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Background and Aims: Fecal incontinence (FI) improvement following injection of autologous skeletal muscle-derived cells has been previously suggested. This study aimed to test the efficacy and safety of said cells through a multicenter, placebo-controlled study, to determine an appropriate cell dose, and to delineate the target patient population that can most benefit from cell therapy. Methods: Patients experiencing FI for at least 6 months were randomized to receive a cell-free medium or low or high dose of cells. All patients received pelvic floor electrical stimulation before and after treatment. Incontinence episode frequency (IEF), FI quality of life, FI burden assessed on a visual analog scale, Wexner score, and parameters reflecting anorectal physiological function were all assessed for up to 12 months. Results: Cell therapy improved IEF, FI quality of life, and FI burden, reaching a preset level of statistical significance in IEF change compared with the control treatment. Post hoc exploratory analyses indicated that patients with limited FI duration and high IEF at baseline are most responsive to cells. Effects prevailed or increased in the high cell count group from 6 to 12 months but pla-teaued or diminished in the low cell count and control groups. Most physiological parameters remained unaltered. No unexpected adverse events were observed. Conclusions: Injection of a high dose of autologous skeletal muscle-derived cells followed by electrical stimulation significantly improved FI, particularly in patients with limited FI duration and high IEF at baseline, and could become a valuable tool for treatment of FI, subject to confirmatory phase 3 trial(s). (ClinicalTrialRegister.eu; EudraCT Number: 2010-021463-32).

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.

BackgroundMuscle 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.MethodsA clamping model of murine (C57b/6J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 hours (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1x106 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.ConclusionWIT-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 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.

Evolution has created a large family of different classes of voltage-gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue-specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Ca(v)2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage-dependent inactivation process and in some channel types by an additional Ca2+-dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore-forming alpha1-subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel alpha1-subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the alpha1-subunits with auxiliary beta-subunits and intracellular regulator proteins. The evidence shows that pore-forming S6 segments and conformational changes in extra- (pore loop) and intracellular linkers connected to pore-forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed.

Until recently, poly(ADP-ribosyl)ation was supposed to be confined only to polymerizing(ADP-ribosyl)transferase/(ADP-ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP-ribosyl)transferase homologues from mouse and man that lack all of the N-terminal DNA binding and BRCA1 C-terminus domains and will be designated polymerizing(ADP-ribosyl)transferase-2 as distinguished from the classical polymerizing(ADP-ribosyl)transferase (polymerizing(ADP-ribosyl)transferase-1). The murine polymerizing(ADP-ribosyl)transferase-2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue (‘APP’, GenBank database AF069298). Expression of the murine polymerizing(ADP-ribosyl)transferase-2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP-ribosyl)ation activity might account for most of the residual poly(ADP-ribosyl)ation observed in polymerizing(ADP-ribosyl)transferase-1−/− mice.

The extracellular domains of the TrkA nerve growth factor (NGF) receptor and its homologs harbor a modular mosaic of potential ligand binding motifs, namely two immunoglobulin (Ig)-like modules and an LRM3 cassette consisting of a tandem array of three leucine-rich motifs (LRMs) flanked by cysteine-rich clusters (Schneider, R., and Schweiger, M. (1991) Oncogene 6, 1807-1811). Identification of a structural motif capable of specifically recognizing the various neurotrophins was achieved by assessing their affinities to isolated recombinant modules of TrkA and TrkB. In both receptors the LRM3 cassette alone could mediate the respective neurotrophin selectivities and affinities. Further tracking down of this NGF-binding site in TrkA strikingly revealed that a single LRM of 24 amino acids could bind NGF selectively with nanomolar affinity. Since this is the first example of a single LRM with a highly specific, well defined function, it might serve as a valuable tool to elucidate the general structural requirements of substrate recognition and high affinity binding in the large superfamily of LRM-containing proteins.

TrkB is a member of the Trk family of neurotrophin receptors. Its extracellular domain exhibits the same modular structure found in its homologs, TrkA and TrkC, consisting of an N-terminal LRM3 cassette and two immunoglobulin-like modules (Ig2 domain) adjacent to the membrane. The LRM3 cassette comprises two cysteine-rich clusters framing a tandem array of three leucine-rich motifs (LRMs). On the basis of the recent identification of a nerve growth factor (NGF) binding site within TrkA, the ability of the different structural entities within the extracellular domain of TrkB to bind the various neurotrophins was determined by using a recombinant receptor approach. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) bound to the LRM3 cassette of TrkB, whereas NGF did not. These binding characteristics evidently reflect in vivo specificities. A more precise mapping of the region(s) responsible for binding BDNF, NT-3, and NT-4 identified the second leucine-rich motif of TrkB as a functional unit capable of binding all three neurotrophins. The affinities and kinetics that this short stretch of amino acids exhibited with respect to the different neurotrophins were clearly akin to those observed for cells ectopically expressing TrkB receptors. With 24 amino acids determining the affinities and kinetics of the interactions with three different partners, the leucine-rich motif is strongly established as one of the most potent and flexible protein--protein interaction motifs.

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.

Objective Transurethral ultrasound-guided injection of autologous myoblasts has recently been shown to cure urinary stress incontinence. In the present study, the dose-dependent changes in maximal urethral closure pressures after application of myoblasts were investigated in a porcine animal model. Methods Myoblast cultures were grown from a porcine muscle biopsy. The biopsy was enzymatically dissociated by using a modified cell dispersion technique. Single myoblasts in suspension were manually collected with a micropipette under microscopic control. Next a clonal myoblast culture was prepared. Before the cells were applied, fluorescence labelling (PKH) was used to assess integration of the injected myoblasts into the rhabdosphincter. With the help of a transurethral ultrasound probe (23 F, 11MHz) and a special injection system, the myoblasts were injected into the rhabdosphincter of five pigs under direct sonographic control. Into two different areas of the rhabdosphincter, increasing different cell counts were injected (total volume 1.5ml). At each area, 10 depots of 150μl volume were injected all along the rhabdosphincter. The following cell counts were used: 1.5 × 10 6 , 2.1 × 10 6 , 4.2 × 10 6 (low range) 5.69 × 10 6 , 8.1 × 10 6 , 1.13 × 10 7 , 1.6 × 10 7 (mid range) 2.26 × 10 7 , 4.4 × 10 7 , and 7.8 × 10 7 (high range). To avoid possible cell rejection, we immunosuppressed the pigs with daily cortisone (1g Solu Dacortin) because allogenic myoblasts were used. Urethral pressure profiles (UPPs) were measured before and 3 wk postoperatively before the pigs were put to sleep. The lower urinary tract was removed in all pigs for histological analysis. Results Histological examination of the specimens revealed that the injected cells had survived at the injection site and had formed new myofibres. Overall the UPP curves revealed dose-dependent changes. Statistically significant increased pressure values of up to more than 300% could be observed in all cases in which higher concentrations of cells had been applied. Increases were also noted in mid range concentrations although not to such a high extent (approximately 150%). Pressure values had even diminished (approximately 50%) after injecting the three lowest concentrations (1.5 × 10 6 , 2.1 × 10 6 , 4.2 × 10 6 ). Conclusions The present results show that the effects after application of myoblasts into the rhabdosphincter are dose-dependent.

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

Light stimuli produce graded hyperpolarizations of the photoreceptor plasma membrane and an associated decrease in a voltagegated calcium channel conductance that mediates release of glutamate neurotransmitter. The Ca v 1.4 channel is thought to be involved in this process. The CACNA1F gene encodes the poreforming subunit of the Ca v 1.4 channel and various mutations in CACNA1F cause X-linked incomplete congenital stationary night blindness (CSNB2). The molecular mechanism of the pathology underlying the CSNB2 phenotype remains to be established. Recent clinical investigations of a New Zealand family found a severe visual disorder that has some clinical similarities to, but is clearly distinct from, CSNB2. Here, we report investigations into the molecular mechanism of the pathology of this condition. Molecular genetic analyses identified a previously undescribed nucleotide substitution in CACNA1F that is predicted to encode an isoleucine to threonine substitution at CACNA1F residue 745. The I745T CACNA1F allele produced a remarkable approximately –30-mV shift in the voltage dependence of Ca v 1.4 channel activation and significantly slower inactivation kinetics in an expression system. These findings imply that substitution of this wild-type residue in transmembrane segment IIS6 may have decreased the energy required to open the channel. Collectively, these findings suggest that a gain-of-function mechanism involving increased Ca v 1.4 channel activity is likely to cause the unusual phenotype.

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.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.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.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.

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.

1. Low threshold, T-type, Ca(2+) channels of the Ca(v)3 family display the fastest inactivation kinetics among all voltage-gated Ca(2+) channels. The molecular inactivation determinants of this channel family are largely unknown. Here we investigate whether segment IIIS6 plays a role in Ca(v)3.1 inactivation as observed previously in high voltage-activated Ca(2+) channels. 2. Amino acids that are identical in IIIS6 segments of all Ca(2+) channel subtypes were mutated to alanine (F1505A, F1506A, N1509A, F1511A, V1512A, F1519A, FV1511/1512AA). Additionally M1510 was mutated to isoleucine and alanine. 3. The kinetic properties of the mutants were analysed with the two-microelectrode voltage-clamp technique after expression in Xenopus oocytes. The time constant for the barium current (I(Ba)) inactivation, tau(inact), of wild-type channels at -20 mV was 9.5 +/- 0.4 ms; the corresponding time constants of the mutants ranged from 9.2 +/- 0.4 ms in V1512A to 45.7 +/- 5.2 ms (4.8-fold slowing) in M1510I. Recovery at -80 mV was most significantly slowed by V1512A and accelerated by F1511A. 4. We conclude that amino acids M1510, F1511 and V1512 corresponding to previously identified inactivation determinants in IIIS6 of Ca(v)2.1 (Hering et al. 1998) have a significant role in Ca(v)3.1 inactivation. These data suggest common elements in the molecular architecture of the inactivation mechanism in high and low threshold Ca(2+) channels.

Ca(v)2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta-subunits regulates Ca2+ entry through Ca(v)2.1. However, the molecular mechanism of this alpha1-beta-subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha(1)2.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (k(inact)) ranging from a 1.5-fold slowing in V1818I (k(inact) = 0.98 +/- 0.09 s(-1) compared with wild type alpha(1)2.1/alpha2-delta/beta1a k(inact) = 1.35 +/- 0.25 s(-1) to a 75-fold acceleration in mutant M1811Q (k(inact) = 102 +/- 3 s(-1). Coexpression of mutant alpha(1)2.1-subunits with beta(2a) resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in I(Ba) inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta2a-subunit-mediated effects on Ca(v)2.1 inactivation are likely to occur via independent mechanisms.

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.