Int J Stem Cells.  2025 Feb;18(1):72-86. 10.15283/ijsc23147.

Assessment of the Therapeutic Effectiveness of Glutathione-Enhanced Mesenchymal Stem Cells in Rat Models of Chronic Bladder Ischemia-Induced Overactive Bladder and Detrusor Underactivity

• Shin JH
• Yu HY
• Kwon H
• Yun HD
• Ryu CM
• Shin DM
• Choo MS

• KMID: 2565198
• DOI: http://doi.org/10.15283/ijsc23147

Abstract

Overactive bladder (OAB) and detrusor underactivity (DUA) are representative voiding dysfunctions with a chronic nature and limited treatment modalities, and are ideal targets for stem cell therapy. In the present study, we investigated the therapeutic efficacy of human mesenchymal stem cells (MSCs) with a high antioxidant capacity generated by the Primed Fresh OCT4 (PFO) procedure in chronic bladder ischemia (CBI)-induced OAB and DUA rat models. Sixteen-week-old male Sprague-Dawley rats were divided into three groups (sham, OAB or DUA, and stem cell groups; n=10, respectively). CBI was induced by bilateral iliac arterial injury (OAB, 10 times; DUA, 30 times) followed by a 1.25% cholesterol diet for 8 weeks. Seven weeks after injury, rats in the stem cell and other groups were injected with 1×10 6 PFO-MSCs and phosphate buffer, respectively. One week later, bladder function was analyzed by awake cystometry and bladders were harvested for histological analysis. CBI with a high-fat diet resulted in atrophy of smooth muscle and increased collagen deposits correlating with reduced detrusor contractility in both rat models. Arterial injury 10 and 30 times induced OAB (increased number of non-voiding contractions and shortened micturition interval) and DUA (prolonged micturition interval and increased residual volume), respectively. Injection of PFO-MSCs with the enhanced glutathione dynamics reversed both functional and histological changes; it restored the contractility, micturition interval, residual volume, and muscle layer, with reduced fibrosis. CBI followed by a high-fat diet with varying degrees of arterial injury induced OAB and DUA in rats. In addition, PFO-MSCs alleviated functional and histological changes in both rat models.

Keyword

Urinary bladder; Overactive; Underactive; Adult stem cell; Glutathoine

Figure

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  Fig. 1 The Primed Fresh OCT4 (PFO) procedure enhances the core functions of human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). (A) Microscopic analysis of normal (naïve) hUC-MSCs and PFO-MSCs. Scale bar=1 mm. (B) Real-time monitoring of the glutathione (GSH)-recovering capacity (GRC) and basal GSH levels in naïve MSCs and PFO-MSCs upon exposure to 200 μM diamide (arrow). The GSH dynamics of each sample were quantified based on the initial F510/F580 fluorescence ratio (FR) (for baseline total GSH) and slope after diamide treatment (for GRC), as previously described (11). (C) Flow cytometry analyses of expression of MSC surface proteins (CD29 and CD90) and hematopoietic lineage markers (CD34 and CD45) in naïve MSCs and PFO-MSCs. An IgG isotype antibody was used as a control. (D) Differentiation into osteogenic (left panel, magnification ×200, scale bar=200 μm), chondrogenic (center panel, magnification ×100, scale bar=200 μm), and adipogenic (right panel, magnification ×400, scale bar=50 μm) lineages, determined using Alizarin Red S, Alcian Blue, and Oil Red O staining, respectively. (E-G) The beneficial effects of the PFO procedure in hUC-MSCs on cell proliferation (n=3, E), the colony forming unit-fibroblast (CFU-F) potency (n=3, F), and chemotaxis (n=7, G) in response to treatment with 10 ng/mL PDGF-AA. Representative results for each assay are shown on the left (magnification ×200, scale bar=100 μm in G). Cell proliferation was determined by the MTT assay. Quantitative data are presented as ratios relative to the naïve control group and are expressed as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001 compared with the naïve group. Statistical analyses were performed by unpaired t-test (F, G) or two-way ANOVA (B, E).

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  Fig. 2 Mesenchymal stem cell enriched by the Primed Fresh OCT4 procedure (PFO-MSC) injection ameliorates bladder function in the overactive bladder (OAB) rat model. (A) Representative awake cystometry results and (B) quantitative analysis of bladder voiding parameters at 1 week post-injection of 1×106 PFO-MSCs into rat bladders. Sham: sham-operated. Quantitative results from ten independent animals per group are presented as the mean±SEM. **p<0.01, ***p<0.001 compared with the OAB group. A one-way ANOVA with the Bonferroni post-hoc test was used for statistical analysis. IAP: intraabdominal pressure, IVP: intravesical pressure.

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  Fig. 3 Histological analysis of the effects of mesenchymal stem cell enriched by the Primed Fresh OCT4 procedure (PFO-MSC) injection on overactive bladder (OAB) bladder injury. (A-C) Representative images of H&E staining (magnification ×100, scale bar=100 μm, A), immunohistochemical staining of α-smooth muscle actin (magnification ×100, scale bar=100 μm, B), and Masson’s trichrome staining (magnification ×200, scale bar=100 μm, C) in bladder tissues at 1 week after injection of PFO-MSCs. (D) Representative images of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to detect apoptotic cells (magnification ×400, scale bar=100 μm) in bladder tissue from the indicated groups. Nuclei were stained with 4’6-diamino-2-phenylindole (DAPI). The percentage of apoptotic cells (green) was derived by calculating the ratio of apoptotic cells (TUNEL positive) to total cells (DAPI-stained nuclei). All quantitative data are presented as the mean±SEM (n=10). **p<0.01, ***p<0.001 compared with the OAB group. A one-way ANOVA with the Bonferroni post-hoc test was used for statistical analysis.

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  Fig. 4 Mesenchymal stem cell enriched by the Primed Fresh OCT4 procedure (PFO-MSC) injection ameliorates bladder function in the detrusor underactivity (DUA) rat model. (A) Representative awake cystometry results and (B) quantitative analysis of bladder voiding parameters at 1 week post-injection of 1×106 PFO-MSCs into rat bladders. Quantitative results from 10 independent animals per group are presented as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001 compared with the DUA group. A one-way ANOVA with the Bonferroni post-hoc test was used for statistical analysis. IAP: intraabdominal pressure, IVP: intravesical pressure.

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  Fig. 5 Histological analysis of the effects of mesenchymal stem cell enriched by the Primed Fresh OCT4 procedure (PFO-MSC) injection on detrusor underactivity (DUA) bladder injury. (A-C) Representative images of H&E staining (magnification ×100, scale bar=100 μm, A), immunohistochemical staining of α-smooth muscle actin (magnification ×100, scale bar=100 μm, B), and Masson’s trichrome staining (magnification ×200, scale bar=100 μm, C) in bladder tissues at 1 week after injection of PFO-MSCs. (D) Representative images (left panel) and quantitation right panel) of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to detect apoptotic cells (green, magnification ×400, scale bar=100 μm) in bladder tissue from the indicated groups. Nuclei were stained with 4’6-diamino-2-phenylindole (DAPI). All quantitative data are presented as the mean±SEM (n=10). **p<0.01, ***p<0.001 compared with the DUA group. A one-way ANOVA with the Bonferroni post-hoc test was used for statistical analysis.

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  Fig. 6 Engraftment of mesenchymal stem cells enriched by the Primed Fresh OCT4 procedure (PFO-MSCs) in chronic bladder ischemia (CBI) induced detrusor underactivity (DUA) bladders. (A, B) Representative images (magnification ×400, scale bar= 100 μm) showing immunofluorescence staining of human B2M (hB2M, green) in overactive bladder (OAB) (A) or DUA (B) bladder tissues from the indicated groups at 1 week post- injection of PFO-MSCs. Nuclei were stained with 4’6-diamino-2-phenylindole (DAPI, blue). It is noteworthy that hB2M＋ cells engrafted primarily as pericytes and located near muscle fibers within the DUA bladders.

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  Fig. 7 Injection of Primed Fresh OCT4 (PFO) induces angiogenesis in bladders with chronic bladder ischemia (CBI). (A, B) Representative images showing immunohistochemical staining of CD31 (magnification ×100, scale bar=100 μm) in overactive bladder (OAB) (A) or detrusor underactivity (DUA) (B) bladder tissues 1 week post-transplantation of mesenchymal stem cells enriched by the PFO procedure (PFO-MSCs). Nuclei were stained with Mayer’s hematoxylin. The number of CD31＋ vessels in the right panel is presented as the mean±SEM (n=5). (C) Changes in the expression of gene transcripts related to angiogenesis in naïve or PFO-MSCs. Expression is presented as the % of GAPDH (n=3). *p<0.05, **p<0.01, ***p<0.001, compared with CBI groups (one-way ANOVA with Bonferroni post-hoc test, C), or compared with naïve cells (unpaired t-test, D). TEK: TEK receptor tyrosine kinase, HGF: hepatocyte growth factor.

Reference

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