Amniotic fluids (10 mL) were obtained from four women undergoing routine amniocentesis at a gestational age of 15 to 19 weeks at Kyungpook National University Hospital, and sent to CORESTEM Inc. (Seoul, Korea) for cell preparation in the Good Manufacturing Practices (GMP) facility. During cell preparation, validation of the manufacturing process, in vitro assessment of genotypic instability, tumorigenicity, and phenotypic profile of the product were conducted. In case #AFSC1301, a cell population with high expression of C-KIT and SSEA4 (stem cell markers) as well as low expression of HLA-DR (immune tolerance marker) was selected and used for this study.

Five-week-old male and female ICR mice were purchased from Orientbio Inc. (Seongnam, Korea). Animal groupings were listed in Table 1. For these experiments, cells from passage 4 were used to ensure a sufficient cell number, and the maximum cell number was 1×10⁶ cells for injection in the mouse urethral sphincter. In the toxicity analysis, animals of both genders were used to eliminate gender differences. ICR mice were prepared for aseptic surgery under general anesthesia with isoflurane. The SUI model was created using a bilateral pudendal nerve transection technique. One week after the incompetent urethral sphincter model was generated, hAFSCs were resuspended in saline (Choongwae Pharma Corp., Seoul, Korea) under aseptic conditions to a final volume of 5 µL and injected at the external sphincter.

Leak point pressure (LPP) and closing pressure (CP) were measured two weeks after cell injection as previously described.13 The intravesical pressure was increased from 0 cm H₂O upward in steps of 1- to 3-cm H₂O until visual identification of the leak point height. The pressure at this leak point was referred to as the LPP. The intravesical pressure was then decreased downward in steps of 1- to 3-cm H₂O until the leak has ceased. The pressure at this leak cessation point was taken as the CP.3 Tissue specimens were prepared for hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining. The possibility of host myogenic, neurogenic, and endothelial genetic conversion induced by the in vivo injection of hAFSCs was analyzed with IHC (antibody information, Table 2). The in vivo immunosuppression effect was examined one week after injection with a cytotoxic T cell marker (CD8). For injected human cell trafficking and analysis of migration to target organs, human nuclear specific antibody (HuNu) was used. The IHC results were confirmed with real-time PCR using human primers and host responses were analyzed with mouse primers (primer sequence information, Table 3). Total RNA was extracted with an RNeasy-kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. A total of 2 µg of RNA was used for cDNA synthesis using cDNA reverse transcription kits (Applied Biosystems, Warrington, UK). The primers were designed with Primer Express Software (Applied Biosystems). Assays were carried out using the ABI Prism Sequence Detection System 7500 with SYBR Green PCR Master Mix (Applied Biosystems). To analyze data, the 2^-ΔΔCt method of relative quantification was adapted to estimate copy numbers.

With optimized cell number and dose, pre-clinical aspects were analyzed. On the day of dosing, general symptoms were examined at each time point. The mortalities were measured and pathological examinations were carried out immediately before treatment and every other week after treatment for four weeks. After the termination of observations, all animal organs and tissues were visually inspected and examined by microscopy for necropsy.

All values are presented as mean±SD. Statistical analysis was undertaken with Student's t-test or one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences v.19 (SPSS, IBM, Armonk, NY, USA). A p-value of less than 0.05 was considered statistically significant. When the value was found to be significant after assessment using the ANOVA statistical test, the Tukey's post-hoc comparison was used.

LPP and CP were measured two weeks after injection of hAFSCs at different passages (Fig. 1A). The mean LPP values for passages 4 and 6 were significantly higher than the mean LPP of the Ctrl(+) group, and the mean LPP of passage 8 was similar to that of the Ctrl(+) group. The mean CP values for passages 4 and 6 were similar to the mean CP value of the Ctrl(+) group. At passage 8, the mean CP value decreased significantly, even though the values were higher than those of the Ctrl(-) group. Real-time PCR analysis for myogenic, neurogenic, and endothelial lineage markers of passage 4 showed significant gene expression, and the degree of expression was inversely proportional to the number of passages (Fig. 1B). With this result, the optimized passage number was selected to be smaller than 6. When using cells prior to passage 4, cell number was limited for several experiments.

LPP and CP were measured two weeks after injection of hAFSCs (Fig. 1C). The mean LPP for the group injected with 1×10⁶ cells was similar to that of the Ctrl(+) group (p=0.874), and it was significantly different from the groups with doses of 1×10⁵ and 1×10⁴ cells (p=0.003). The mean CP for the 1×10⁶ dose group was significantly different from the 1×10⁵ and 1×10⁴ dose groups (p=0.012 and p=0.003, respectively). Real-time PCR analysis for myogenic, neurogenic, and endothelial lineage markers showed that gene expression for the 1×10⁶ dose group was significant and directly proportional to the number of cells (Fig. 1D). With this result, the optimized cell dose for injection was selected to be 1×10⁶. This cell number is the maximum in this animal model because of the limited size of the urethral sphincter.

The therapeutic efficacy of hAFSCs was evaluated using cells obtained at passage 6 and a cell number of 1×10⁶ at two weeks after injection. Histological examination revealed no apparent sign of inflammation or tissue damage at the injection sites (Fig. 2A). Compared with the Ctrl(+) group, the Ctrl(-) group showed only a scant muscle layer at the atrophic urethral sphincter. In the hAFSCs group, a normal appearance of circular muscle mass regeneration and a number of capillaries were identified at the urethral sphincter region. This histological result was confirmed by IHC staining using myogenesis-, neurogenesis-, and endothelial genesis-related antibodies. The hAFSCs group was positive for the antibodies and the intensity was higher in this group compared with the Ctrl(-) group. In immunogenicity analysis, CD8 signals for cytotoxic T cells were not detected in the hAFSCs group. The histologic results were confirmed with real-time PCR analysis. The evaluated genes were related to myogenesis, neurogenesis, and endothelial lineage differentiation. In mouse primer analysis at week 2, most of the genes were expressed at significantly higher levels in the hAFSCs group than in the Ctrl(-) group, with the exception of Nestin and CD34 (Fig. 2B). In contrast, human gene expressions were relatively decreased compared with mouse gene expression (Fig. 2C).

At week 2 after cell injection, one female mouse from the control group had corneal opacity, and a male mouse had secondary wound closure. However, these symptoms were within the tolerance levels and were not considered significant. With respect to weight change, food consumption, ophthalmology test, urine test, hematological test, clinical chemistry, estrous cycle, sperm motility, and organ weight, no significant changes were found between the experimental groups and the control group. At necropsy, there were no meaningful changes observed by histopathological examination in all animal groups, and there were no significant changes related to the injections in the brain, lungs, liver, kidney, and spinal cord. Histopathological findings showed no connection to the test materials (Table 4).

In vivo histological and IHC analysis with HuNu antibody identified the injected hAFSCs in situ. With H&E staining, the experimental groups showed normal organ histology compared to controls (Fig. 3A and B). IHC staining revealed focal detection of injected cells at the injection site and target organs two weeks after cell injection (Fig. 3C and D). Upon analysis of cell migration to other organs, cells were detected in the brain, heart, kidney, lung, spleen, prostate, thymus, testis, ovary, and urethra. The kidney (one case), spleen (four cases), prostate (one case), thymus (one case), and urethra (three cases) showed resident injected cells, which indicated that the injected cells migrated into neighboring organs. In the male experimental group, 7/24 (29.17%) cases showed HuNu positive cells in the kidney (2 cells in one case), spleen (3, 6, 2, 2 cells in four cases), prostate (1 cell in one case), and urethra (1 cell in one case). In the female experimental group, 2/24 (16.6%) cases showed HuNu positive cells in the thymus and urethra (1 cell at each organ in 1 case) and urethra (1 cell in 1 case). When compared with the injected cell number (1×10⁶), the positive cell number was significantly lower, and the positive cells were not detected at week 4 (data not shown). Therefore, injected cell migration and resident cell influence were not considered a factor for hAFSC therapy.