloride), into the host TA muscle, to trigger concerted muscle degeneration and regeneration. In lieu of MIME, we performed a SHAM procedure on the right TA muscle of the same set of animals. RESULTS: In MIME-treated muscles, 22% ± 7% and 78% ± 7% muscle fibers were RFP+ and GFP+, respectively (mean ± standard deviation); and all RFP+ fibers were positive for desmin and dystrophin. Conclusion. We conclude that MIME helps generate muscle fibers of donor origin, in host muscle.

Use of uniformly sized muscle fiber fragments for restoration of muscle tissue function.

Treatment of extensive muscle loss due to traumatic injury, congenital defects, or tumor ablations is clinically challenging. The current treatment standard is grafting of autologous muscle flaps; however, significant donor site morbidity and graft tissue availability remain a problem. Alternatively, muscle fiber therapy has been attempted to treat muscle injury by transplanting single fibers into the defect site. However, irregularly organized long fibers resulted in low survivability due to delay in vascular and neural integration, thus limiting the therapeutic efficacy. Therefore, no effective method is available to permanently restore extensive muscle injuries. To address the current limitations, we developed a novel method that produces uniformly sized native muscle fiber fragments (MFFs) for muscle transplantation. We hypothesized that fragmentation of muscle fibers into small and uniformly sized fragments would allow for rapid reassembly and efficient engraftment within the defect site, resulting in accelerated recovery of muscle function. Our results demonstrate that the processed MFFs have a dimension of approximately 100 µm and contain living muscle cells on extracellular matrices. In preclinical animal studies using volumetric defect and urinary incontinence models, histological and functional analyses confirmed that the transplanted MFFs into the injury sites were able to effectively integrate with host muscle tissue, vascular, and neural systems, which resulted in significant improvement of muscle function and mass. These results indicate that the MFF technology platform is a promising therapeutic option for the restoration of muscle function and can be applied to various muscle defect and injury cases.

Skeletal muscle-derived cell implantation for the treatment of sphincter-related faecal incontinence.

BACKGROUND: In an earlier pilot study with 10 women, we investigated a new approach for therapy of faecal incontinence (FI) due to obstetric trauma, involving ultrasound-guided injection of autologous skeletal muscle-derived cells (SMDC) into the external anal sphincter (EAS), and observed significant improvement. In the current study, we tested this therapeutic approach in an extended patient group: male and female patients suffering from FI due to EAS damage and/or atrophy. Furthermore, feasibility of lower cell counts and cryo-preserved SMDC was assessed. METHODS: In this single-centre, explorative, baseline-controlled clinical trial, each patient (n = 39; mean age 60.6 ± 13.81 years) received 79.4 ± 22.5 × 106 cryo-preserved autologous SMDC. Changes in FI parameters, Fecal Incontinence Quality of Life (FIQL), anorectal manometry and safety from baseline to 1, 6 and 12 months post implantation were evaluated. RESULTS: SMDC used in this trial contained a high percentage of myogenic-expressing (CD56+) and muscle stem cell marker-expressing (Pax7+, Myf5+) cells. Intervention was well tolerated without any serious adverse events. After 12 months, the number of weekly incontinence episodes (WIE, primary variable), FIQL and patient condition had improved significantly. In 80.6% of males and 78.4% of females, the WIE frequency decreased by at least 50%; Wexner scores and severity of FI complaints decreased significantly, independent of gender and cause of FI. CONCLUSIONS: Injection of SMDCs into the EAS effectively improved sphincter-related FI due to EAS damage and/or atrophy in males and females. When confirmed in a larger, placebo-controlled trial, this minimal invasive procedure has the potential to become first-line therapy for FI. TRIAL REGISTRATION: EU Clinical Trials Register, EudraCT 2010-023826-19 (Date of registration: 08.11.2010).

Allogenic Myocytes and Mesenchymal Stem Cells Partially Improve Fatty Rotator Cuff Degeneration in a Rat Model.

PURPOSE: Rotator cuff (RC) tears result not only in functional impairment but also in RC muscle atrophy, muscle fattening and eventually to muscle fibrosis. We hypothesized that allogenic bone marrow derived mesenchymal stem cells (MSC) and myocytes can be utilized to improve the rotator cuff muscle fattening and increase the atrophied muscle mass in a rat model. METHODS: The right supraspinatus (SSP) tendons of 105 inbred rats were detached and muscle fattening was provoked over 4 weeks; the left side remained untouched (control group). The animals (n = 25) of the output group were euthanized after 4 weeks for reference purposes. The SSP-tendon of one group (n = 16) was left unoperated to heal spontaneously. The SSP-tendons of the remaining 64 rats (4 groups with n = 16) were repaired with transosseous sutures. One group received a saline solution injection in the SSP muscle belly, two other groups received 5 × 106 allogenic myocytes and 5 × 106 allogenic MSC injections from donor rats, respectively, and one group received no additional treatment. After 4 weeks of healing, the supraspinatus muscle mass was compared quantitatively and histologically to all the treated groups and to the untreated contralateral side. RESULTS: In the end of the experiments at week 8, the myocyte and MCS treated groups showed a significantly higher muscle mass with 0.2322 g and 0.2257 g, respectively, in comparison to the output group (0.1911 g) at week 4 with p < 0.05. There was no statistical difference between the repaired, treated, or spontaneous healing groups at week 8. Supraspinatus muscle mass of all experimental groups of the right side was significantly lower compared to the untreated contralateral muscle mass. CONCLUSION: This defect model shows that the injection of allogenic mycocytes and MSC in fatty infiltrated SSP muscles is better than no treatment and can partially improve the SSP muscle belly fattening. Nevertheless, a full restoration of the degenerated and fattened rotator cuff muscle to its original condition is not possible using myocytes and MSC in this model.

Development of an in vitro potency assay for human skeletal muscle derived cells.

BACKGROUND: Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals. METHODS: CD56+ SMDCs were separated from CD56- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of in vitro differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence. RESULTS: CD56- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56+ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56+ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected. DISCUSSION: AChE activity was found to truly reflect the in vitro differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful in vitro validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal incontinence. Thus, we recommend using AChE activity of in vitro differentiated SMDCs as a potency measure in end stage (phase III) clinical trials using SMDCs for skeletal muscle regeneration and subsequent market approval application (MAA).

Timed Delivery of Therapy Enhances Functional Muscle Regeneration.

Cell transplantation is a promising therapeutic strategy for the treatment of traumatic muscle injury in humans. Previous investigations have typically focused on the identification of potent cell and growth factor treatments and optimization of spatial control over delivery. However, the optimal time point for cell transplantation remains unclear. Here, this study reports how myoblast and morphogen delivery timed to coincide with specific phases of the inflammatory response affects donor cell engraftment and the functional repair of severely injured muscle. Delivery of a biomaterial-based therapy timed with the peak of injury-induced inflammation leads to potent early and long-term regenerative benefits. Diminished inflammation and fibrosis, enhanced angiogenesis, and increased cell engraftment are seen during the acute stage following optimally timed treatment. Over the long term, treatment during peak inflammation leads to enhanced functional regeneration, as indicated by reduced chronic inflammation and fibrosis along with increased tissue perfusion and muscle contractile force. Treatments initiated immediately after injury or after inflammation had largely resolved provided more limited benefits. These results demonstrate the importance of appropriately timing the delivery of biologic therapy in the context of muscle regeneration. Biomaterial-based timed delivery can likely be applied to other tissues and is of potential wide utility in regenerative medicine.

Contribution of minced muscle graft progenitor cells to muscle fiber formation after volumetric muscle loss injury in wild-type and immune deficient mice.

Volumetric muscle injury (VML) causes an irrecoverable loss of muscle fibers, persistent strength deficits, and chronic disability. A crucial challenge to VML injury and possible regeneration is the removal of all of the in situ native elements necessary for skeletal muscle regeneration. Our first goal was to establish a reliable VML model in the mouse tibialis anterior (TA) muscle. In adult male wild-type and nude mice, a non-repaired ≈20% VML injury to the TA muscle resulted in an ≈59% loss in nerve evoked muscle strength, ≈33% loss in muscle mass, and ≈29% loss of muscle fibers at 28 day post-injury. Our second goal was to investigate if minced muscle grafts (≈1 mm3 tissue fragments) promote recovery of muscle fibers after VML injury and to understand if the graft-derived progenitor cells directly contribute to fiber regeneration. To assess donor cell contribution, donor muscle tissue was derived from UBC-GFP mice in a subset of experiments. Minced grafts restored ≈34% of the lost fibers 28 days post-injury. The number of GFP+ fibers and the estimated number of regenerated fibers were similar, regardless of host mouse strain. The muscle tissue regeneration promoted by minced grafts did not improve TA muscle strength at this time post-injury. These findings demonstrate the direct contribution of minced muscle graft-derived myogenic stem/progenitor cells to recovery of muscle fibers after VML injury and signify the utility of autologous myogenic stem cell therapies for this indication.

Transplantation of Skeletal Muscle Stem Cells.

Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .

Examinations of a new long-term degradable electrospun polycaprolactone scaffold in three rat abdominal wall models.

Alternative approaches to reinforce native tissue in reconstructive surgery for pelvic organ prolapse are warranted. Tissue engineering combines the use of a scaffold with the regenerative potential of stem cells and is a promising new concept in urogynecology. Our objective was to evaluate whether a newly developed long-term degradable polycaprolactone scaffold could provide biomechanical reinforcement and function as a scaffold for autologous muscle fiber fragments. We performed a study with three different rat abdominal wall models where the scaffold with or without muscle fiber fragments was placed (1) subcutaneously (minimal load), (2) in a partial defect (partial load), and (3) in a full-thickness defect (heavy load). After 8 weeks, no animals had developed hernia, and the scaffold provided biomechanical reinforcement, even in the models where it was subjected to heavy load. The scaffold was not yet degraded but showed increased thickness in all groups. Histologically, we found a massive foreign body response with numerous large giant cells intermingled with the fibers of the scaffold. Cells from added muscle fiber fragments could not be traced by PKH26 fluorescence or desmin staining. Taken together, the long-term degradable polycaprolactone scaffold provided biomechanical reinforcement by inducing a marked foreign-body response and attracting numerous inflammatory cells to form a strong neo-tissue construct. However, cells from the muscle fiber fragments did not survive in this milieu. Properties of the new neo-tissue construct must be evaluated at the time of full degradation of the scaffold before its possible clinical value in pelvic organ prolapse surgery can be evaluated.

Quality of life assessment in female patients 2 and 4 years after muscle-derived cell transplants for stress urinary incontinence treatment.

INTRODUCTION: Regenerative medicine for the treatment of urinary incontinence has become a popular area of focus in the search for therapies for this disease. The paper focused on women's quality of life assessment who were subjected to transplantation of MDSC (autologous muscle derived stem cells) to the urethral sphincter. METHODS: The procedure was conducted in 16 female patients who completed the observation stage. Assessment of quality of life before and after the treatment (two and four years post-operation) was conducted based on the validated I-QOL questionnaire (the Polish language version). RESULTS: The questionnaire study showed that autologous cell therapy significantly improves quality of life in female patients suffering from stress urinary incontinence (SUI). The total I-QOL score increased from 49 (SD ± 7.7) before therapy to 77 (SD ± 5.4) two years post-operation. Four years after the procedure, quality of life remained at a higher level than before therapy, although quality of life decreased by several points when compared with the results from the two-year follow-up - 63 (SD ± 7.2). Patients reported significantly less concern related to their ability to reach the toilet to avoid incontinence, improved sleep at night, a higher level of satisfaction with life, and more satisfaction with their sexual lives (p<0.05). CONCLUSION: The MDSC injection procedure for SUI treatment has significant improved quality of life in the majority of our patients in 2 and 4 year follow-up.

Innervation of dystrophic muscle after muscle stem cell therapy.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated. METHODS: Green fluorescent protein (GFP)-expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. RESULTS: The percent overlap between pre- and postsynaptic immunolabeling was greater in donor-derived GFP(+) myofibers, and fewer GFP(+) myofibers were identified as denervated compared with control GFP(-) fibers (P = 0.001 and 0.03). GFP(+) fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). CONCLUSION: It is unclear whether GFP(+) fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor-derived fibers demonstrate clear histological innervation. Muscle Nerve 54: 763-768, 2016.

Direct Insertion of Muscle Fibers is not Restricted to Facial Skin.

Facial musculature is divided into masticatory muscles, i.e. M. masseter and M. buccalis, with bony insertions and smaller facial muscles involved in facial expression, which insert into bone and skin. There are four fixed osteocutaneous points of the face, i.e. zygomatic (Mac Gregor), mandibular (Furnas), orbital (Psillakis), and masseteric with an antigravitational effect and functional role in facial expression (1,2). In other body parts, the direct insertion of muscle fibers into skin has not been reported. In the neck-shoulder region, direct insertion of skeletal muscles into the skin can be observed during surgery in this area. We observed this phenomenon in 3 adult male patients (51-65 years old) during lipoma surgery. In our case series, lipomas were of the superficial subcutaneous type. After local anesthesia with 1% ropivacain, a fusiform skin incision was followed by en bloc resection of the lipoma. During resection, we became aware that muscle fibers of M. levator scapulae inserted into the back skin (Figure 1). This has practical implications for surgery because of possible pain when these fibers need to be cut to deliver a lipoma or other subcutaneous tumors. Lipomas are a common benign tumor of the subcutaneous adipose tissue, known as the superficial type. However, lipomas may occur intramuscularly, intermuscularly, parostealy and intra-osseously (3). In the deep subtypes of lipoma, pain and discomfort may be a leading clinical symptom whereas superficial lipomas often are asymptomatic (4,5). Surgery is the treatment of choice, while liposuction and laser-lipolysis are alternatives (6). During surgery of lipomas of the shoulder/neck region, direct insertion of muscle fibers of M. levator scapulae into skin was noted - an observation possibly underreported in surgical literature.

Open surgical treatment for chronic midportion Achilles tendinopathy: faster recovery with the soleus fibres transfer technique.

PURPOSE: The study aimed to compare two methods of open surgical treatment for midportion Achilles tendinopathy in sportsmen. A novel technique consisting in transferring some soleus fibres into the degenerated tendon to improve its vascularization and longitudinal tenotomies are evaluated and compared. METHODS: From 2006 to 2011, fifty-two competitive and noncompetitive athletes affected by midportion Achilles tendinopathy were surgically treated and prospectively evaluated at 6 months and at a final 4-year mean follow-up. Twenty patients had longitudinal tenotomies, and thirty-two had soleus fibres transfer. Clinical outcome was evaluated by the American Orthopaedic Foot and Ankle Society (AOFAS) score and the Victorian Institute of Sports Assessment-Achilles (VISA-A) score. Time to return to walk and to run and tendon thickening were also recorded. RESULTS: Patients in the soleus transfer group had a higher increase in AOFAS and VISA-A score at 6 months and at the mean 4-year final follow-up (by 5.4 points, 95 % CI 2.9-7.9, p < 0.001 and by 5.7 points, 95 % CI 2.5-8.9, p = 0.001, for AOFAS and VISA, respectively). They also needed less time to return to run: 98.9 ± 17.4 days compared to 122.2 ± 26.3 days for the longitudinal tenotomies group (p = 0.0019). The soleus transfer group had a greater prevalence of tendon thickening (59.4 % compared to 30.0 % in the longitudinal tenotomies group, p = 0.037). CONCLUSIONS: Open surgery for midportion Achilles tendinopathy is safe and effective in medium term. Despite similar outcomes in postoperative functional scores, soleus transfer allows a faster recovery but has a higher incidence of tendon thickening. These results should suggest the use of the soleus graft technique in high-level athletes. LEVEL OF EVIDENCE: Prospective comparative study, Level II.

Muscle fragments on a scaffold in rats: a potential regenerative strategy in urogynecology.

INTRODUCTION AND HYPOTHESIS: The use of permanent synthetic meshes to improve the outcome of pelvic organ prolapse (POP) repair causes frequent and serious complications. The use of the synthetic, biodegradable scaffold methoxypolyethyleneglycol-polylactic-co-glycolic acid (MPEG-PLGA) seeded with autologous muscle fiber fragments (MFF), as an adjunct to native tissue POP repair, is a potential new alternative. METHODS: A rat abdominal wall model of native repair was used with six animals in each of three groups: native repair, native repair + MPEG-PLGA, and native repair + MPEG-PLGA + MFF. MFF were labeled with PKH26-fluorescence dye. After 8 weeks labeled cells were identified in tissue samples and histopathological and immunohistochemical analyses of connective tissue organization and desmin reactivity of muscle cells were performed. Fresh tissue samples were subjected to uniaxial biomechanical testing. Statistical analyses were performed using one-way analysis of variance (ANOVA). RESULTS: MPEG-PLGA was fully degraded after 8 weeks. Desmin-immunopositive (6/6) and PKH26-positive cells (6/6) were found only after native repair + MPEG-PLGA + MFF, indicating survival, proliferation, and integration of cells originating from the MFF. This group also showed significantly increased stiffness in the high stiffness zone compared with native repair + MPEG-PLGA (p = 0.032) and borderline significantly higher stiffness compared to native repair (p = 0.054). CONCLUSIONS: In this pilot study, MPEG-PLGA scaffolds seeded with autologous MFF affected some histological and biomechanical properties of native tissue repair in an abdominal wall defect model in rats. The method thus appears to be a simple tissue engineering concept with potential relevance for native tissue repair of POP.

Transplantation of devitalized muscle scaffolds is insufficient for appreciable de novo muscle fiber regeneration after volumetric muscle loss injury.

Volumetric muscle loss (VML) is a traumatic and functionally debilitating muscle injury with limited treatment options. Developmental regenerative therapies for the repair of VML typically comprise an ECM scaffold. In this study, we tested if the complete reliance on host cell migration to a devitalized muscle scaffold without myogenic cells is sufficient for de novo muscle fiber regeneration. Devitalized (muscle ECM with no living cells) and, as a positive control, vital minced muscle grafts were transplanted to a VML defect in the tibialis anterior muscle of Lewis rats. Eight weeks post-injury, devitalized grafts did not appreciably promote de novo muscle fiber regeneration within the defect area, and instead remodeled into a fibrotic tissue mass. In contrast, transplantation of vital minced muscle grafts promoted de novo muscle fiber regeneration. Notably, pax7+ cells were absent in remote regions of the defect site repaired with devitalized scaffolds. At 2 weeks post-injury, the devitalized grafts were unable to promote an anti-inflammatory phenotype, while vital grafts appeared to progress to a pro-regenerative inflammatory response. The putative macrophage phenotypes observed in vivo were supported in vitro, in which soluble factors released from vital grafts promoted an M2-like macrophage polarization, whereas devitalized grafts failed to do so. These observations indicate that although the remaining muscle mass serves as a source of myogenic cells in close proximity to the defect site, a devitalized scaffold without myogenic cells is inadequate to appreciably promote de novo muscle fiber regeneration throughout the VML defect.

The effect of endoscopic administration of autologous porcine muscle-derived cells into the urethral sphincter.

OBJECTIVE: To verify the fate of autologous porcine myogenic cells after endoscopic administration into the urethral sphincter. METHODS: This study was performed on pig animal models. The muscle-derived cells (MDCs) were isolated and identified. After the third passage, the 6 × 10(7) of PKH26 labeled cells were injected into the urethral sphincter using a urethrocystoscope. The urethras were collected after 28 days. To analyze the fate of injected cells, the PKH26 presence, the desmin expression, and the distribution of acetylcholine receptors were evaluated in the tissue sections. Moreover, the maximal urethral closure pressure (MUCP) was assessed in experimental and control groups at day 1 and day 28. RESULTS: The isolated porcine MDCs expressed desmin and were able to differentiate into myotubes in vitro. At day 28 after the transplantation, the depots of PKH26-positive cells were observed in the muscular layer, but also in the submucosa. The staining for desmin revealed that cells located in the muscle layer were integrated with muscle fibers that possessed acetylcholine receptors. However, cells administered into nonmuscle tissue did not express desmin. Urethral pressure profilometry demonstrated no significant differences between MUCP in the transplanted group in comparison to the control group at day 28. CONCLUSION: The present study demonstrates the successful endoscopic transplantation of myogenic cells into the urethral sphincter. The experiments indicated the key importance of precise cell administration in terms of their fate after the injection.

Separate or combined treatments with daily sildenafil, molsidomine, or muscle-derived stem cells prevent erectile dysfunction in a rat model of cavernosal nerve damage.

INTRODUCTION: Long-term daily administration of phosphodiesterase type 5 (PDE5) inhibitors in the rat prevents or reverses corporal veno-occlusive dysfunction (CVOD) and smooth muscle cell (CSMC) loss and fibrosis, in both aging and bilateral cavernosal nerve resection (BCNR) models for erectile dysfunction. In the aging rat model, corporal implantation of skeletal muscle-derived stem cells (MDSC) reverses CVOD. Nitric oxide (NO) and cyclic guanosine monophosphate can modulate stem cell lineage. AIM: To investigate in the BCNR model the effects of sildenafil at lower doses, alone or in combination with MDSC or the NO donor molsidomine, on CVOD and the underlying corporal histopathology. MAIN OUTCOMES MEASURES: CVOD, histological, and biochemical markers in rat corporal tissue. Methods. Rats subjected to BCNR were maintained for 45 days either untreated, or received sildenafil in the water or retrolingually at 10, 2.5, and 1.25 mg/kg/day (medium, low, and very low doses), or intraperitoneal molsidomine, or MDSC implantation into the corpora cavernosa separately or in combination. Cavernosometry evaluated CVOD. Histopathology was assessed on penile sections by Masson trichrome, immunohistochemistry for α-smooth muscle actin (ASMA), or immunofluorescence for neuronal nitric oxide synthase (nNOS)/neurofilament 70, and in fresh tissue by Western blot for various markers and picrosirius red for collagen. RESULTS: All treatments normalized erectile function (drop rate), and most increased the CSMC/collagen ratio and ASMA expression in corporal tissue sections, and reduced collagen content in the penile shaft. MDSC also increased nNOS and brain-derived neurotrophic factor. The combination treatment was not superior to MDSC or sildenafil given alone, and upregulated PDE5. CONCLUSIONS: Lowering the dose of a continuous long-term sildenafil administration still maintained the prevention of CVOD in the BCNR rat previously observed, but it was less effective on the underlying histopathology. As in the aging rat model, MDSC also counteracted CVOD, but supplementation with very low-dose sildenafil did not improve the outcome.

ß4 integrin marks interstitial myogenic progenitor cells in adult murine skeletal muscle.

Skeletal muscle growth and its regeneration following injury rely on myogenic progenitor cells, a heterogeneous population that includes the satellite cells and other interstitial progenitors. The present study demonstrates that surface expression of ß4 integrin marks a population of vessel-associated interstitial muscle progenitor cells. Muscle ß4 integrin-positive cells do not express myogenic markers upon isolation. However, they are capable of undergoing myogenic specification in vitro and in vivo: ß4 integrin cells differentiate into multinucleated myotubes in culture dishes and contribute to muscle regeneration upon delivery into diseased mice. Subfractionation of ß4 integrin-expressing cells based on CD31 expression does not further enrich for myogenic precursors. These findings support the expression of ß4 integrin in interstitial, vessel-associated cells with myogenic activity within adult skeletal muscle.

Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies.

Muscular dystrophies (MDs) are a heterogeneous group of inherited disorders characterized by progressive muscle wasting and weakness likely associated with exhaustion of muscle regeneration potential. At present, no cures or efficacious treatments are available for these diseases, but cell transplantation could be a potential therapeutic strategy. Transplantation of myoblasts using satellite cells or other myogenic cell populations has been attempted to promote muscle regeneration, based on the hypothesis that the donor cells repopulate the muscle and contribute to its regeneration. Embryonic stem cells (ESCs) and more recently induced pluripotent stem cells (iPSCs) could generate an unlimited source of differentiated cell types, including myogenic cells. Here we review the literature regarding the generation of myogenic cells considering the main techniques employed to date to elicit efficient differentiation of human and murine ESCs or iPSCs into skeletal muscle. We also critically analyse the possibility of using these cellular populations as an alternative source of myogenic cells for cell therapy of MDs.