Yonsei Med J.  2015 May;56(3):648-657. 10.3349/ymj.2015.56.3.648.

Pre-Clinical Efficacy and Safety Evaluation of Human Amniotic Fluid-Derived Stem Cell Injection in a Mouse Model of Urinary Incontinence

• Choi JY
• Chun SY
• Kim BS
• Kim HT
• Yoo ES
• Shon YH
• Lim JO
• Yun SJ
• Song PH
• Chung SK
• Yoo JJ
• Kwon TG

• KMID: 2450337
• DOI: http://doi.org/10.3349/ymj.2015.56.3.648

Abstract

PURPOSE
Stem cell-based therapies represent new promises for the treatment of urinary incontinence. This study was performed to assess optimized cell passage number, cell dose, therapeutic efficacy, feasibility, toxicity, and cell trafficking for the first step of the pre-clinical evaluation of human amniotic fluid stem cell (hAFSC) therapy in a urinary incontinence animal model.
MATERIALS AND METHODS
The proper cell passage number was analyzed with hAFSCs at passages 4, 6, and 8 at week 2. The cell dose optimization included 1x10(4), 1x10(5), and 1x10(6) cells at week 2. The in vivo cell toxicity was performed with 0.25x10(6), 0.5x10(6), and 1x10(6) cells at weeks 2 and 4. Cell tracking was performed with 1x10(6) cells at weeks 2 and 4.
RESULTS
The selected optimal cell passage number was smaller than 6, and the optimal cell dose was 1x10(6) for the mouse model. In our pre-clinical study, hAFSC-injected animals showed normal values for several parameters. Moreover, the injected cells were found to be non-toxic and non-tumorigenic. Furthermore, the injected hAFSCs were rarely identified by in vivo cell trafficking in the target organs at week 2.
CONCLUSION
This study demonstrates for the first time the pre-clinical efficacy and safety of hAFSC injection in the urinary incontinence animal model and provides a basis for future clinical applications.

Keyword

Preclinical evaluation; amniotic fluid; stem cells; urinary incontinence

MeSH Terms

Amniotic Fluid/*cytology
Animals
Cell Movement
Disease Models, Animal
Humans
Injections
Mice
Stem Cell Transplantation/*methods
Stem Cells/*cytology
Treatment Outcome
Urinary Incontinence/*therapy

Figure

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  Fig. 1 Effect of hAFSC passage numbers and doses for urodynamics. (A) The mean leak point pressures (LPPs) for the groups of passages 4, 6, and 8 were similar to those of the Ctrl(+) group, while the closing pressure (CP) was significantly lower at passage 8 at two weeks after cell injection. (B) The urodynamic results according to the hAFSC passage numbers were confirmed with real-time PCR analysis at the same period. (C) The mean LPP and CP values for the 1×106 dose group was similar only to Ctrl(+) at two weeks after cell injection. (D) The urodynamic results were confirmed with real-time PCR analysis at the same period. The different letters on top of the bars show significant differences at p<0.05 by ANOVA Tukey's post-hoc test. Ctrl(+): sham operated normal control group, P4, P6, and P8: passages 4, 6, and 8 cell injected group after bilateral pudendal nerve transaction, respectively, 106, 105, and 104: cell injected group with number of 106, 105, and 104, respectively, Ctrl(-): PBS-injected (without cell) negative control group after bilateral pudendal nerve transaction. hAFSC, human amniotic fluid stem cell.

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  Fig. 2 Evaluation of efficacy at passage 6 with 1×106 hAFSCs. (A) Histological and immunohistochemical analysis at two weeks after cell injection. hAFSC-injected groups showed circular muscle mass regeneration, enhanced myogenesis, neurogenesis and endothelial genesis, and non-immunogenicity. (B) Real-time PCR analysis of the expression of myogenesis, neurogenesis, and endothelial genesis lineage markers with mouse primer at the same period. Most of genes were expressed significantly higher in hAFSC groups than in the Ctrl(-) group, except for Nestin and CD34. The different letters on top of the bars show significant differences at p<0.05. (C) Real-time PCR analysis with human primer at week 2. The human gene expressions were relatively decreased compared with mouse gene expression. Magnification at ×100. Ctrl(+): sham operated normal control group, Ctrl(-): PBS-injected (without cell) negative control group after bilateral pudendal nerve transaction, AFSC: hAFSC-injected group after bilateral pudendal nerve transaction. hAFSC, human amniotic fluid stem cell; H&E, hematoxylin and eosin.

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  Fig. 3 Histological and immunohistochemical analysis for in vivo cell trafficking. (A and B) Representative images of histological analysis at two weeks after cell injection. With H&E staining, target organs of the experimental groups (A, cell-injected) showed normal histology as the control groups (B, PBS-injected). (C and D) Representative images of immunohistochemical analysis at two weeks after cell injection. The spleen and urethra showed resident injected cells in the experiment group (D). The nucleus is shown as a blue dot with DAPI staining, and yellow arrows indicate detected hAFSCs with human nucleus-specific antibody in target organs. Magnification at ×100. hAFSC, human amniotic fluid stem cell; H&E, hematoxylin and eosin.

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