Combined injection of three different lineages of early-differentiating human amniotic fluid-derived cells restores urethral sphincter function in urinary incontinence.

To investigate whether a triple combination of early-differentiated cells derived from human amniotic fluid stem cells (hAFSCs) would show synergistic effects in urethral sphincter regeneration. We early-differentiated hAFSCs into muscle, neuron and endothelial progenitor cells and then injected them into the urethral sphincter region of pudendal neurectomized ICR mice, as single-cell, double-cell or triple-cell combinations. Urodynamic studies and histological, immunohistochemical and molecular analyses were performed. Urodynamic study showed significantly improved leak point pressure in the triple-cell-combination group compared with the single-cell- or double-cell-combination groups. These functional results were confirmed by histological and immunohistochemical analyses, as evidenced by the formation of new striated muscle fibres and neuromuscular junctions at the cell injection site. Molecular analysis showed higher target marker expression in the retrieved urethral tissue of the triple-cell-combination group. The injection of early-differentiated hAFSCs suppressed in vivo host CD8 lymphocyte aggregations and did not form teratoma. The nanoparticle-labelled early-differentiated hAFSCs could be tracked in vivo with optical imaging for up to 14 days after injection. Our novel concept of triple-combined early-differentiated cell therapy for the damaged sphincter may provide a viable option for incontinence treatment.