Urinary Stem Cells and Their Therapeutic Potential


Yuanyuan Zhang, assistant professor of regenerative medicine at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine, has extended earlier work on stem cells from urine that suggests that these cells might be more therapeutically useful than previously thought.

These urinary stem cells can be isolated from a patient’s urine sample, and they can be induced, in the laboratory, to form bladder-type cells; smooth muscle and urothelial (bladder-lining) cells. Such stem cells could certainly be used to treat urinary tract problems, even though a good deal more work is required to confirm that they can do just that.

Nevertheless, Zhang and his co-workers have discovered that these urinary tract stem cells are much more plastic than previously thought. In the laboratory, Zhang and others have managed to differentiate urinary tract stem cells into bone, cartilage, fat, skeletal muscle, nerve, and endothelial cells (the cells that line blood vessels). This suggests that urine-derived stem cells could be used in a variety of therapies.

USCs undergo multipotential differentiation in vitro. (a-c) endothelial differentiation of USCs. USCs (p3) were induced to endothelial lineage by culture in EBM-2 medium containing VEGF 50 ng/ml for 14 days. (a) In vitro vessel formation. Endothelial differentiated USCs were cultured on Matrigel for 18h to form branched networks (angiogenesis) and tubular structures. Scale bar = 100μm. (b) Expression analysis of endothelial-specific transcripts by RT-PCR. (c) Immunofluorescence staining using endothelial-specific markers revealed enhanced staining of the markers with differentiation (middle row) compared to the non-treated control (top row). Scale bar = 50μm.
USCs undergo multipotential differentiation in vitro. (a-c) endothelial differentiation of
USCs. USCs (p3) were induced to endothelial lineage by culture in EBM-2 medium containing
VEGF 50 ng/ml for 14 days. (a) In vitro vessel formation. Endothelial differentiated USCs were
cultured on Matrigel for 18h to form branched networks (angiogenesis) and tubular structures. Scale
bar = 100μm. (b) Expression analysis of endothelial-specific transcripts by RT-PCR. (c)
Immunofluorescence staining using endothelial-specific markers revealed enhanced staining of the
markers with differentiation (middle row) compared to the non-treated control (top row). Scale bar =
50μm.

Zhang said that urinary tract stem cells could be used to treat urological disorders such a kidney disease, urinary incontinence, and erectile dysfunction. However, Zhang is optimistic that they can also be used to treat a wider variety of treatment options, such as making replacement bladders, urine tubes, and other urologic organs.

Since these stem cells come from the patient’s own body, they can have a low chance of being rejected by the immune system. Also, they do not cause tumors when implanted into laboratory animals.

In their latest work, Zhang and his colleagues obtained urine samples from 17 healthy individuals whose ages ranged from five to 75 years old. Even though these stem cells are only one of a large collection of cells in urine, isolating urinary stem cells from urine only requires minimal processing.

A single USC (inset) is followed through different passages (p0-p12). The cells were expanded to a colony were cultured in KSFM-EFM medium with 5% serum and images recorded with passage. Images shown at x100
A single USC (inset)
is followed through different passages (p0-p12). The cells were expanded to a colony were cultured in
KSFM-EFM medium with 5% serum and images recorded with passage. Images shown at x100

In the laboratory, Zhang and his team differentiated the cells into derivatives of all three embryological layers (endoderm – skin and nervous tissue; mesoderm – bone, muscle, glands, and blood vessels; and endoderm – digestive system).

Differentiation of one USC clone into UCs and SMCs. (a) USCs (p3) t were used to differentiate into two distinct lineages. Culture in SMCs-lineage differentiation (2.5 ng/ml TGF-􀈕1 and 5 ng/ml PDGF-BB) and UCs-lineage differentiation (30 ng/ml EGF) medium was used for 14 days.
Differentiation of one USC clone into UCs and SMCs. (a) USCs (p3) t were used to
differentiate into two distinct lineages. Culture in SMCs-lineage differentiation (2.5 ng/ml TGF-􀈕1 and
5 ng/ml PDGF-BB) and UCs-lineage differentiation (30 ng/ml EGF) medium was used for 14 days.

After showing the multipotent nature of urinary tract stem cells in the laboratory, Zhang and others took smooth muscle cells and urothelial cells made from urinary tract stem cells and transplanted them into mice with tissue scaffolds that had been made from decellularized pig intestine. The scaffolds only had extracellular molecules and not cells. After one month, the implanted cells had formed multi-layered, tissue-like structures.

USCs were infected with BMP9 or control GFP and were injected subcutaneously into nude mice. i) Bony masses were only observed in mice implanted with BMP-transduced USCs at week 4. ii) The harvested bony masses were subjected to microCT imaging revealing the isosurface (left) and density heat maps (right).
USCs were infected with BMP9 or control GFP and were
injected subcutaneously into nude mice. i) Bony masses were only observed in mice implanted with
BMP-transduced USCs at week 4. ii) The harvested bony masses were subjected to microCT imaging
revealing the isosurface (left) and density heat maps (right).

Urinary tract stem cells or as Zhang calls them, urine-derived stem cells or USCs, have many cell surface characteristics of mesenchymal stem cells from bone marrow, but they are also like pericytes, which are cells on the outside of small blood vessels. Zhang and others suspect that USCs come from the upper urinary tract, including the kidney. Patients who have had kidney transplants from male donors have USCs with a Y chromosome in them, which suggests that the kidney is a source or one of the sources of these cells.

Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for expression of kidney-lineage marker. (a) FISH (left) and amelogenin gene PCR analysis (right) analysis of USCs isolated from urine obtained from a male donor-to-female recipient kidney transplant for presence of Y-chromosome (L: DNA ladder, M: male control, F: female control, A4: USC from male donor-to-female recipient urine sample, N: negative control).
Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for
expression of kidney-lineage marker. (a) FISH (left) and amelogenin gene PCR analysis (right)
analysis of USCs isolated from urine obtained from a male donor-to-female recipient kidney transplant
for presence of Y-chromosome (L: DNA ladder, M: male control, F: female control, A4: USC from
male donor-to-female recipient urine sample, N: negative control).

Even more work needs to be done before we can truly become over-the-moon excited about these cells as a source of material for regenerative medicine, Zhang’s work is certainly an encouraging start.

See Shantaram Bharadwaj, et al., Multi-Potential Differentiation of Human Urine-Derived Stem Cells: Potential for Therapeutic Applications in Urology. Stem Cells 2013 DOI: 10.1002/stem.1424.

Cultured Human Kidney Cells Improve Chronic Kidney Injury


Chronic kidney disease (CKD) is extremely expensive to treat and also leads to additional complications, such as heart and circulatory troubles. In general, when you have CKD, your life is a drag. ~13% of the worldwide population has CKD and in the US alone, the estimated Medicare costs for the treatment of this disease is $42 million.

There are drugs that can treat CKD, but these drugs (statins, angiotensin 2 receptor blockers and angiotensin converting enzyme (ACE) inhibitors, and erythropoietin to improve anemia) must be given for some time and at high doses before their effects become apparent.

The hormone erythropoietin (EPO) is made by the kidneys, and EPO signals to the bone marrow to produce more red blood cells. Recombinant versions of EPO are given to anemia patients, and have also been used illicitly in aerobic athletics to artificially boost red blood cell production (e.g., Floyd Landis, Lance Armstrong, etc.). However, EPO has another function in CKD in that EPO administration seems to protect the kidney from damage caused by low oxygen delivery. EPO production is quite low in CKD patients and this might play a role in the problems encountered by CKD patients.

The laboratory of James Yoo from the Institute for Regenerative Medicine at Wake Forest University has investigated the ability of cultured, human kidney cells that express EPO to improve kidney structure and function in a rodent model of CKD.

In this experiment, Yoo and his coworkers cut off the blood supply to the kidneys of hairless rats and then fed them the antibiotic gentimicin for a day (five doses). 8-10 weeks after this treatment, kidney function was reduced and the rats had all the signs of CKD.

Next, Yoo and others injected into the kidneys of these rats cultured human kidney cells. One group of rats received injections of buffer into their kidneys, some received cultured human kidney cells, and another group received cultured kidney cells that had been engineered to express EPO. These kidney cells came from a discarded organ from a 51-year-old human organ donor.

The kidneys of these rats were assayed for function and structure. One of the features of CKD is lots of protein in the urine. When the levels of protein in the urine of these rats was examined 1, 4, and 12 weeks after they had received infusions of the kidney cells, along with other markers of kidney damage, the levels of protein in the urine were high in the rats injected with buffer, lower in those injected with cultured kidney cells, and much mower in those injected with the EPO-expressing kidney cells. Also, hemoglobin levels (hemoglobin is the protein in red blood cells that ferries oxygen from the lungs to the tissues) were significantly higher in the rats injected with EPO-expressing kidney cells.

Next, Yoo and his colleagues examined the kidneys for inflammation and scarring. Scarring is relatively easy to detect because there are tissue stains that will highlight scarring (e.g., Masson’s Trichome stain). Once again, the buffer injected kidneys were loaded with scars, the kidney cell-injected kidneys had much less scarring and the rats injected with EPO-expressing kidney cells had even less scarring in most of the kidney. Also the presence of inflammatory cells in the kidney, which is indicative of cell damage, was significantly lower in kidneys injected with either type of cultured kidney cell. As an added bonus, Yoo’s group examined the markers of kidney cell damage (8-OHdG) and these were lower in the kidneys injected with cultured human kidney cells.

Did the injected cells hang around in the kidneys and contribute to the kidney? The answer seems to be, only a bit. When the rat kidneys were checked for human cells 12 weeks after injection, very few human kidney cells were found.

These experiments suggest that cultured kidney cells, particularly EPO-expressing ones, can initiate regeneration in damaged kidneys. While this experimental protocol requires adjustment and tweaking, it suggests a potential therapeutic strategy for treating CKD patients.

Bone Marrow Stem Cells Can Become Kidney Stem Cells and Heal Acute Renal Injury.


Patients with failing kidneys often suffer from chronic kidney disease or end-stage renal disease. These two conditions are associated with a substantial amount of suffering and death, and current treatments from chronic kidney disease and end-stage renal disease do virtually nothing to halt the progression of these diseases.

Fortunately there has been a respectable amount of recent work on kidney regeneration after kidney injury, but these new discoveries have not led to therapeutic advances. The shortage of kidneys for transplantation and the structural complexity of the kidney have slowed the development of therapeutic strategies for the kidney.

Stem cell-based therapy for damaged kidneys is a distinct possibility for several reasons. First, kidneys do seem to possess resident stem cells and extra renal stem cells also seem to reside in the kidney. Several studies have confirmed the presence of cells in the kidneys that possess stem cell-specific proteins (Sca-1, c-Kit, and CD133). When isolated and tested in the laboratory, these renal stem cells can differentiate, proliferate, and eventually reline denuded renal tubules, and thereby restore the structural and functional integrity of the kidney (See Yeagy BA, Cherqui S, Pediatr Nephrol 2011, 26:1427–1434; Parikh CR, et al., Ann Clin Biochem 2010, 47:301–312; Lee P-T, et al., Stem Cells 2010, 28:573–584; Bussolati B, et al., Am J Pathol 2005, 166:545–555; Dekel B, et al., J Am Soc Nephrol 2006, 17:3300–3314; Gupta S, et al., J Am Soc Nephrol 2006, 17:3028–3040; Lazzeri E, et al., J Am Soc Nephrol 2007, 18:3128–3138; and Kitamura S, et al., FASEB J 2005, 19:1789–1797). Unfortunately, the exact role of renal stem cells and their functional limitations and physiological niche are all subjects that are still being investigated.

Other work has shown that bone marrow stem cells can contribute to kidney repair after kidney injury (see Park HC, et al., Am J Physiol Renal Physiol 2010, 298:F1254–F1262; Cheng Z, et al., Mol Ther 2008, 16:571–579; and Qian H, et al., Int J Mol Med 2008, 22:325–332). It is unclear however, if bone marrow stem cells can trans-differentiate into renal stem cells.

To this end, a Chinese group has examined if bone marrow stem cells can actually trans-differentiate into renal stem cells after acute kidney injury. This work resulted from collaboration between the laboratories of Yong Xu at the Urology department at the Second Hospital of Tianjin Medical University, in Tianjin, China, and Zongjin Li at the School of Medicine at Nankai University in Tianjin, China.

In this study, workers from Xu’s and Li’s laboratories transplanted bone marrow stem cells from mice that expressed a glowing protein in their cells into mice that had been subjected to radiation. Radiation treatment wipes out the bone marrow of the mouse, and the transplantation reconstitutes the bone marrow. Therefore, the mice that were treated with radiation now have bone marrow stem cells that glow in the dark and anywhere those cells go, they will be traceable.

Once it was clear that the irradiated mice that had received the bone marrow transplantations had normal blood work (5 weeks later), their kidneys were subjected to acute damage by being deprived of sufficient blood flow for a short period of time. Four weeks later, the kidneys of these animals were examined in order to determine if the transplanted bone marrow stem cells had migrated to the kidneys to help heal them. A second experiment utilized a small protein called a “cytokine,” which acts as a powerful signal to stem cells. This particular cytokine, granulocyte colony stimulating factor (G-CSF), mobilizes stem cells from bone marrow such that the bone marrow stem cells move from their comfortable, leisurely existence to the bloodstream where they can go to help heal other tissues. By giving some of the transplanted mice doses of G-CSF, Xu and Li and their co-workers were able to determine if the bone marrow stem cells moved from the bone marrow to the kidney to take up residence in the kidney as the new renal stem cell population.

The results clearly showed that bone marrow stem cells moved from the bone marrow to the kidney to participate in kidney healing. However, it did not end there. These same labeled, glowing bone marrow stem cells expressed the proteins normally found in resident renal stem cells. While these bone marrow stem cells only constituted a small proportion of the renal stem cell population, they were clearly a part of the Sca-1+ or c-Kit+ renal progenitor cell population. Secondly, treatment with G-CSF almost doubled the frequency of bone marrow-derived renal stem cells in the kidney. G-CSF treatment also increased the capillary density in the injured kidney, which is significant, because bone marrow stem cells are rich in a population of blood vessel-making stem cells. Furthermore, the new blood vessels all glowed in the dark, which shows that they were made by the bone marrow-derived stem cells that moved to the kidney are contributed to the resident renal stem cell population that participated in kidney repair.

Thus, these data in this study establish that stem cells from bone marrow can trans-differentiate into cells that share many of the properties of renal resident stem cells. Furthermore, mobilization of these stem cells with cytokines like G-CSF mobilization can enhance the healing effects of these cells and might provide the basis for a new therapeutic strategy for end-stage renal disease or chronic kidney disease.

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.