Stem cell therapies have been proposed for SUI treatment as a way to overcome the limited efficacy and adverse reactions attributed to therapies involving bulking agents. However, most stem cell harvesting protocols require invasive procedures and/or result in harvesting of low cell numbers. Recently, hAFSCs have been proposed as a promising stem cell source for various cell therapies and tissue engineering. These cells can be obtained non-invasively and can differentiate into multiple cell lineages, such as adipocytes , osteoblasts , chondrocytes , renal cells , hepatocytes  and cardiomyocytes .
In the present study, we obtained a homogeneous genotypic cell profile using a double sorting procedure with C-KIT antibody. FACS analysis showed the hAFSCs were positive for mesenchymal stem cell markers, including SSEA4, CD44, CD73, CD90 and CD105, and the expression levels were similar to previous reports [7, 16]. The hAFSCs showed negative expression for hematopoietic stem cell marker CD45. These results suggest that the hAFSCs were of the mesenchymal stem cell and not hematopoietic stem cell lineage.
When cultured in myogenic induction media, hAFSCs differentiated into muscle progenitor cells. During differentiation, expression of early myogenic differentiation markers (PAX7 and MYOD) decreased gradually, and expression of the middle (DESMIN) and late (DYSTROPHIN) differentiation markers increased over time.
Culture media containing 5-azaC and TGF-β induced a similar level of myogenic differentiation as did CM treatment. However, cell viability was significantly enhanced with CM treatment. These results suggest that hAFSCs have myogenic potential and that CM might be the best medium for induction of myogenic differentiation.
We also evaluated the therapeutic feasibility of periurethral injection of hAFSCs in an SUI animal model. When hAFSCs were injected into the animal, IHC staining with HuNu confirmed that the injected cells were able to survive in the host environment. They integrated into the mouse sphincter muscle layer and survived within these in vivo conditions for 14 days. Real-time PCR gave us valuable information on the interaction between human cells and mouse cells. Human myogenic gene expression gradually decreased over time, while mouse gene expression steadily increased. These results indicate the grafted hAFSCs might have undergone in situ myogenic differentiation and induced host muscle regeneration. These findings are similar to other reports of human stem cell transplantation into animals [17–19]. The details underlying the specific mechanism of action need to be investigated.
Clinically, SUI can occur acutely or chronically in humans. Chronic SUI, which usually occurs in the female population, is commonly caused by weakness of urethral sphincter muscle owing to vaginal delivery, for example. Meanwhile, acute SUI can be induced by urethral sphincter muscle resection or atrophy, after prostate surgery in males or perineal trauma. In all cases, we can expect improvements of incontinence if urethral sphincter muscle is restored morphologically and functionally. We observed sphincter muscle atrophy after bilateral pudendal nerve transection in this study and we confirmed the regeneration of urethral sphincter muscle after cell injection in vivo. Although our animal model is closer to acute SUI rather than chronic SUI, we believe this therapeutic method can improve both chronic and acute SUI by functional restoration of urethral sphincter muscle.
The wide use of human stem cells for therapeutic application has prompted the search for non-invasive methods for tracking injected cells. For example, Delo et al. developed an MRI-based cell tracking method , and were able to detect injected hAFSCs up to four weeks. However, this method requires expensive high resolution MRI and has a potential radiation hazard. In the present study, we established an optical imaging-based cell tracking method by labelling cells with MNPs@SiO2 (RITC). AFSCs were labeled with nanoparticles without signs of cytotoxicity. The labeled cells were detected for up to 10 days after injection using optical imaging. At Day 14, the signal strength was under the detection range. These results suggest that MNPs@SiO2 (RITC) can be used for noninvasive in vivo tracking of injected hAFSCs. A limitation of optical imaging is that the detector could not catch the signal when the signal strength was under the detection range. We assumed the signal strength at Day 14 was out of the optical imaging range. We, therefore, performed real-time PCR to confirm longer detection of muscle regeneration.
Histological and IHC analysis showed that periurethral injection of hAFSCs into denervated urethral sphincter stimulated normal-appearing sphincter muscle regeneration over time. These results correlated well with the real-time PCR analysis for myogenic gene expressions over time. The functional analysis of the sphincters showed that LPP and CP of the hAFSC injected group were restored to nearly normal values, while the Cell (-) group values remained low throughout the study period. These results indicate that periurethral injection of hAFSCs into a denervated urethral sphincter can restore apparently normal urethral sphincter histology and function.
Restoration of sphincter function requires regeneration of a neuronal component (neuromuscular junction and nerve regeneration) as well as muscle regeneration. In this study, we found similar expression levels in the Cell (+) group and the normal control, whereas the Cell (-) group showed a higher expression level. This indicates that the transplanted hAFSCs can control damaged tissue regeneration by harmonizing the physical environment . Over-proliferation or unwanted differentiation can cause malignant formation. In addition, significantly enhanced neurogenic gene expression was seen in the Cell (+) group compared with the Cell (-) group. This result suggests that injected cells can have a physiologic effect on the neuromuscular junction re-formation and nerve regeneration.
Recently, several reports have suggested that stem cells may have a low immunogenicity and immunomodulatory function [22, 23]. In this study, we found that hAFSCs have a lower expression of HLA-DR compared with isotope control. Furthermore, injection of hAFSCs into ICR mice also did not stimulate CD8+ T cell infiltration into the injected area. These findings suggest that hAFSCs have an immune tolerance and/or immunosuppression effect, similar to that reported for other stem cells. When injected into the renal subcapsule, hAFSCs did not cause teratomas after eight weeks. This result supports the use of hAFSCs as a safe for cell therapeutic application in terms of tumorigenicity.
The limitations of this study include the lack of identification of a precise mechanism for a paracrine effect, nerve regeneration, and the inability to track the metabolism or fate of the injected cells in vivo.