Autologous mesenchymal stem cells from different origins, including adipose tissue, have been proposed alone or in combination with surgery for the treatment of anorectal incontinence with encouraging preclinical and clinical outcomes(1). Cell transplantation as tridimensional structures instead of individual cell suspension seems to increase cell viability and implantation(2). We previously developed a protocol for mesenchymal stem cell isolation directly from haemorrhoidal tissue in pigs and humans and demonstrated that the isolated cells fulfill mesenchymal stem cell criteria(3). We aim to evaluate the characteristics and secretome of haemorrhoidal tissue mesenchymal stem cells in both two-dimensional and tri-dimensional cultures as spheroids for the treatment of anorectal incontinence and compare them with adipose tissue-derived mesenchymal stem cells.

Informed consent was obtained from all patients. Tissue samples were procured from haemorrhoidectomy specimens or liposuction waste products and processed and characterized according to previously reported methods. Haemorrhoidal tissue mesenchymal stem cells were cultured as spheroids using the “hanging-drop technique” or agarose pits. Immunofluorescence was performed to assess the expression of mesenchymal proteins and cell viability. Conditioned media of 2D cultures or spheroids in suspension were generated using low serum culture medium and analysed with semi-quantitative cytokine array (120 cytokines) and ELISA assays.

Cultured cells demonstrated expression of vimentin and good viability, even as spheroids (2.6±2% cell death). Cytokine profile in the secretome of haemorrhoidal tissue mesenchymal stem cells was similar to that of adipose tissue-derived mesenchymal stem cells, with shared core cytokines (FGF-9, OPG, CCL2, CCL11, CCL13, IGFBP-4, IGFBP-6). Concentration of HGF (hepatocyte growth factor) measured by ELISA was higher in haemorrhoidal tissue mesenchymal stem cells conditioned medium than in adipose tissue-derived mesenchymal stem cells conditioned medium (19613±3528 versus 179±84 pg/ml p<0.001) whereas VEGF (vascular endothelial growth factor) concentration followed an opposite trend (7627±947pg/ml versus 181±74 pg/ml p<0.001). Production of mesenchymal proteins and growth factors was not affected by the spheroid configuration of cells.

We demonstrated that haemorrhoidal tissue mesenchymal stem cells exhibit a similar secretome profile compared to adipose tissue-derived mesenchymal stem cells. However, by producing more HGF and less VEGF, haemorrhoidal tissue mesenchymal stem cells might be better candidates. Moreover, generation of spheroids did not compromise cell viability or their ability to produce structural proteins and growth factors.

Haemorrhoidal tissue mesenchymal stem cells could be used in autotransplantation protocols for the treatment of anal incontinence as cell suspensions or as spheroids. In vivo studies evaluating these approaches alone or in combinaison with surgery (sphincteroplasty) are necessary prior to clinical validation.

Balaphas, A.; Meyer, J.; Meier, R.P.H.; Liot, E.; Buchs, N.C.; Roche, B.; Toso, C.; Bühler, L.H.; Gonelle-Gispert, C.; Ris, F. Correction: Balaphas et al. Cell Therapy for Anal Sphincter Incontinence: Where Do We Stand? Cells 2021, 10, 2086. Cells 2023, 12, 2857.
Yen BL, Hsieh CC, Hsu PJ, Chang CC, Wang LT, Yen ML. Three-Dimensional Spheroid Culture of Human Mesenchymal Stem Cells: Offering Therapeutic Advantages and In Vitro Glimpses of the In Vivo State. Stem Cells Transl Med. 2023 May 15;12(5):235-244. Balaphas A, Meyer J, Buchs NC, Modarressi A, Bühler LH, Toso C, Gonelle-Gispert C, Ris F. Isolation and Characterization of Stem Cells from the Anal Canal Transition Zone in Pigs. Dig Dis Sci. 2023 Feb;68(2):471-477. doi: 10.1007/s10620-022-07690-7.
Balaphas A, Meyer J, Buchs NC, Modarressi A, Bühler LH, Toso C, Gonelle-Gispert C, Ris F. Isolation and Characterization of Stem Cells from the Anal Canal Transition Zone in Pigs. Dig Dis Sci. 2023 Feb;68(2):471-477.

Continence 12S (2024) 101677
DOI: 10.1016/j.cont.2024.101677