ADSC-AI · Use of Adipose Derived Stem Cells (ADSC) for Anal Incontinence Cell Therapy

Final Activity Report Summary - ADSC-AI (Use of adipose derived stem cells (ADSC) for anal incontinence cell therapy)

The possibility of improving the function of organs or structures damaged in numerous pathological scenarios by the use of cellular therapies is a true clinical reality. Stem cells isolated from adult tissues are one of the tools for this new therapeutic approach. 'Adipose derived stem cells' (ADSC) have shown multipotency in vitro (differentiation into fat, bone, cartilage, muscle). Several pre-clinical studies point to the use of ADSC in cell therapies. Anal incontinence (AI) impacts severely the quality of life of sufferers, about 2-8% of the adult population. Injury to the pudendal nerve and/or to anal sphincter muscle from prior obstetric trauma is described as the primary risk factor to develop AI. The overall aim of this project is to develop a cell therapy for AI using ADSC in an animal model of anal sphincter deficiency. We will develop a rat model of AI by simulated birth trauma (vaginal distension + episiotomy), and will characterise the structural and functional changes of the anal sphincter by in vitro, in vivo and ex vivo studies. Then we will test the effect of cells injected in the anal sphincter in the structural and functional restoration of the anal sphincter, comparing multipotent, undifferentiated ADSC with cells differentiated into a myogenic lineage. Results: The initial data showed that there was no need to perform the vaginal distension (simulated birth) to damage the anal sphincter. Therefore, we performed direct sectioning of both internal and external anal sphincter, in the midline between the vaginal meatus and the anus, as in ano-rectal surgery for ano-rectal cancer. Standard histology (hematoxilin/eosin) on paraffin-embedded sections of perianal region showed smooth muscle and skeletal muscle corresponding to internal and external anal sphincter, respectively. To evaluate anal sphincter function in vivo, we tried several approaches but the most successful is inserting a micro-tip transducer a few millimetres right into the anal canal in the so called High Pressure Zone, recording anal pressure: an intact sphincter, i.e. continent, shows periods of closure (24.48 +- 5.48 secs) with an average maximal pressure of 63.77 +- 1.19 mm Hg, while a damaged sphincter stays closed for short periods of time (7.94 +- 2.76 secs) and with lower maximal pressure (49.35 +- 2.73 mm Hg). We are evaluating the effect of ADSC injection in the restoration of anal pressure at different time points (up to 4 weeks). We label the cells with a lentiviral vector encoding EGFP, for which there are commercially available antibodies. These cells retain positivity for EGFP and membrane markers of mesenchymal stem cell up to at least until passage 17. By immunohistochemistry, we can detect presence of ADSC-EGFP at the injury site. We obtained 2 funded projects (from the Spanish Health Research Fund) to complete the experiments proposed in this Marie Curie International Reintegration Grant.

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