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

括约肌缺陷和各种形式的尿失禁是那些受影响的人和他们的环境的主要负担。超过10%的人口患有不同形式的尿失禁。对于许多患者来说，仍然没有令人满意的治疗方法。因此，我们在这个方案中比较了同种脂肪前体细胞(ADSCs)和来自肌肉组织的同源脂肪前体细胞(MPC)在我们已建立的经尿动力学证实为括约肌缺陷的猪大动物模型中的再生效率。对括约肌复合体的损伤是由球囊扩张和远端烧灼引起的，并分别通过使用标准和高清晰度尿路侧貌测量法在长达12周的随访期内进行壁压测量来证实。诱导不全后，将雄性动物的荧光标记细胞注射到雌性动物的尿路中。通过体内壁压测量，与未注射细胞的动物相比，接受细胞治疗的动物随着时间的推移记录了括约肌的再生。所用细胞的定位由经尿道内窥镜荧光术记录。在实验结束时，通过不同的方式探索细胞疗法的效果：a)通过定量的尿动力学测量在活体动物中进行。B)通过对尿路的荧光成像，对注射的标记细胞进行体外定位。C)经过长达12周的随访后，处死动物，用(免疫)组织化学方法检查治疗区域和一般括约肌组织的冰冻切片。这些细胞及其精确的注射部位通过它们的荧光标记和融合蛋白的表达来可视化，也可以通过Y染色体的原位杂交来显示。采用免疫组织化学方法对其进行鉴定，并通过TUNEL染色和激活的caspase-3检测凋亡细胞。D)用激光切割法从冰冻切片中分离出注入的荧光细胞，进行基因标记和重组载体DNA的聚合酶链式反应分析。E)用RT-PCR检测关键的再生促进因子在处理后组织中的表达。通过这些方法，在活体动物、器官外植体、组织切片和组织提取液中监测细胞治疗的效果。我们调查ADSC和MPC在注射部位停留多长时间，如果它们是通过迁移分布的，保持完好，以及它们对不足的括约肌复合体的再生做出了什么贡献。我们探索ADSCs和MPC之间在括约肌再生方面的可测量差异是否可检测到。这项临床前研究将提供证据，证明使用自体ADSCs或MPC治疗是否能促进尿失禁患者的尿道括约肌功能再生。

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.