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.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).