Autologous mesenchymal stem cells from different origins have been proposed for the treatment of anal incontinence and encouraging preclinical and clinical results have been reported. Isolation of anal canal transition zone mesenchymal stem cells was previously described in pigs and it was hypothesized that these cells might have different properties than stem cells from other sources regarding anal sphincters healing. Excess tissue from haemorrhoid surgery could constitute an accessible source of anal canal transition zone tissue in humans. We aimed to develop a protocol for mesenchymal stem cell isolation directly from hemorrhoidal tissue in human.

After patients’ informed consent, tissue samples were procured from haemorrhoid surgery specimens. Transmural hemorrhoidal tissue samples were rinsed and digested with 0.1% type I collagenase for 60 minutes at 37°C and mechanically disrupted. After differential centrifugation, the retrieved cells were plated on dishes with mesenchymal stem cell medium and dissociated with trypsin when confluent. Cells were used from passage 3 to 5 for flow cytometry analysis, cytology and differentiation assays.

The isolation procedure was repeated 10 times and allowed to obtain a mean of 232’250 +/- 83’448 primary cells per sample. Expanded cells exhibited a typical “fibroblast-like” morphology and it was possible to culture them both in two and three dimensions, under specific conditions. Almost all cells were double positive for CD90/CD73 (99.0 +/- 0.7 %), positive for CD105 (99.8 +/- 0.1 %) and were negative for CD19, CD14, CD34, CD45 and HLA-DR, matching the definition of mesenchymal stem cell according to the international consensus. Trilineage differentiation assay demonstrated transformation of the isolated cells in adipocytes, osteoblasts and chondrocytes and thereby confirmed this phenotype.

We herein report the successful establishment of a protocol for hemorrhoidal tissue mesenchymal stem cell isolation and differentiation. This represents a key step in the establishment of an autologous mesenchymal stem cell therapy for patients suffering from anal incontinence. Further functional characterization of the isolated cells represents the next step before clinical validation.