Rat mesenchymal stem cell secretome promotes elastogenesis and facilitates recovery from simulated childbirth injury

Abstract

Vaginal delivery is a risk factor for stress urinary incontinence (SUI). Mesenchymal stem cells (MSCs) home to injured organs and can facilitate repair. The goal of this study was to determine if MSCs home to pelvic organs after simulated childbirth injury and facilitate recovery from SUI via paracrine factors. Three experiments were performed. Eighteen female rats received vaginal distension (VD) or sham VD and labeled intravenous (IV) MSCs to investigate if MSCs home to the pelvic organs. Whole-organ imaging and immunofluorescence were performed 1 week later. Thirty-four female rats received VD and IV MSCs, VD and IV saline, or sham VD and IV saline to investigate if MSCs accelerate recovery of continence. Twenty-nine female rats received VD and periurethral concentrated conditioned media (CCM), VD and periurethral control media, or sham VD and periurethral control media to investigate if factors secreted by MSCs accelerate recovery from VD. Urethral histology and function were assessed 1 week later. Significantly more MSCs were observed in the urethra, vagina, and spleen after VD compared to sham VD. Continence as measured by leak point pressure (LPP) was significantly reduced after VD in rats treated with saline or control media compared to sham VD but not in those given MSCs or CCM. External urethral sphincter (EUS) function as measured by electromyography (EMG) was not improved with MSCs or CCM. Rats treated with MSCs or CCM demonstrated an increase in elastin fibers near the EUS and urethral smooth muscle more similar to that of sham-injured animals than rats treated with saline or control media. MSCs homed to the urethra and vagina and facilitated recovery of continence most likely via secretion of paracrine factors. Both MSCs and CCM have promise as novel noninvasive therapies for SUI.

Figure 1

Characterization of mesenchymal stem cells (MSCs). (A) Example flow cytometry results showing positivity for cluster of differentiation 29 (CD29), CD90, and CD54, as well as negativity for CD45. Blue represents MSCs at P8, 0.0139% of which were green fluorescent protein positive (GFP⁺), since this was prior to GFP labeling. Red represents MSCs at P15, 90.1% of which were GFP⁺, after GFP labeling. Green and orange are negative controls; 93.8% of P15 cells were CD29⁺, CD90⁺, CD54⁺, and CD45⁻. (B) Adipogenic and osteogenic differentiation of MSCs. Adipogenic-induced cells contained intracellular lipid droplets, stained orange by oil red O (scale bar: 10 μm). Osteogenic-induced cells are red as assessed with alizarin red staining (scale bar: 100 μm).

Figure 2

Homing of mesenchymal stem cells (MSCs) to pelvic organs 1 week after vaginal distension (VD) and sham VD. (A) Example ex vivo whole organ imaging results. Yellow oval indicates region of interest inside of which fluorescent flux was calculated. Pseudocolor scale indicates the quantification of level of fluorescent flux, with red being the most intense and purple being the least intense. Rats that received no cells had only nominal total fluorescent flux and are not shown. (B) Fluorescent flux of organs 1 week after VD or sham VD. Each bar indicates mean ± standard error of the mean of organs from nine rats. *A statistically significant difference compared to the same organ from the sham VD group with p < 0.05. **A statistically significant difference compared to the same organ from the sham VD group with p < 0.005. (C) Example immunofluorescence from the urethra and vagina 1 week after VD or sham VD. Blue indicates nuclei with 4′,6-diamidino-2-phenylindole (DAPI) stain; green indicates MSCs labeled with green fluorescent protein (GFP); red indicates smooth muscle with α-actin immunolabeling. Rows 1–4 are at the same magnification (scale bar: 100 μm). The last row shows an example immunofluorescence at higher magnification (scale bar: 10 μm) demonstrating dual labeling of blue and green to show association of nuclei with MSCs. Rats that did not receive cells did not show fluorescence (data not shown).

Figure 3

Functional testing after vaginal distension and treatment. Results of functional testing after vaginal distension (VD) or sham VD and treatment with mesenchymal stem cells (VD + MSC) or saline (A–C) or concentrated conditioned media (CCM) or control media (D–F). Bladder pressure during filling and leak point pressure (LPP) testing (A and D). External urethral sphincter electromyogram (EMG) firing rate during filling and LPP testing (B and E). External urethral sphincter EMG amplitude (C and F). Each bar indicates mean ± standard error of the mean of from five to seven rats. *A significant difference compared to the same outcome for the sham VD group with p < 0.05.

Figure 4

Urethral histology 1 week after vaginal distension and treatment. Examples of urethral histology 1 week after vaginal distension (VD) or sham VD and treatment with mesenchymal stem cells (VD + MSC) or saline (A) or concentrated conditioned media (CCM) or control media (B). Results of semiquantitative grading of urethral histology after VD or sham VD and treatment with MSCs or saline (C) or CCM or control media (D). Each bar indicates mean ± standard error of the mean from five rats. Semiquantitative data was analyzed with a Kruskal–Wallis one-way ANOVA on ranks followed by a Dunn’s post hoc test. *A significant difference compared to the same outcome for the sham VD group with p < 0.05. +A significant difference compared to the same outcome for VD + MSC group with p < 0.05. sm, smooth muscle; EUS, external urethral sphincter.

Figure 5

Histological staining of urethral sections after vaginal distension and treatment. Example urethral cross sections stained with Masson’s trichrome (upper panels) and near sections stained with elastin von Giesson stain (EVG; lower panels) 1 week after vaginal distension (VD) or sham VD and treatment with mesenchymal stem cells (VD + MSC) or saline (A) or concentrated conditioned media (CCM) or control media (B). Inset in upper panels indicates approximate area of higher magnification EVG example placed just below it. Note that anatomical structures do not align exactly in near sections. Masson’s trichrome scale bar: 100 μm. EVG scale bar: 10 μm. Black arrows indicate disorganized elastin fibers in EVG examples.