Stem Cell-Derived Smooth Muscle Cells Help Restructure Urethral Sphincter Muscles in Rats


Stress urinary incontinence affects 25%-50% of the female population and is defined as the leakage of the bladder upon exertion. The exertions that can cause the bladder to leak can be as simple as laughing, coughing, sneezing, hiccups, yelling, or even jumping up and down. Stress urinary incontinence costs Americans some $12 billion a year and also causes a good deal of embarrassment and compromises quality of life. Unsurprisingly, stress urinary incontinence also is associated with an increased incidence of anxiety, stress, and depression.

In most cases of stress urinary incontinence, injury to the internal sphincter muscles of the urethra or to the nerves that innervate these muscles (both smooth and voluntary muscles) significantly contribute to the condition. Conservative management of stress urinary incontinence can work at first, but can fail later on. The other option is corrective surgery that reconstructs the urethral sphincter and increases urethral support. However, even though such surgeries can and often do work, recurrence of the incontinence is rather common. Is there a better way?

Yan Wen from Stanford University School of Medicine and colleagues and collaborators from College of Medicine of Case Western Reserve in Cleveland, Ohio, Southern Medical University in Guangzhou, China, and Montana State University have used a novel stem cell-based technique to treat laboratory Rowett nude rats that had a surgically-induced form of stress urinary incontinence. While the results are not overwhelming, they suggest that a stem cell-based approach might be a step in the right direction.

Wen and others used a human embryonic stem cell line called H9 and two different types of induced pluripotent stem cell lines to make, in culture, human smooth muscle progenitor cells (pSMCs). Fortunately, protocols for differentiating pluripotent stem cells into smooth muscle cells is well worked out and rather well understood. These pSMCs were also tagged with a firefly luciferase gene that allowed visualization of the cells after implantation.

Six groups of rats were treated in various ways. The first group had stress urinary incontinence and were only treated with saline solutions. The second group of animals also had stress urinary incontinence and were treated with cultured human pSMCs that were derived from human bladders. The third group of animals also had stress urinary incontinence and were treated with pSMCs made from H9 human embryonic stem cells. The next two groups also had stress urinary incontinence and were treated with two different induced pluripotent stem cell lines; one of which was induced with a retroviral vector and the second of which was made with episomal DNA. Both lines were originally derived from dermal fibroblasts. The final group of rats did not have stress urinary incontinence and were used as a control group.

The cells were introduced into the mice by means of injections into the urethra under anesthesia. Two million cells were introduced in each case, three weeks after the induction of stress urinary incontinence. All animals were examined five weeks after the cells were injected into the animals.

Because the cells were tagged with firefly luciferase, the animals could be given an injection of luciferin, which is the substrate for luciferase. Luciferase catalyzes a reaction with luciferin, and the cells glow. This glow is easily detected by means of a machine called the Xenogen Imaging System. Such experiments showed that the injected cells did not survive terribly well, and by 9 days after the injections, they were usually not detectable. Two rats that had been injected with retrovirally-induced induced pluripotent stem cell-derived pSMCs lasted until 35 days after injection, but these rats were the exception and not the rule.

Did the cells integrate into the urethral sphincter by the signal is too low to be detected using luciferase? The answer to this question was certainly yes, but the amount of integration was nothing to write home about. Small patches of cells showed up in the urethra sphincters that expressed human gene products, and therefore, had to be derived from the injected cells.

In vivo survival of transplanted pSMCs in RNU rats. (A): The RV-iPSC pSMCs were periurethrally injected into the rats and monitored with BLI. (B): At day 12, a small number of the transplanted cells were detected in the proximal rat urethra. The transplanted human cells were determined by positive staining of HuNuclei and smoothelin. (C): Gene expression of human ERV-3 in rat urethras 5 weeks after cell transplantation. Y-axis on left shows the scale for ERV-3 copy numbers from tissue samples. Each human cell contains one copy of ERV-3 transcript; hence, the number of copies is equal to the number of cells. Y-axis on right shows the ERV-3 copy numbers of the standard cell samples. The cell numbers in the standard graph are 0.5 × 103, 1 × 103, 2 × 103, 4 × 103, 8 × 103, and 10 × 103 (red dots). ERV-3 amplifications in all pSMC-treated groups were very low. Abbreviations: BLI, bioluminescent imaging; bSMC, bladder smooth muscle cell; DAPI, 4′,6-diamidino-2-phenylindole; Epi, episomal plasmid; ERV-3, endogenous retrovirus group 3; H&E, hematoxylin and eosin; Hu, human; HuNuclei, human nuclei; iPSC, induced pluripotent stem cell; pSMCs, smooth muscle progenitor cells; RNU, Rowett Nude; RV, retrovirus vector.
In vivo survival of transplanted pSMCs in RNU rats. (A): The RV-iPSC pSMCs were periurethrally injected into the rats and monitored with BLI. (B): At day 12, a small number of the transplanted cells were detected in the proximal rat urethra. The transplanted human cells were determined by positive staining of HuNuclei and smoothelin. (C): Gene expression of human ERV-3 in rat urethras 5 weeks after cell transplantation. Y-axis on left shows the scale for ERV-3 copy numbers from tissue samples. Each human cell contains one copy of ERV-3 transcript; hence, the number of copies is equal to the number of cells. Y-axis on right shows the ERV-3 copy numbers of the standard cell samples. The cell numbers in the standard graph are 0.5 × 103, 1 × 103, 2 × 103, 4 × 103, 8 × 103, and 10 × 103 (red dots). ERV-3 amplifications in all pSMC-treated groups were very low. Abbreviations: BLI, bioluminescent imaging; bSMC, bladder smooth muscle cell; DAPI, 4′,6-diamidino-2-phenylindole; Epi, episomal plasmid; ERV-3, endogenous retrovirus group 3; H&E, hematoxylin and eosin; Hu, human; HuNuclei, human nuclei; iPSC, induced pluripotent stem cell; pSMCs, smooth muscle progenitor cells; RNU, Rowett Nude; RV, retrovirus vector.

The exciting part about these results, however, was that when Wen and others examined the rat urethral sphincters for the presence of things like elastin and other proteins that make for a healthy urethral sphincter, there was a good deal of elastin, but it was not human elastin but rat elastin. Therefore, this elastin synthesis was INDUCED by the implanted cells even though it was not made by the implanted cells. Instead, the implanted cells seemed to signal to the native cells to beef up their own production of sphincter-specific gene products, which made from a better sphincter. This was not the case in animals that received injections of human pSMCs derived from human bladders.

Assessment of elastin fibers in the proximal urethra of the rat. (A): Representative images of cross-section of proximal urethra with Weigert’s Resorcin-Fuchsin’s elastin and van Gieson’s collagen staining. Elastic fiber shown as dark blue, collagen as red pink, and other tissue elements as yellow. Scale bars = 100 µm. (B): Quantification of elastin fibers was assessed using Image-Pro Plus software and expressed as a percentage of the arbitrary ROIs. Each bar represents the mean value ± SEM. Abbreviations: bSMC, human bladder smooth muscle cells; H9-pSMC, surgery plus H9-pSMC injection; Hu-bSMC, surgery plus injection of human bladder smooth muscle cells; pSMCs, smooth muscle progenitor cells; Pure control, no surgery and no treatment; ROIs, regions of interest; Sham saline, surgery plus saline injection.
Assessment of elastin fibers in the proximal urethra of the rat. (A): Representative images of cross-section of proximal urethra with Weigert’s Resorcin-Fuchsin’s elastin and van Gieson’s collagen staining. Elastic fiber shown as dark blue, collagen as red pink, and other tissue elements as yellow. Scale bars = 100 µm. (B): Quantification of elastin fibers was assessed using Image-Pro Plus software and expressed as a percentage of the arbitrary ROIs. Each bar represents the mean value ± SEM. Abbreviations: bSMC, human bladder smooth muscle cells; H9-pSMC, surgery plus H9-pSMC injection; Hu-bSMC, surgery plus injection of human bladder smooth muscle cells; pSMCs, smooth muscle progenitor cells; Pure control, no surgery and no treatment; ROIs, regions of interest; Sham saline, surgery plus saline injection.

Because these mice were sacrificed five weeks after the injections, Wen and others could not assess the urethral function of these animals. Therefore, it is uncertain if the improved tissue architecture of the urethral sphincter properly translated into improved function even though it is reasonable to assume that it would. Having said that, it is possible that the experiments that detected the presence of increased amounts of elastin and collagen in the sphincters of these rats was complicated by the presence of bladder tissue in the preparations. Since bladder tissue was included in all trials of this experiment, it is unlikely that bladder tissue is the sole cause of increase elastin and collagen in the stem cell-treated rats. Secondly, rat regenerative properties may not properly match the regenerative properties in older human patients. Here again, unless such an experiment is attempted in larger animal models and then in human patients, we will never know if this procedure is viable for regenerative treatments in the future.

For now, it is an interesting observation, and perhaps a promising start to might someday become a viable regenerative treatment for human patients.

This paper appeared in Stem Cells Translational Medicine, vol 5, number 12, December 2016, pp. 1719-1729.

Mesenchymal Stem Cell Transplantation to Heal Mother’s Childbirth Injury


Occasionally. vaginal birth can lead to injury in the mother. Some of these injuries are relatively light and the mother heals rather quickly, but others can be more severe. Stress urinary incontinence (SUI) affects 4-35% of women who have given birth via vaginal delivery. SUI causes unintentional leakage from the bladder during heavy exercise, laughter, coughing, sneezing, heavy lifting, or jumping. SUI can cause discomfort, embarrassment, and some degree of social isolation. Unfortunately the treatments for SUI range from surgery to physiotherapy and they do little good.

In order to provide better options for mothers, researchers at the Cleveland Clinic’s Department of Biomedical Engineering have used female rats with birth-induced injuries as a model system. In this model system, injections of mesenchymal stem cells improved recovery from childbirth-induced injuries.

Previous work by this research group showed that injected mesenchymal stem cells tended to move into the spleen. However, if the urethra and vagina were damaged by childbirth trauma prior to mesenchymal stem cell injections, the cells targeted the damaged tissues and secreted trophic factors, which stimulated the differentiation and survival of remaining cells, and also induced the mesenchymal stem cells to engraft into the smooth muscles around the urethra and vagina. These activities accelerated and improved recovery of the animals from SUI.

Margot S. Damaser from the Cleveland Clinic said, “Stem cell-based therapy has recently gained attention as a promising treatment for SUI. Stem cell therapies may be more feasible and less invasive than current therapies.”

Other kinds of stem cells have been used to experimentally treat SUI in laboratory animals. Autologous or self-donated muscle stem cells have been used to treat SUI in animals and in human clinical trials. Fat-based stem cells have also been used, but only in animal models.

Damaser believes that mesenchymal stem cells have the added advantage of not being recognized by the immune system and therefore the possibility to implanting stem cells from an unrelated donor is a possibility for older patients.

“Since rat MSCs were used in this study, the results can only be applied to rat models of injury-treated rats,: said Damaser. “Human adult stem cells need to be investigated in future studies to see if these findings also apply to humans.”

Other researchers think that this procedure might serve as a treatment for SUI in older women. “This study provides evidence that mesenchymal stem cell transplantation could favorably impact a side effect of delivery and aging by releasing factors that can influence the urethra and vagina to treat stress urinary incontinence,” said Amit N Patel, director of cardiovascular regenerative medicine at the University of Utah. “Further studies are required to confirm that this animal study translates to humans.”

Human Amniotic Stem Cells Treat Stress Urinary Incontinence in Animals


Stress Urinary Incontinence (SUI) refers to the involuntary leakage of urine during physical activity, sneezing, or coughing. This is an extremely embarrassing problem for women and some men. Treatments for this condition includes various types of tapes (tension free vaginal tape) and slings (transobturator slings or pubovaginal slings) can help somewhat. Nonsurgical treatments include bulking agents (polytetrafluoroethylene, bovine collagen, silicone particles, carbon beads, and autologous fat or chondrocytes. The non-surgical procedures have only had limited success, and even the present surgical procedures are largely unsatisfactory.

Therefore, to design better treatments for SUI, researchers have turned to stem cells. In this present paper, Bum Soo Kim and his colleagues from the Departments of Urology, Physiology, Plastic and Reconstructive Surgery in the School of Medicine at Kyungpook National University in Daegu, South Korea, in collaboration with Anthony Atala from the Wake Forest University of Medicine in Winston-Salem, North Carolina have injected human amniotic stem cells into the urethras of laboratory animals with SUI. Their results are very positive and interesting.

Mice can be given SUI by means of bisecting the nerve bundle (pudendal nerve) that serves the urethra. By crushing this nerve bundle, the mice lose control of their bladders and show all the signs of SUI.

One week after the mice were confirmed have SUI, they were given an injection of about half a million human amniotic stem cells into the muscle that cinches down the urethra. Cells were injected into either side of the urethral sphincter muscle. The injected cells were labeled with tiny dye-laced beads to watch them during their sojourn inside the animals.

Measurements of the bladder control of the injected mice was determined with a catheter that measured LPP or leak point pressure and the closing pressure or CP. After these measurements, the animals were sacrificed and their bladders were examined to determine if the injected stem cells integrated into urethral sphincter.

The results showed that the injected cells had the characteristics of mesenchymal stem cells. In culture, they were able to differentiate into smooth muscle tissue.

In the laboratory animals, the injected cells had migrated into the urethral sphincter and surrounded it. Optical tracking injected cells showed that they were alive and localized in the urethral area. LPP and CP measurements also showed that these measurements were significantly higher in the injected animals 2 weeks and 4 weeks after stem cell injections relative to those animals that were not injected.

Tissue sampling of the urethral sphincters from injected animals showed that the sphincter muscles were loaded with dye-containing beads, indicating that the injected amniotic stem cells integrated into the sphincter muscle and stayed there.  Secondly, the cells expressed a boat-load of genes involved in the formation of muscle (PAX7, MYF5 and
MYOD), and MYOGENIN, MEF2 and MLP).  They also expressed genes that encode proteins that allow smooth muscle cells to respond to impulses from nerve cells.  This shows that the injected cells responded to the environment into which they were placed and differentiated accordingly, in this case, into smooth muscle.  Also, none of the injected animals showed any signs of rejection of the injected stem cells by the immune system.  Also, none of the animals showed any tumors from the injected stem cells.

This study shows several positive results.  First of all, the injected amniotic stem cells were able to nicely survive in the urethral sphincter.  Secondly, they were not rejected by the host immune system and did not form tumors.  Third, these cells also differentiated into smooth muscle cells and properly integrated into the urethral sphincter muscle.  Finally, these cells partially restored function to the urethral sphincter.

Is this procedure feasible in humans?  In theory, it is certainly possible.  Injections into the human urethral sphincter are difficult and require high resolution imaging procedures to properly guide the physician during the injection.  High resolution MRI-guided injections are possible, but this exposes the patient to large quantities of radiation.   This present paper shows that it is entirely possible to mark the cells in a manner that is not toxic to them in order to properly trace the injected cells.  Such labeling would allow physicians to deliver the cells with lower-resolution techniques that do not expose the patient to such high levels of radiation.

This paper provides a remarkable example of how non-embryonic stem cells can provide a treatment for a troublesome and embarrassing medical problem.  The paper is Bum Soo Kim, et al.,  “Human amniotic fluid stem cell injection therapy for urethral sphincter regeneration in an animal model,” BMC Medicine 2012, 10:94 doi:10.1186/1741-7015-10-94.