Injection of progenitor cells from adipose tissue and muscle tissue for regeneration of urethral sphincter deficiency in a large animal model

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Sphincter deficiency and various forms of urinary incontinence are a major burden for those affected and for their environment. More than 10% of the population suffer from different forms of urinary incontinence. For many patients, there is still no satisfactory therapy. We therefore investigate in this proposal the regenerative efficacy of homologous adipose progenitor cells (ADSCs) in comparison to homologous progenitor cells from muscle tissue (MPCs) upon local injection in our established porcine large animal model with urodynamically proven sphincter deficiency. Damage to the sphincter complex is induced by balloon dilatation and distal cauterisation, and confirmed by wall pressure measurement using standard and high definition urethral profilometry methods, respectively, over a follow-up period of up to twelve weeks. After induction of insufficiency, fluorescent labeled cells of male animals are injected into the urethra of female animals. The regeneration of the sphincter is recorded in animals with cell therapy over time compared to animals without cell injection by wall pressure measurements in vivo. The localisation of the cells applied is recorded by transurethral endoscopic fluorimetry. At the end of the experiment, the effect of cell therapy is explored in different ways: A) In living animals by quantitative urodynamic measurements. B) The injected labeled cells are localised ex vivo by fluorescence imaging of the urethra. C) After a follow-up of up to 12 weeks animals are sacrificed, and cryosections of the treated areas and the sphincter tissue in general are examined by (immuno-) histochemistry. The cells and their precise injection site are visualised by their fluorescence label and the expression of a fusion protein, optionally also by in situ hybridisation of the Y chromosome. They are characterized immunohistochemically, and apoptotic cells are detected by TUNEL staining and activated caspase-3. D) Using laser dissection, the injected fluorescent cells are isolated from cryosections to detect male gene markers and the recombinant vector DNA by PCR analysis. E) The expression of key regeneration-promoting factors in the treated tissue is evaluated by RT-PCR. With these methods the efficacy of cell therapy is monitored in living animals, in organ explants, tissue section and tissue extracts. We investigate how long ADSCs and MPCs remain at the injection sites, if they are distributed e.g., through migration, remain intact, and which contribution they do make to the regeneration of an insufficient sphincter complex. We explore whether measurable differences in the sphincter regeneration between ADSCs and MPCs are detectable. This preclinical study will provide evidence if a therapy with autologous ADSCs or MPCs facilitates a functional regeneration of the urethral sphincter of patients suffering from urinary incontinence.