Australian Researchers Make A Kidney in the Laboratory With Stem Cells


Stem cell researchers from the University of Queensland in Australia have successfully grown a kidney in the laboratory with stem cells. This new breakthrough will almost certainly open the door to improved treatments for patients with kidney disease, and bodes well for the future of organ bioengineering.

Mini-kidney in dish. (Source: University of Queensland)
Mini-kidney in dish. (Source: University of Queensland)

The principal investigator of this research project, Professor Melissa Little, from University of Queensland’s Institute for Molecular Bioscience (IMB), said that new treatments for kidney disease were urgently needed.

“One in three Australians is at risk of developing chronic kidney disease and the only therapies currently available are kidney transplant and dialysis,” Little said. “Only one in four patients will receive a donated organ, and dialysis is an ongoing and restrictive treatment regime. We need to improve outcomes for patients with this debilitating condition, which costs Australia $1.8 billion a year.”

Little’s research team designed a new step-wise protocol to coax embryonic stem cells to gradually form all the required kidney-specific cell types and to induce them to “self-organize” into a mini-kidney in a dish.  The embryonic stem cell line HES3 was used in this work, which derived by Reubinoff and others in the laboratory of Alan Trounson in 2000.

“During self-organization, different types of cells arrange themselves with respect to each other to create the complex structures that exist within an organ, in this case, the kidney,” Little said. “The fact that such stem cell populations can undergo self-organization in the laboratory bodes well for the future of tissue bioengineering to replace damaged and diseased organs and tissues. It may also act as a powerful tool to identify drug candidates that may be harmful to the kidney before these reach clinical trial.”

Despite the success of this research, Little cautioned that she and other kidney researchers had a great deal of work to do to before this protocol might be ready for human trials. Regardless, it is a very exciting step forward.

The Queensland Minister for Science and Innovation Ian Walker congratulated Little and her co-workers for their advances, and added that biomedical research was crucial in ensuring a healthier future for Queenslanders.

“The work by the IMB research team is an important milestone in developing improved treatments for chronic kidney disease and will ensure those with the condition can continue to live fulfilling and productive lives,” Walker said.

Little’s research team included Dr. Minoru Takasato, Pei Er, Melissa Becroft, Dr. Jessica Vanslambrouck, from IMB, and her collaorators, Professors Andrew Elefanty and Ed Stanley, from the Murdoch Children’s Research Institute and Monash University.

The research is published in the scientific journal Nature Cell Biology and supported by the Queensland Government, the Australian Research Council, as part of the Stem Cells Australia Strategic Research Initiative, and the National Health and Medical Research Council of Australia.

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.

FGF Proteins Maintain Stem Cells in Developing Kidney


The digestion of food and other metabolic processes necessary for the maintenance of our bodies produce waste products that must be routinely removed. Without proper removal of these metabolic waste products, they will build up in concentration and poison the organs and tissues. In the human body, two organs work to dispose of wastes. The liver processes wastes in order to make them more water-soluble. Waste products that are not water soluble are excreted into the bile. Bile is made by the liver and stored in the gall bladder. When we eat fat-soluble food molecules, bile helps solubilize the fatty materials so that enzymes can degrade them. Bile is also used to dump things the body no longer wants into the intestine so that they can be excreted rom the body. The second organ that excreted water-soluble materials is the kidneys. The kidneys filter the blood and concentrate the waste products into a fluid called urine. Urine is excreted by means of the urinary tract.

Kidneys are composed of 800,000 to 1.5 million tiny units called nephrons. Nephrons are small structures that are composed of a series of tubes. The front end of the nephron is inflated into a cup-shaped structure that is fed by a knot of tiny blood vessels. The walls of these blood vessels is very porous and the fluid of the blood and many things dissolved in it move from the blood vessels into the nephron, The long tubules of the nephron recapture water and precious ions while leaving the wastes. Eventually the concentrated fluid, urine, is deposited into a tube called the ureter that connects the kidneys to the urinary bladder.

In the United States, 10% of the population is afflicted with some kind of chronic kidney disease. Regenerative treatments for the kidneys are in their infancy. The developing kidney contains a robust stem cell population, but soon after birth, the stem cells form nephrons and are essentially all used up. However, researchers at Washington University School of Medicine in St. Louis, Missouri have identified two proteins in mice and humans that are required to maintain a supply of stem cells in the developing kidney. Manipulation of these proteins might provide a means to maintain or even renew a stem cell population in the kidney.

These two proteins, Fibroblast Growth Factor-9 (FGF-9) and Fibroblast Growth Factor-20 (FGF-20), allow mouse kidney stem cells to survive in culture longer than previously reported. Though the cells were maintained only five days (up from about two), this work is a small step toward the future goal of growing kidney stem cells in the lab.

In the developing embryo, these early stem cells give rise to adult cells called nephrons, the blood filtration units of the kidneys. Raphael Kopan from Washington University said, “When we are born, we get a certain allotment of nephrons. Fortunately, we have a large surplus. We can donate a kidney – give away 50 percent of our nephrons – and still do fine. But, unlike our skin and gut, our kidneys can’t build new nephrons.”

Herein lies the reason why regenerative medicine in the kidney is behind other tissues: the skin and the gut have small pools of local stem cells that continually renew these organs throughout life. A term used by scientists for such a pool of local stem cells that support a system is a “stem cell niche.” During early development, the embryonic kidney has a stem cell niche, but later in development, possibly shortly before or shortly after birth; all stem cells in the kidney differentiate to form nephrons. This leaves no stem cell niche.

According to Kopan: “In other organs, there are cells that specifically form the niche, supporting the stem cells in a protected environment. But in the embryonic kidney, it seems the stem cells form their own niche, making it a bit more fragile. And the signals and conditions that lead the cells to form this niche have been elusive.”

David M. Ornitz and his laboratory provide a surprising clue to the signals that maintain the embryonic kidney’s stem cell niche. Earlier this year, Ornitz, who studies FGF signaling during the development of the mouse ear, published a paper showing that FGF20 plays an important role in inner ear development. However, even though mice that lack FGF20 “are profoundly deaf,” explained Ornitz, “they are otherwise viable and healthy,” but “in some cases we noticed that their kidneys looked small.”

Past work from his own lab and others suggested that FGF9, a closely-related cousin of FGF20, might also participate in kidney development. In general, members of the FGF family are known to play important and broad roles in embryonic development, tissue maintenance, and wound healing. Mice that lack FGF9 have defects in development of the male urogenital tract and die after birth due to defects in lung development.

Since FGF9 and 20 are so closely related, Ornitz and Sung-Ho Huh, Ph.D., a postdoctoral research associate, and former postdoctoral researcher Hila Barak, Ph.D., thought the two proteins might serve overlapping, redundant functions in the developing kidney. Essentially, the two proteins might compensate for each other if one is missing. The next logical question, according to Ornitz, was what happens when both are missing.

The results confirmed these hypotheses. “When we examined mice lacking both FGF9 and FGF20, we saw that the embryos had no kidneys,” Ornitz said.

In humans, the role of these proteins came from genetic studies in families that showed a marked propensity for their children to have kidney problems. Collaborators in France identified two families that lost more than one pregnancy because the fetuses developed without kidneys (renal agenesis).

Analyses of DNA from the fetuses that had died from renal agenesis showed mutations in four genes known to be involved in kidney development. One of these mutations resulted in the total loss of FGF20. This analysis confirmed the mouse studies, but also underscored an important difference between humans and mice: in mice, FGF9 appears to compensate for a total loss of FGF20, but in humans, fetuses that develop without kidneys had normal FGF9. This suggests that the two genes do not completely compensate for each other in human kidney development.

Now that at least some of the more crucial proteins necessary to support the kidney stem cell niche have been identified, Kopan and Ornitz both look to future work to further extend the life of such cells in the lab. Kopan noted, “The holy grail would be to deliver these cells back to a diseased kidney,” Kopan said. “This is a very small step. But we hope this will be a stimulus to the field, for us and for others to continue thinking about how to convince these cells to stick around longer.”