J Korean Med Sci.  2012 Nov;27(11):1300-1307. 10.3346/jkms.2012.27.11.1300.

Human Amniotic Fluid Stem Cell-derived Muscle Progenitor Cell Therapy for Stress Urinary Incontinence

• Chun SY
• Cho DH
• Chae SY
• Choi KH
• Lim HJ
• Yoon GS
• Kim BS
• Kim BW
• Yoo JJ
• Kwon TG

• KMID: 2157938
• DOI: http://doi.org/10.3346/jkms.2012.27.11.1300

Abstract

The most promising treatment for stress urinary incontinence can be a cell therapy. We suggest human amniotic fluid stem cells (hAFSCs) as an alternative cell source. We established the optimum in vitro protocol for the differentiation from hAFSCs into muscle progenitors. These progenitors were transplanted into the injured urethral sphincter and their therapeutic effect was analyzed. For the development of an efficient differentiation system in vitro, we examined a commercial medium, co-culture and conditioned medium (CM) systems. After being treated with CM, hAFSCs were effectively developed into a muscle lineage. The progenitors were integrated into the host urethral sphincter and the host cell differentiation was stimulated in vivo. Urodynamic analysis showed significant increase of leak point pressure and closing pressure. Immunohistochemistry revealed the regeneration of circular muscle mass with normal appearance. Molecular analysis observed the expression of a larger number of target markers. In the immunogenicity analysis, the progenitor group had a scant CD8 lymphocyte. In tumorigenicity, the progenitors showed no teratoma formation. These results suggest that hAFSCs can effectively be differentiated into muscle progenitors in CM and that the hAFSC-derived muscle progenitors are an accessible cell source for the regeneration of injured urethral sphincter.

Keyword

Amniotic Fluid; Stem Cells; Cell Differentiation; Urinary Sphincter; Regeneration; Urinary Incontinence

MeSH Terms

Amniotic Fluid/*cytology
Animals
Biological Markers/metabolism
Cell Differentiation
Cell Lineage
Cell Transformation, Neoplastic
Cells, Cultured
Coculture Techniques
Female
Gene Expression Regulation
Humans
Immunohistochemistry
Mice
Mice, Inbred ICR
Regeneration
*Stem Cell Transplantation
Stem Cells/*cytology/metabolism
Urethra/physiology
Urinary Incontinence, Stress/pathology/*therapy
Urodynamics
Biological Markers

Figure

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  Fig. 1 Stem cell and myogenic lineage markers expression of undifferentiated hAFSCs. Undifferentiated hAFSCs produced significantly higher expression of stem cell markers compared to myogenic lineage cell markers.

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  Fig. 2 Expression of myogenic markers in hAFSCs-derived muscle progenitor cells. (A) Representative immunocytochemical (at day 7) and real-time PCR analysis of the expression of muscle progenitor markers. Conditioned medium treated cells showed relatively elongated morphology and enhanced myogenic gene expression for PAX7 and MYOD compared with the control and co-culture systems. Ctrl, commercial medium; Co, co-culture; CM, conditioned medium. (B) The growth rate of hAFSCs-derived muscle progenitor cells in CM measured for 14 days. The growth rate of hAFSCs-derived muscle progenitor cells in CM was inhibited because the differentiating cells are usually postmitotic (*P < 0.001; †P < 0.001). Ctrl, undifferentiated hAFSCs; Muscle, hAFSCs-derived muscle progenitor cells.

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  Fig. 3 Therapeutic property of muscle progenitor cells for urethral sphincter regeneration. In urodynamic study (A), leak point pressure (LPP) and closing pressure (CP) improved significantly in animals with pudendal neurectomy in the muscle progenitor group at week 2 and 4, compared with those in the hAFSC group (*P = 0.003, †P = 0.003, ‡P = 0.008, §P = 0.008). The representative H&E and IHC images (B) and real-time PCR (C) results of the muscle progenitor group exhibited the accelerated sphincter regeneration with myogenic lineage markers expression. The results of injected human cell detection, immune responses and a safety study in vivo (D) showed that hAFSCs-derived muscle progenitor cells showed their integration into the host tissue at week 2, scant CD8 lymphocyte aggregation in the urethral sphincter at week 2 and no teratoma formation under renal capsule at week 4. Ctrl (-), bilateral pudendal nerve transected group; hAFSC, bilateral pudendal nerve transected and hAFSC injected group; Muscle, bilateral pudendal nerve transected and hAFSCs-derived muscle progenitor cell injected group; Ctrl (+), sham operated group.

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