Human umbilical cord mesenchymal stem cells-derived extracellular vesicles as a therapeutic approach to ameliorate bladder injury in animal models of radiation cystitis

Abstract

Background: Radiation cystitis (RC) is a major complication of pelvic radiotherapy, leading to inflammation, vascular damage, and fibrosis. While mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show promise in regenerative medicine, their therapeutic effects on RC remain unclear. This study evaluates the efficacy of human umbilical cord MSC-EVs (huMSC-EVs) in mitigating Radiation-induced bladder damage.

Methods: Animal models of RC were established using 20 Gy pelvic irradiation. Forty-four female Sprague-Dawley rats were divided into four groups: negative control (NC, no radiation), positive control (PC, radiation only), local treatment (LT, huMSC-EVs injected into the bladder wall), and systemic treatment (ST, intravenous huMSC-EVs). Bladder function and compliance were assessed via metabolic cage and urodynamic studies (UDS) at 6 and 24 weeks. Histopathological changes and inflammatory cytokine levels were evaluated at multiple time points.

Results: Administration of huMSC-EVs significantly improved urinary frequency and hematuria. Histological analysis showed reduced urothelial disintegration and edema in the early phase, and improved urothelial integrity, reduced hyperplasia and vascular lesions, and restored bladder architecture in the late phase, in the treated groups. UDS demonstrated preserved bladder compliance and voiding efficiency in LT rats, with significantly higher voided volume (p < 0.01) and lower post-voiding residue (p < 0.05) compared to the PC group 24 weeks post-irradiation. Pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 were markedly lowered and their levels were similar to the non-radiated NC group in LT-treated rats (p = 1.00, p = 0.22, p = 0.16), 24 weeks post-irradiation.

Conclusions: Local huMSC-EVs therapy effectively reduces RC-related bladder injury and preserves function, likely by modulating inflammatory response and epithelial regeneration. These findings highlight huMSC-EVs as a promising strategy to prevent chronic RC, warranting further clinical exploration.

Keywords: Animal model; Mesenchymal stem cells; Radiation cystitis; Regenerative medicine.

Fig. 1

Establishment of the radiation cystitis animal models. Dosimetry and radiotherapy contouring of the bladder based on the coronal, transversal, and sagittal views on the CT-scan imaging (a). The animals were positioned to receive the planned single radiotherapy dose of 20 Gy, as demonstrated. After receiving the radiation dose, animals were grouped based on whether they received huMSC-EVs and the route of injection to positive control (PC), local treatment (LT), and systemic treatment (ST) (b). The timeline of the study (c). Cystography of rats in the LT group with urographin before (left) and after (right) voiding to check for bladder perforation or fistula after the surgery and local administration to the bladder wall (d). Figure 1b was created in BioRender. Ganjaroudi, N. (2025) https://BioRender.com/b03b502

Fig. 2

Characterization of the human umbilical mesenchymal stem cells (huMSCs) and huMSC-derived extracellular vesicles (huMSC-EVs). Flow cytometry analyses revealed the positive expression of surface markers CD73 (100%), CD90 (100%), and CD105 (99.5%) and the absence of HLA-DR (0.1%), CD34 (0.036%), and CD45 (0.078%) on huMSCs (a). Transmission electron microscopy (TEM) of the huMSC-EVs revealed their cup-shaped morphology (b). Dynamic light scattering (DLS) revealed the size distribution and measured the size of huMSC-EVs to be about 126 nm (c). The scale bar is 100 nm

Fig. 3

The accumulation of the human umbilical mesenchymal stem cells-derived extracellular vesicles (huMSC-EVs) in both local and systemic treatment groups was confirmed by the detection of the DiL-stained huMSC-EVs in the bladder tissue 2 weeks after huMSC-EVs injection (a). The quantitative analysis of DiL-labeled huMSC-EVs demonstrates a higher retention of huMSC-EVs in the LT group compared to the ST group (N = 3 per group) (b). Error bars demonstrate standard deviations (SD). The scale bar is 10 μm in the original and 20 μm in the magnified images

Fig. 4

Urinary frequency in negative control (NC), positive control (PC), local treatment (LT), and systemic treatment (ST) with human umbilical mesenchymal stem cells-derived extracellular vesicles (huMSC-EVs) groups (a). Hematuria as scored by the urine strip instructions in ST, LT, and PC groups (b). The weight gain plot of three representative rats per group within 24 weeks post-radiation, demonstrating the impaired weight gain in the PC group (c). P-values were calculated using two-way ANOVA post-hoc analysis of Tukey’s Honest Significant Difference (HSD) test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. Error bars demonstrate standard deviations (SD). Each group included four animals per time point (N = 4)

Fig. 5

Macroscopic and pathological changes in the acute and chronic phases of radiation cystitis 2 weeks and 24 weeks after irradiation (a). Macroscopic images from freshly harvested bladder 24 weeks after irradiation revealed bladder deformity and areas of telangiectasia and hemorrhage (indicated by the arrow) in the positive control (PC) and systemic treatment (ST) groups. In 2 weeks, compared to the normal bladder in the negative control (NC) group, in the PC group, severe urothelial thinning (indicated by the pointer) in almost 80% of the urothelial surface and edema in the submucosal layer (indicated by the asterisk) can be seen. The severity of the urothelial damage and edema is less, and evidence of mitosis and regeneration in the basal layer can be seen in the local treatment (LT) and ST groups. In 24 weeks post-irradiation, edema (indicated by the asterisk), hemorrhagia (indicated by the pointer), and heterogenous areas of urothelial hyperplasia and urothelial thinning were indicative of the chronic radiation cystitis in the PC group. The LT and ST groups can show evidence of the regeneration and restoration of the urothelium and the amelioration of the edema and telangiectasias. However, some vascular lesions are still present in the ST group. The percentage of urothelium thinning in each radiated group, as scored by pathologists, is demonstrated (N = 3 per group) (b). Hyperplasia assessed by the percentage of ki67-stained cells in the urothelium basal layer at 24 weeks post-irradiation is also demonstrated (a) and quantified by a pathologist as the ratio of Ki67-stained cells to the total number of cells in the urothelial layer (c), as a measure of urothelium hyperplasia. Error bars demonstrate standard deviations (SD). The scale bar is 200 μm

Fig. 6

Urodynamics studies (UDS) in rats. Rats were positioned as demonstrated (a) and catheterized to perform UDS. A representative recording of detrusor pressure (Pdet) and electromyography (EMG) from each group at 6 and 24 weeks is demonstrated (b). The green arrow, red arrow, and asterisk demonstrate base Pdet at the onset of the filling phase, max Pdet, and Pdet at the end of the filling phase, respectively. Leak point pressure (LPP) (c), voided volume per void (d), and post-void residue (PVR) (e) are also demonstrated in negative control (NC), positive control (PC), local treatment (LT), and systemic treatment (ST) groups. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. Error bars demonstrate standard deviations (SD). Each group included three animals per time point (N = 3). Figure 6a was created in BioRender. Ganjaroudi, N. (2025) https://BioRender.com/4ziyr7h

Fig. 7

The infiltration of inflammatory cells identified by IHC markers of CD45, a pan-leukocyte marker expressed on all immune cells, and CD3, a T-cell marker (a). The quantification of the percentage of the stained area using imageJ for CD45 (b) and CD3 (c) shows that local huMSC-EVs therapy ameliorated the local infiltration of the inflammatory cells, while the positive control group had the highest infiltration of the immune cells, and T cells in the bladder stroma. Serum levels of pro-inflammatory cytokines, interleukin 1 β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-𝛼 (TNF-𝛼) in negative control, positive control, local treatment, and systemic treatment groups (d). *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. Error bars demonstrate standard deviations (SD). Each group included three animals per time point (N = 3). The scale bar is 400 μm