UC Davis Stem Cell Scientists Make Bladder Cells from Pluripotent Stem Cells


Patients who suffer from malformation of the spinal cord or have suffered a severe spinal cord injury sometimes have bladder malfunction as well. Replacing a poorly functioning bladder is a goal of regenerative medicine, but it is not an easy goal. The bladder is lined with a special cell population called “urothelium.” Urothelium is found throughout the urinary tract and it is highly elastic. Persuading stem cells to form a proper urothelium has proved difficult.

Urothelium
From http://ocw.tufts.edu/data/4/221158/221174_xlarge.jpg

Now scientists from the University of California, Davis (my alma mater), have succeeded in devising a protocol for differentiating human pluripotent stem cells into urothelium. The laboratory of Eric Kurzock, chief of the division of pediatric urologic surgery at UC Davis Children’s Hospital, published this work in the journal Stem Cells Translational Medicine. This work is quite exciting, since it provides a way to potentially replace bladder tissue for patients whose bladders are too small or do not function properly.

Kurzock explained: “Our goal is to use human stem cells to regenerate tissue in the lab that can be transplanted into patients to augment or replace their malfunctioning bladders,”

In order to make bladder cells in the laboratory, Kurzrock and his coworkers used two different types of human pluripotent stem cells. First, they used two types of induced pluripotent stem cells (iPS cells). The first came from laboratory cultures of human skin cells that were genetically engineered and cultured to form iPS cultures. The second iPS line was derived from umbilical cord blood cells that had been genetically reprogrammed into an embryonic stem cell-like state.

Even though further work is needed to establish that bladder tissues made from such stem cells are safe or effective for human patients, Kurzrock thinks that iPS cell–derived bladder grafts made from a from a patient’s own skin or umbilical cord blood cells represent the ideal tissue source for regenerative bladder treatments. This type of tissue would be optimal, he said, because it lowers the risk of immunological rejection that typifies most transplants.

One of the truly milestone developments in this research is the protocol Kurzrock and his colleagues developed to direct pluripotent stem cells to differentiate into bladder cells. This protocol was efficient and, most importantly, allowed the stem cells to proliferate in culture over a long period of time. This is crucial in order to have enough material for therapeutic purposes.

“What’s exciting about this discovery is that it also opens up an array of opportunities using pluripotent cells,” said Jan Nolta, professor and director of the UC Davis Stem Cell program and a co-author on the new study. “When we can reliably direct and differentiate pluripotent stem cells, we have more options to develop new and effective regenerative medicine therapies. The protocols we used to create bladder tissue also provide insight into other types of tissue regeneration.”

To hone their urothelium-differentiation protocol, Kurzrock and his colleagues used human embryonic stem cells obtained from the National Institutes of Health’s human stem cell repository. These cells were successfully differentiated into bladder cells. Afterwards, the Kurzrock group used the same protocol to coax iPS cells made from skin and umbilical cord blood into urothelium. Not only did these cells look like urothelium, but they also expressed the protein “uroplakin,” which is unique to the bladder and helps make it impermeable to toxins in urine.

In order to bring this protocol to the clinic, the cells must proliferate, differentiate and express bladder-specific proteins without depending on any animal or human products. They must do all these things independent of signals from other human cells, said Kurzrock. Therefore, for future research, Kurzrock and his colleagues plan to modify their laboratory cultures so that they will not require any animal and human products, which will allow use of the cells in patients.

Kurzrock’s primary goal as a physician is with children who suffer from spina bifida and other pediatric congenital disorders. Currently, when he surgically reconstructs a child’s defective bladder, he must use a segment of their own intestine. Because the function of intestine, which absorbs food, is almost the opposite of bladder, bladder reconstruction with intestinal tissue may lead to serious complications, including urinary stone formation, electrolyte abnormalities and cancer. According to Kurzrock, developing a stem cell alternative not only will be less invasive, but should prove to be more effective, too, he said.

Another patient group who might benefit from this research is bladder cancer patients. More than 70,000 Americans each year are diagnosed with bladder cancer, according to the National Cancer Institute. “Our study may provide important data for basic research in determining the deviations from normal biological processes that trigger malignancies in developing bladder cells,” said Nolta. More than 90 percent of patients who need replacement bladder tissue are adults with bladder cancer. Kurzrock said “cells from these patients’ bladders cannot be used to generate tissue grafts because the implanted tissue could carry a high risk of becoming cancerous. On the other hand, using bladder cells derived from patients’ skin may alleviate that risk. Our next experiments will seek to prove that these cells are safer.”

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.