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Schematic overview of cell injections. For cell injections in cadaveric urethra samples, the urethra was opened and placed on a sponge with the epithelium (ie, urothelial cell layer) facing up. (A) Injections in the submucosal layer by WN were performed at an angle of approximately 30° to 45°. For cell injections, the tip of the WN was inserted in the tissue for a few millimeters. Injections by WJ were performed vertically. The tip of the WJ lance was lowered by a gauge to the surface of the urothelial layer and moved 2 mm down without tissue penetration to avoid loss of cells by splash to the side caused by the Bernoulli effect. The X- and Y-dimensions for histologic evaluation are explained in the inserts on top. (B) For transurethral cell injections in living animals by aid of cystoscope under visual control, WJ injections were performed. After slightly tilting the device in the urethra, the flexible tip of the injection lance enabled angulated WJ injections in the urethra without penetration of the urothelium. (C) Schematic overview for determination of the distribution of cells in the tissue targeted and determination of DISIC. WN: Williams needle; WJ: waterjet; DISIC: distance between sphincter muscle and injected cells.
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Current regimen to treat patients suffering from stress urinary incontinence often seems not to yield satisfactory improvement or may come with severe side effects. To overcome these hurdles, preclinical studies and clinical feasibility studies explored the potential of cell therapies successfully and raised high hopes for better outcome. However,...
Citations
... Seven days after cell injection, gilts were sedated and sacrificed (KCl i.v. lethal), bladders and urethrae were prepared, and the area of injection was determined by imaging (IVIS Spectrum; PerkinElmer) 46 . Areas of injected pMSCs and PS-FluoGrün-Fi364-labeled particles are visualized by IVIS as false-color red-to-yellow heatmaps. ...
... Cryosections (20 µm, CM1860 UV; Leica) were generated from the area of cell injection, mounted, and stained by HE chemistry to visualize the tissue targeted 41,46 . Cryosections of porcine spleen and lymph node tissues served as positive controls. ...
... To detect infiltrating leukocytes, cryosections were blocked (5% milk powder in 0,1% Tween/PBS), incubated with anti-CD45 antibodies (1:200 in 1% BSA/PBS, 37°C, 90 min, black humidified box, Invitrogen), washed 3 times (0,1% Tween 20 in PBS), reacted with secondary AF555labeled anti-ms antibodies (1:200 in 1% BSA/PBS, 37°C, 45 min, black humidified box, Invitrogen), washed again, counterstained by DAPI, and recorded by fluorescence microscopy (DMi8; Leica with LAS X software). To detect the human male pMSCs injected in the porcine urethra, tissue was scratched off from eight consecutive crysections to isolate DNA (DNeasy extraction kit, Qiagen) 46 . Male chromosomal DNA was detected in cryosections by a hot start PCR of the SRY gene (Table S2) employing 50 cycles for DNA amplification (denaturation: 94°C for 30s, annealing: 65°C for 30s, extension:72°C for 20s) followed by primer extension (72°C for 5 min.; ...
In animal models, cell therapies for different diseases or injuries have been very successful. Preclinical studies with cells aiming at a stroke, heart attack, and other emergency situations were promising but sometimes failed translation in clinical situations. We, therefore, investigated if human placenta-derived mesenchymal stromal cells can be injected in pigs without provoking rejection to serve as a xenogenic transplantation model to bridge preclinical animal studies to more promising future preclinical studies. Male human placenta-derived mesenchymal stromal cells were isolated, expanded, and characterized by flow cytometry, in vitro differentiation, and quantitative reverse-transcription polymerase chain reaction to prove their nature. Such cells were injected into the sphincter muscle of the urethrae of female pigs under visual control by cystoscopy employing a Williams needle. The animals were observed over 7 days of follow-up. Reactions of the host to the xenogeneic cells were explored by monitoring body temperature, and inflammatory markers including IL-1ß, CRP, and haptoglobin in blood. After sacrifice on day 7, infiltration of inflammatory cells in the tissue targeted was investigated by histology and immunofluorescence. DNA of injected human cells was detected by PCR. Upon injection in vascularized porcine tissue, human placenta-derived mesenchymal stromal cells were tolerated, and systemic inflammatory parameters were not elevated. DNA of injected cells was detected in situ 7 days after injection, and moderate local infiltration of inflammatory cells was observed. The therapeutic potential of human placenta-derived mesenchymal stromal cells can be explored in porcine large animal models of injury or disease. This seems a promising strategy to explore technologies for cell injections in infarcted hearts or small organs and tissues in therapeutically relevant amounts requiring large animal models to yield meaningful outcomes.
Muscular insufficiency is observed in many conditions after injury, chronic inflammation, and especially in elderly populations. Causative cell therapies for muscle deficiencies are not state of the art. Animal models to study the therapy efficacy are, therefore, needed. We developed an improved protocol to produce myoblasts suitable for pre-clinical muscle therapy studies in a large animal model. Myoblasts were isolated from the striated muscle, expanded by employing five different protocols, and characterized on transcript and protein expression levels to determine procedures that yielded optimized regeneration-competent myoblasts and multi-nucleated myotubes. We report that swine skeletal myoblasts proliferated well under improved conditions without signs of cellular senescence, and expressed significant levels of myogenic markers including Pax7, MyoD1, Myf5, MyoG, Des, Myf6, CD56 (p ≤ 0.05 each). Upon terminal differentiation, myoblasts ceased proliferation and generated multi-nucleated myotubes. Injection of such myoblasts into the urethral sphincter complex of pigs with sphincter muscle insufficiency yielded an enhanced functional regeneration of this muscle (81.54% of initial level) when compared to the spontaneous regeneration in the sham controls without myoblast injection (67.03% of initial level). We conclude that the optimized production of porcine myoblasts yields cells that seem suitable for preclinical studies of cell therapy in a porcine large animal model of muscle insufficiency.
The leading cause of stress urinary incontinence in women is the urethral sphincter muscle deficiency caused by mechanical stress during pregnancy and vaginal delivery. In men, prostate cancer surgery and injury of local nerves and muscles are associated with incontinence. Current treatment often fails to satisfy the patient's needs. Cell therapy may improve the situation. We therefore investigated the regeneration potential of cells in ameliorating sphincter muscle deficiency and urinary incontinence in a large animal model. Urethral sphincter deficiency was induced surgically in gilts by electrocautery and balloon dilatation. Adipose tissue-derived stromal cells and myoblasts from Musculus semitendinosus were isolated from male littermates, expanded, characterized in depth for expression of marker genes and in vitro differentiation, and labelled. The cells were injected into the deficient sphincter complex of the incontinent female littermates. Incontinent gilts receiving no cell therapy served as controls. Sphincter deficiency and functional regeneration were recorded by monitoring the urethral wall pressure during follow-up by two independent methods. Cells injected were detected in vivo during follow-up by transurethral fluorimetry, ex vivo by fluorescence imaging, and in cryosections of tissues targeted by immunofluorescence and by PCR of the SRY gene. Partial spontaneous regeneration of sphincter muscle function was recorded in control gilts, but the sphincter function remained significantly below levels measured prior to induction of incontinence (67.03±14.00%, n=6, p<0.05). Injection of myoblasts yielded an improved sphincter regeneration within five weeks of follow-up but did not reach significance compared to control gilts (81.54±25.40%, n=5). A significant and full recovery of the urethral sphincter function was observed upon injection of adipose tissue-derived mesenchymal stromal cells within five weeks of follow-up (100.4±23.13%, n=6, p<0.05). Injection of stromal cells provoked slightly stronger infiltration of CD45pos leukocytes compared to myoblasts injections and controls. The data of this exploratory study indicate that adipose tissue-derived mesenchymal stromal cells inherit a significant potential to regenerate the function of the urethral sphincter muscle.
