Isolation of internal and external sphincter progenitor cells from the human anal sphincter with or without radiotherapy.

AIM: We aimed to isolate and propagate internal and external anal sphincter progenitor cells from the human anal sphincter, with or without radiotherapy, for tailored cell therapy of faecal incontinence.

METHODS

Sphincter progenitor cells were isolated from normal internal and external anal sphincters collected from 10 patients with rectal cancer who had undergone abdominoperineal resection with (n = 6) or without (n = 4) preoperative chemoradiotherapy. The isolated cells and differentiated muscle fibres were identified using immunofluorescence assay, western blotting and reverse transcription polymerase chain reaction (RT-PCR). The proliferation of progenitor cells with and without radiotherapy was compared by quantitative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

RESULTS

The immunofluorescence assay before differentiation confirmed that the internal anal sphincter progenitor cells expressed CD34 and neural-glial antigen 2 (NG2), whereas the external anal sphincter progenitor cells expressed CD34 and PAX7. After differentiation, the internal anal sphincter progenitor cells expressed desmin, calponin and α-smooth muscle actin, whereas the external anal sphincter progenitor cells expressed desmin, myogenic factor 4 and myosin heavy chain. The differential expression profiles of both cell types were confirmed by western blotting and RT-PCR. MTT assays showed that the viability of internal and external anal sphincter progenitor cells was significantly lower in the radiotherapy group than that in the nonradiotherapy group.

CONCLUSIONS

This study describes the differential harvest internal and external sphincter muscle progenitor cells from human anal sphincters. We confirm that radiotherapy decreases the viability of internal and external anal sphincter progenitor cells.