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Background Muscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration. Methods A clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration. Results 2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI. Conclusion WIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

Background Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. Methods Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. Results MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. Conclusions Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

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).

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).

We assessed the efficacy and safety of the application of autologous fibroblasts and myoblasts for treatment in post-prostatectomy urinary incontinence after a minimal followup of 1 year.Sixty-three patients with stress urinary incontinence after radical prostatectomy were treated with transurethral ultrasound guided injections of autologous fibroblasts and myoblasts obtained from skeletal muscle biopsies. All subjects were evaluated preoperatively and 12 months postoperatively in terms of incontinence and Quality of Life Instrument scores, urodynamic parameters, and morphology and function of the urethra and rhabdosphincter.Of the 63 patients 41 were continent 12 months after implantation of cells, 17 showed improvement and 5 did not show any improvement. Incontinence and Quality of Life Instrument scores as well as thickness and contractility of the rhabdosphincter were significantly improved postoperatively.The use of myoblast and fibroblast therapy represents a minimally invasive, safe and effective treatment for post-prostatectomy incontinence after a followup of 1 year.

In the last years preclinical studies have paved the way for the use of adult muscle derived stem cells for reconstruction of the lower urinary tract. Between September 2002 and October 2004, 42 women and 21 men suffering from urinary stress incontinence (age 36-84 years) were recruited and subsequently treated with transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. In parallel, 7 men and 21 women (age 39-83 years) also diagnosed with urinary stress incontinence were treated with standard transurethral endoscopic injections of collagen. Patients were randomly assigned to both groups. After a follow-up of 12 months incontinence was cured in 39 women and 11 men after injection of autologous myoblasts and fibroblasts. Mean quality of life score (51.38 preoperatively, 104.06 postoperatively), thickness of urethra and rhabdosphincter (2.103 mm preoperatively, 3.303 mm postoperatively) as well as contractility of the rhabdosphincter (0.56 mm preoperatively, 1.462 mm postoperatively) were improved postoperatively. Only in two patients treated with injections of collagen incontinence was cured. The present clinical results demonstrate that, in contrast to injections of collagen, urinary incontinence can be treated effectively with ultrasonography-guided injections of autologous myo- and fibroblasts.

Objective: Recent progress in the field of cellular cardiomyoplasty has opened new prospects for the treatment of ischemic heart disease and currently moves from bench to bedside. The aim of the present study was to develop a novel cell delivery technique, reducing target tissue damage and improving cell dispersion and engraftment. Methods: In 30 male Fischer F344 rats an infarction of the left ventricle was generated by ligation of the left anterior descendent artery. Seven days after infarction, either 15 microdepots of 10 ml myoblast cell suspension (microdepot group) or culture medium (control group) were injected into the infarcted region using an automatic pressure injection device, or three depots of 50 ml myoblast cell suspension (macrodepot group) were injected using the standard surgical technique. Echocardiography was performed in all rats before and 6 weeks after cell injection. In all groups the perioperative mortality was below 20%. Six weeks after cell transplantation, a significant improvement of ejection fraction was seen in both myoblast treated groups compared to controls (macrodepot, microdepot, control; 53.7G11.9, 70.7G2.0, 39.1G6.4; PZ0.026, P!0.001). The microdepot group showed a more decent improvement than the macrodepot group (70.7G2.0 vs. 53.7G11.9, PZ0.013). In both treated groups, grafted myoblasts differentiated into multinucleated myotubes within host myocardium, however, the engraftment pattern was different and angiogenesis was enhanced in the microdepot group. Conclusions: Intramyocardial multisite pressure injection allows the safe and reliable transplantation of several myoblast microdepots into an infarcted myocardium and improves the efficacy of myoblast transplantation compared to the standard technique. Q 2005 Elsevier B.V. All rights reserved.

Experimental and clinical studies investigated whether urinary incontinence can be effectively treated with transurethral ultrasound-guided injections of autologous myoblasts and fibroblasts.This new therapy was performed in eight female pigs. It could be shown that the injected cells survived well and that new muscle tissue was formed. Next, 42 patients (29 women, 13 men) suffering from urinary stress incontinence were treated. The fibroblasts were mixed with a small amount of collagen as carrier material and injected into the urethral submucosa to treat atrophies of the mucosa. The myoblasts were directly injected into the rhabdosphincter to reconstruct the muscle and to heal morphological and functional defects. In 35 patients urinary incontinence could be completely cured. In seven patients who had undergone multiple surgical procedures and radiotherapy urinary incontinence improved. No side effects or complications were encountered postoperatively. The experimental as well as the clinical data clearly demonstrate that urinary incontinence can be treated effectively with autologous stem cells. The present data support the conclusion that this new therapeutic concept may represent a very promising treatment modality in the future.

1. Introduction and rationaleThe increasing incidence in urinary incontinence (UI)with advancing age is directly correlated to spontaneousapoptosis of the muscle cells of the rhabdosphincter, thestriated urethral sphincter. As life expectancy of the wholesociety is growing, significance of UI as a disease of theelderly will increase further. Age-dependent reduction in thenumber of striated muscle cells is now held the highestranking cause of urinary stress incontinence in elderlywomen and men (Strasser et al., 1999, 2000a,b).The technical availability of autologous satellite cellsobtained from skeletal muscle enabled the immediateapplication of muscle progenitor cells in the treatment ofUI by means of tissue engineering techniques in patients ofany age, including elderly (Strasser et al., 2004).This review focuses on the theoretical assumptions andexperimental findings on muscle derived stem cells (MDSC)justifying their straightforward application in urology. Onlyquite recently, the regenerative function of mammaliantissues has become understood as ubiquitous (Deans andMoseley, 2000; Moore, 2002). Not only rapidly renewingtissues like skin, bone marrow or blood were found topossess a stem cell reserve: even apparently quiescenttissues, like nerve tissue or heart muscle disclosed—duringcellular stress or tissue loss—a tissue inherent renewingpotential (Hanashima et al., 2004). Tissue renewal dependson the persistence of so-called adult stem cells or precursorsof mature cells.Moreover, not only acute processes like injury orintoxication activate stem cell directed tissue regeneration.Cellular aging, cell death (apoptosis and necrosis) andrenewal continue throughout life and through all tissues.Imbalances between cell death and renewal are now thoughtto occur during aging (when apoptosis supersedes renewingcapacity) and during malignant transformation (whenregular cell death is hindered by apoptosis inhibition)(Hanahan and Weinberg, 2000).According to this rationale, the higher prevalence ofUI among elderly persons of both sexes can beinterpreted as a symptom of increasingly poor andeventually failing tissue regeneration in the vesico-urethral apparatus.A direct correlation between age and the number ofapoptotic cells in the rhabdomyosphincter on the one hand,and impaired detrusor contractility and weak closurepressure caused by structural deficits of the rhabdosphincteron the other hand, suggest that stress incontinence ispredominantly associated with a dysfunction of the striatedurethral sphincter (Frauscher et al., 1998, Strasser et al.,1999).Yiou and co-workers have shown that the adultrhabdosphincter in mice regenerates after an injury bymeans of intrinsic satellite cells (Yiou et al., 2003). Thisfinding is interesting per se, because rhabdosphincter andstriated skeletal muscles have a different embryologicalorigin. Muscle cells of the external sphincter arethought to arise via transdifferentiation of urethral smoothmuscle cells (Borirakchanyavat et al., 1997)—and thus

We have analysed the voltage-gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP(+)-PKH(+) myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage-gated sodium, calcium and potassium channels. A down-regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down-regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down-regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects expression of voltage-gated ion channels of skeletal myoblasts.

BACKGROUND The electrophysiological properties of myoblast cultures established from the human and porcine rhabdosphincter (RS) and porcine lower limb muscle (LLSKM) were studied to elucidate their potential for tissue engineering applications in the lower urinary tract. METHODS Muscle biopsies were collected from the prostatic part of the RS, the RS of male pigs, and the porcine LLSKM. Ion channels were studied by means of the patch-clamp technique. RESULTS Only one subtype each of voltage gated Na+ and Ca2+ channels was observed in porcine RS and LLSKM. Two types of voltage gated Ca2+ channels were identified in human RS cells. The porcine RS and LLSKM myoblasts displayed similar fusion competence. CONCLUSIONS Porcine RS and LLSKM myoblasts and human RS and human skeletal muscle cells show a high degree of similarity. Injection of autologous skeletal muscle myoboblasts in the lower urinary tract might, therefore, represent a promising approach to treat stress incontinence after radical prostatectomy. © 2004 Wiley-Liss, Inc.

To evaluate the cellular survival of donor fibroblasts after transplantation at the vesico-ureteral junction (VUJ) and to analyse their potential for reconstructive cell replacement in an animal model as autologous fibroblasts have been used as soft tissue augmentation material for scared and damaged tissue.Muscles biopsies were procured from the lower limb muscles of 4 pigs; cytoplasm of fibroblasts was labelled with nano-sized iron oxide particles. Six weeks after taking of the muscle biopsies, fibroblast transplantation was performed, 3 × 10(6) cells suspended in transplantation medium (in 1-ml syringes) were injected at the VUJ using the modified STING technique. Animals were killed 8 weeks later; seeded fibroblasts were identified using prussian blue staining protocol; histological evaluation and morphological analysis were performed by light microscopy (Mayer's haematoxylin-eosin staining); and bladders were scanned by MRI for visualization and localization of the iron-labelled donor cells.Donor fibroblast cell colonization and cellular viability at the VUJ was demonstrated by MRI and histochemically indicating cellular uptake of iron particles at the VUJ. It was also evident that transplanted fibroblasts integrate into the extracellular matrix of the distal ureter augmenting ureteral host tissue.Labelled implanted autologous fibroblasts were visualized by staining procedure as well as MRI scan demonstrating persistence at the VUJ, suggesting that in vitro expanded fibroblasts survived in vivo after transplantation.

OBJECTIVE To assess the efficacy and safety of the application of autologous myoblasts and fibroblasts for treating female stress urinary incontinence (SUI) after a follow-up of ≥1 year. PATIENTS AND METHODS In all, 123 women with SUI (aged 36–84 years) were treated with transurethral ultrasonography-guided injections with autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were suspended in a small amount of collagen as carrier material and injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. All patients were evaluated before and 12 months after the injection using the Incontinence and Quality of Life Instrument (I-QOL) scores, urodynamic variables, and morphology and function of the urethra and rhabdosphincter. RESULTS At 1 year after implanting the cells, 94 of the 119 women (79%) were completely continent, 16 (13%) had a substantial improvement and nine (8%) a slight improvement. Four patients were lost to follow-up.The incontinence and I-QOL scores, and the thickness, contractility and electromyographic activity of the rhabdosphincter were significantly improved after treatment. CONCLUSIONS These results show the efficacy and safety of transferring autologous myoblasts and fibroblasts in the treatment of female SUI, after a follow-up of 1 year.

Preclinical studies have suggested that transurethral injections of autologous myoblasts can aid in regeneration of the rhabdosphincter, and fibroblasts in reconstruction of the urethral submucosa. We aimed to compare the effectiveness and tolerability of ultrasonography-guided injections of autologous cells with those of endoscopic injections of collagen for stress incontinence.Between 2002 and 2004, we recruited 63 eligible women with urinary stress incontinence. 42 of these women were randomly assigned to receive transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts, and 21 to receive conventional endoscopic injections of collagen. The first primary outcome measure was an incontinence score (range 0-6) based on a 24-hour voiding diary, a 24-hour pad test, and a patient questionnaire. The other primary outcome measures were contractility of the rhabdosphincter and thickness of both the urethra and rhabdosphincter. Analysis was by intention to treat. This trial is registered with Controlled-Trials.com, number CCT-NAPN-16630.At 12-months' follow-up, 38 of the 42 women injected with autologous cells were completely continent, compared with two of the 21 patients given conventional treatment with collagen. The median incontinence score decreased from a baseline of 6.0 (IQR 6.0-6.0; where 6 represents complete incontinence), to 0 (0-0) for patients treated with autologous cells, and 6.0 (3.5-6.0) for patients treated with collagen (p0.0001). Ultrasonographic measurements showed that the mean thickness of the rhabdosphincter increased from a baseline of 2.13 mm (SD 0.39) for all patients to 3.38 mm (0.26) for patients treated with autologous cells and 2.32 mm (0.44) for patients treated with collagen (p0.0001). Contractility of the rhabdosphincter increased from a baseline of 0.58 mm (SD 0.32) to 1.56 mm (0.28) for patients treated with autologous cells and 0.67 mm (0.51) for controls (p0.0001). The change in the thickness of the urethra after treatment was not significantly different between treatment groups. No adverse effects were recorded in any of the 63 patients.Long-term postoperative results and data from multicentre trials with larger numbers of patients are needed to assess whether injection of autologous cells into the rhabdosphincter and the urethra could become a standard treatment for urinary incontinence.

Objectives To investigate the efficacy of transurethral ultrasound (TUUS)-guided injections of autologous myoblasts and fibroblasts in women with incontinence. Methods Between January and June 2005, 20 female patients suffering from stress urinary incontinence (SUI) were included. Skeletal muscle biopsies were taken from the left arm to obtain cultures from autologous fibroblasts and myoblasts. By TUUS guidance the fibroblasts were injected into the urethral submucosa and the myoblasts were injected into the rhabdosphincter. A defined incontinence score, quality-of-life score and urodynamic, electromyographic, and laboratory parameters, as well as morphology and function of urethra and rhabdosphincter were evaluated before and up to 2 yr after therapy. Results Eigtheen of 20 patients were cured 1 yr after injection of autologous stem cells and in 2 patients SUI was improved. Two years after therapy 16 of the 18 patients presented as cured, 2 others were improved, and 2 were lost to follow-up. Incontinence and quality-of-life scores were significantly improved postoperatively. The thickness of urethra and rhabdosphincter as well as activity and contractility of the rhabdosphincter were also statistically significantly increased after therapy. Conclusions Clinical results demonstrate that SUI can be treated effectively with autologous stem cells. The present data support the conclusion that this therapeutic concept represents an elegant and minimally invasive treatment modality to treat SUI.

Defects caused by traumatic or postsurgical loss of muscle mass may result in severe impairments of the functionality of skeletal muscle. Tissue engineering represents a possible approach to replace the lost or defective muscle. The aim of this study was to compare the suitability of three different biomaterials as scaffolds for rat myoblasts, using a new animal model. PKH26-fluorescent-stained cultured rat myoblasts were either seeded onto polyglycolic acid meshes or, alternatively, suspended in alginate or in hyaluronic acid-hydrogels. In each of the eight Fisher CDF-344 rats, four capsule pouches were induced by subcutaneous implantation of four silicone sheets. After two weeks the silicone sheets were removed and myoblast-biomaterial-constructs were implanted in the preformed capsules. Specimens were harvested after four weeks and examined histologically by H&E-staining and fluorescence microscopy. All capsules were well-vascularized. Implanted myoblasts fused by forming multinucleated myotubes. This study demonstrates that myoblasts seeded onto different biomaterials can be successfully transplanted into preformed highly vascularized capsule pouches. Our animal model has paved the way for studies of myoblast-biomaterial transplantations into an ectopic non-muscular environment.

Objectives: To prove whether intramyocardial transplantation of combined skeletal myoblasts (SM) and mononuclear bone marrow stem cells is superior to the isolated transplantation of these cell types after myocardial infarction in rats. Methods: In 67 male Fischer rats myocardial infarction was induced by direct ligature of the LAD. Seven days postinfarction baseline echocardiography and intramyocardial cell transplantation were performed. Via lateral thoracotomy 200 ml containing either 10 7 SMs or 10 7 bone marrow-derived mononuclear cells (BM-MNC) or a combination of 5 £ 10 6 of both cell types (MB) were injected in 10 –15 sites in and around the infarct zone. In controls (C) 200 ml of cell-free medium were injected in the same manner. Before injection both cell types were stained using a fluorescent cell linker kit (PKH, Sigma). In addition, SMs were transfected with green fluorescent protein. Nine weeks postinfarction follow-up echocardiography was performed and animals were sacrificed for further analysis. Results: At baseline echocardiography there was no difference in left ventricular ejection fraction (LVEF; C, SM, BM-MNC, MB: 60.1 ^ 3.2, 53.3 ^ 10.2, 53.1 ^ 8.7, 49 ^ 9.0%) and left ventricular end diastolic diameter (LVEDD; C, SM, BM-MNC, MB: 6.5 ^ 0.8, 5.17 ^ 0.8, 5.77 ^ 1.4, 6.25 ^ 0.8 mm) between the different therapeutic groups. Eight weeks after cell transplantation LVEDD was significantly increased in all animals except those that received a combination of myoblasts and bone marrow stem cells (MB; C, SM, BM-MNC, MB: 7.7 ^ 0.6 mm, P ¼ 0:001; 7.7 ^ 1.5 mm, P , 0:001; 7.7 ^ 1.1 mm, P ¼ 0:005; 6.6 ^ 1.7 mm, P ¼ 0:397). At the same time LVEF decreased significantly in the control group (C), stayed unchanged in animals that received bone marrow stem cells (BM-MNC) and increased in animals that received myoblasts (SM) and a combination of both cell types (MB; C, SM, BM-MNC, MB: 45.3 ^ 7.0%, P ¼ 0:05; 63.9 ^ 15.4%, P ¼ 0:044; 54.3 ^ 6.3%, P ¼ 0:607; 63.0 ^ 11.5%, P ¼ 0:039). Conclusions: The present data show that the concept of combining SMs with bone marrow-derived stem cells may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity. Further studies are required to identify the exact mechanisms underlying this synergy and to allow full exploitation of its therapeutic potential. q 2004 Elsevier B.V. All rights reserved.

Purpose Myoblast transplantation has been shown to augment damaged tissue. Moreover it could be used as an autologous bulking agent in refluxing vesicoureteral junctions in order to restore the damaged muscular integrity. But graft viability and the behaviour of the donor myoblasts at the ureterovesical junction have not been demonstrated yet. Material and methods Muscle biopsies from the back muscles of 5 pigs were obtained. Quality of the cells was evaluated by electrophysiological and immunohistochemical tests. Additionally they were labelled with a PKH fluorescent membrane dye to provide fluorescent marking of live cells. 5 weeks after muscle biopsies all animals underwent cell transplantation with 300 × 106 cells suspended in transplantation medium loaded into five 1-cc tuberculin syringes and injected into the ureterovesical junction. Animals were sacrificed 8 weeks later, the donor cells were identified using PKH26 labelling and fluorescence-activated cell-sorter analysis. Moreover histopathology and evaluation concerning tissue reaction at the donor site was also carried out. Results 2-3 intact autologous skeletal myoblast–derived skeletal muscle cells/visual field were found in all injected animals. However, immunohistochemistry demonstrated viability of transplanted myoblasts only at the borders of the bulking area wheras central widespread necrosis with red fluorescent cell detritus could be observed. We did not observe marked signs of an inflammatory response, even though a fibrotic scar was found at the site of injection. Conclusions These results highlight the inability of the vast majority of transplanted myoblasts to survive. Only a small portion of the cells survive when transplanted into a well vascularized area which is in favour of the assumption of an indirect impairment such as paracrine effects. Failure of myoblast transplantation in this animal study does not prove that the procedure itself is ineffective, but these results do caution against the application of in vivo myoblasts as a bulking agent.