Hypothesis / aims of study
Urinary incontinence (UI) is a common problem the older adults affecting to 200 million people worldwide, and it affects about 25% of adult women. There are three types of UI: stress UI, urgency UI, and mixed UI. Stress urinary incontinence (SUI) is the most common type among women and it affects to 50% of women with UI. Several non-surgical and surgical treatment options are available for SUI, such as pelvic floor muscle training, vaginal pessary, urethral bulking agents, and surgery. For severe SUI, surgical interventions is the most recommended treatment option, although it present post-operative complications. In this context, the search and development of less invasive therapies as treatment for SUI continues to be a main requirement and stem cell-based therapy may be an important option for treating these disorders. Mesenchymal stem cells (MSC) are adult stem cells and one of the most attractive sources for stem cell research and therapy. The aim of this study is to use MSc obtained from perinatal tissues and study their use of the treatment of SUI using in vitro and in vivo models.
Study design, materials and methods
Cell cultures: SUI patients and pregnant women were recruited at the Department of Obstetrics and Gynecology under informed consent approved by the Ethics Committee. Suburethral tissues were obtained by biopsy and cell cultures were established by enzymatic digestion. Isolated cells were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. Human placentas were obtained during natural or cesarean births and decidua-derived mesenchymal stromal cells (DMSC) were isolated from placental membranes by enzymatic digestion. Isolated cells were cultured in DMEM supplemented with 2 mM glutamine, 0.1 mM sodium pyruvate, 55 μM β-mercaptoethanol, 1% non-essential amino acids, 1% penicillin/streptomycin, 10% fetal bovine serum and 10 ng/ml epidermal growth factor (EGF). All steps of tissue processing were performed in a biosafety cabinet using appropriate aseptic techniques. For trans-well co-culture assays, DMSC were placed on top of the trans-wells and SUI-isolated cells were placed at the bottom of the well at a ratio 1:2 or 1:5. After 24-48 hours of co-culture, migration, proliferation and the cytokines present in the coculture supernatants were studied. Migration was evaluated by the CytoSelectt 24-Well Cell Migration Assay. Proliferation was evaluated by Alamar blue assay. Cell culture supernatants were analyzed by the ProcartaPlex™ Multiplex Immunoassays.
Animal model of stress UI caused by childbirth: Vaginal distention in female Sprague‐Dawley rats was used to simulate the maternal injuries of childbirth. Animal experiments were performed meeting the animal protection requirements and the approval of the Animal Experimentation Ethics Committee. The animals were divided in two groups, control and DMSC-treated animals. Two doses of DMSC (1 million DMSCs suspended in phosphate-buffered saline) were injected into the periurethral area of the rats. The sham injection was performed with the same volume of phosphate buffered saline without cells. UI was assessed by measurement of the leak point pressure (LPP). To evaluate the engrafment of DMSCs, cells were labeled with VivoTrack before injection. Labeled cells were infused into the periurethral area and visualized by Bruker In Vivo Xtreme animal imaging system once a week.
Data analysis: The data were analyzed using the t-Student test to obtain the significance of the data between groups and data in which a p<0.05 is shown as statistically significant.
We have developed a SUI rat model consisting of vaginal distension which mimics the maternal injuries of childbirth. In this model there is a consistently decrease in the abdominal leak point pressure (LPP) indicative of SUI. After periurethral injection of saline or DMSC, LPP value was significantly higher in the DMSC group when compared to that of the control group (p＜0.05). The existence of VivoTrack-stained DMSCs in the injected periurethral tissue was verified by using an animal imaging system. We have observed that the cells remain in the ventral area, that they are still alive at least for 14 days after the injection, and they do not produce tumors or adverse reactions such as graft-versus-host syndrome.
To assess the possible mechanism involved in the regenerative potential of DMSC in SUI, we performed several in vitro assays by coculturing DMSC and SUI isolated cells. It is important to note that DMSCs are able to migrate and home at injured sites with high efficiency. The results showed that DMSC have a significant increase in their in vitro migration toward SUI cells when compared with control media used as negative control.
To examine whether DMSC can modify SUI cell proliferation, coculture of both cell types was stablished. DMSC significantly increased the proliferation of SUI cells as compared with untreated cells at the two examined ratios.
The possible interaction between DMSC and SUI cells was examined by evaluating cytokines, chemokines, and growth factors expression on coculture supernatant using multiplex immunoassays that allows to simultaneously quantitate many proteins. Preliminary results showed that anti-inflammatory cytokines such as IL6, IL8, and MCP3 were significantly reduced in coculture, when expressed as a relative ratio to the secretion of those proteins by both cells cultured separately. In addition, angiogenic growth factors such as VEGF and metalloproteinases MMP-2, MMP-3 and MMP-9 were significantly decreased in the coculture supernatant.
Interpretation of results
This original work shows that DMSC injection into periurethral tissue after vaginal distension in rats led to increased LPP suggesting that DMSC can be used as one of the possibly effective cell therapies for SUI. This effect is likely to be mediated by cellular secretions acting through fibroblasts proliferation from pelvic floor tissue and modifying the fibroblast microenvironment after vaginal distension which may lead to improved continence.