Mesenchymal Stem Cells Assist Kidney Transplants in Cats


Dr. Chad Schmiedt, a veterinary surgeon from the University of Georgia (UGA) Veterinary Teaching Hospital, and his colleagues have used mesenchymal stem cells from the fat of cats to optimize the acceptance of a new kidney in cats.

The recipient of this kidney transplant was a four-year-old flame point Siamese male cat named Arthur. Arthur’s owners brought him from Virginia to the University of Georgia after he was diagnosed with chronic renal failure about a year ago. Two other veterinary hospitals declined to operate on Arthur, since they did not deem this cat an optimal candidate for a kidney transplant. As it turns out, Arthur has trouble absorbing cyclosporine, which is the anti-rejection drug used to prevent the recipient of the kidney transplant from rejecting it.

Arthur
Arthur

In his initial consultation with Arthur’s owners, Schmiedt had the idea of using adult feline stem cells as a part of Arthur’s immunosuppressive protocol. There was precedent for this, since a cat that was operated on at University of Georgia Veterinary Teaching Hospital in 2013 had received a kidney transplant with doses of its own mesenchymal stem cells to prevent rejection of the transplanted organ. This cat was doing well one year after surgery.

“To the best of my knowledge, UGA is the only veterinary facility in the world to use adult stem cells in feline kidney transplantation,” said Schmiedt, who actually heads UGA’s feline kidney transplant program.”

Schmiedt continued: “We used feline adult stem cells in one other transplant that we did last year. A study published in 2012 found that the use of MSCs during renal transplant surgery i humans lowered the risk of acute organ rejection, decreased the risk of infection, and the patients had better estimated renal function one year after surgery.”

Mesenchymal stem cells can be harvested fat, bone marrow, and umbilical cord or placenta. Before the transplant surgery, Schmiedt isolated mesenchymal stem cells (MSCs) from Arthur’s fat and the UGA Regenerative Medicine Service grew the stem cells from the fat sample for use in Arthur after his surgery.

Arthur has his kidney transplant on May 15, 2014. The first surgery harvests a kidney from the donor cat (named Joey) and the second surgery transplants the donated kidney into Arthur. The UGA transplant program for cats requires that the donor cat be adopted by the recipient family’s family, which means that Joey and Arthur will become lifelong playmates.

“Cat owners who seek kidney transplants for their sick cats have to be very dedicated,” said Schmiedt. “They will give their car medication twice a day for the rest of its life. They also must be willing to take their cats to the veterinarian for frequent check-ups… a significant amount of time and expense is involved in keeping the recipient and donor cats healthy. But cat lovers who will go to this extent are willing to extend this kind of care to all cats they own.”

Apparently, Joey will be joined by Arthur and five other felines as well.

Stem cells do not replace the need for antirejection medication, and since Arthur’s body poorly absorbs cyclosporine, he will need to take a second antirejection drug as well called mycophenolate. Schmiedt, however, and his colleague stem cell scientist Dr. John Peroni sees MSCs making an important contribution to transplant medicine: “MSCs in veterinary species have been primarily used to treat musculo-skeletal injury – problems with bones, tendons, and joints – and those are our most frequent uses here at the UGA College of Veterinary Medicine. But there is good evidence to support using stem cells to modulate the immune system and regulate inflammation. So, the transplant setting might be another optimal use for these types of stem cells.”

In order to access the efficacy of MSCs in a transplant setting, controlled studies must be done. It is clear that transplanted MSCs do not improve kidney function, but they do seem to slow down the progression of kidney disease. Schmiedt thinks that benefits to patients are possible: “The only down side is harvesting the cells seven to 10 days ahead of the surgery, which adds to the cost of transplant procedure.”

Adaptation of this procedure to animals could smooth the path to making this procedure readily available in humans as well.

Amniotic Fluid Stem Cells Aid Kidney Transplantation Success in a Pig Model


When a kidney patient receives a new kidney, the donated kidney undergoes a brief loss of blood supply followed by a restoration of the blood supply. This phenomenon is called ischemia/reperfusion (IR), and IR tends to cause cell death, followed by rather extensive scarring. Tissue scarring is called tissue fibrosis and a scarred kidney can lead to so-called transplant dysfunction, which means that the transplanted kidney does not work terrible well, and this can cause transplant failure.

Previous studies in laboratory rodents have shown that mesenchymal stem cells from amniotic fluid (afMSCs) are beneficial in protecting against transplant-induced fibrosis (Perin L, et al. PLoS One 2010;5:e9357; Hauser PV, et al. Am J Pathol 2010;177:2011-2021).

Now a research group at INSERM, France led by Thierry Hauet has developed a pig-based model of kidney autotransplantation that is comparable to the human situation with regards to the structure of the kidney and the damage that results from renal ischemia (for papers, see Jayle C, et al. Am J Physiol Renal Physiol 2007; 292: F1082-1093; and Rossard L, et al. Curr Mol Med 2012; 12: 502-505). On the strength of these previous experiments, Hauet’s group has published a new paper in Stem Cells Translational Medicine in which they report that porcine afMSCs can protect against IR-related kidney injuries in pigs.

Hauet and others showed that porcine afMSCs could be easily collected at birth and cultured. These cells showed the ability to differentiate into fat, and bone cells, made many of the same cell surface markers as other types of mesenchymal stem cells (e.g., CD90, CD73, CD44, and CD29), but showed a diminished ability to differentiate into blood vessel cells. When afMSCs are added to extirpated kidneys during the reperfusion (reoxygenation) process in an “in vitro” (fancy way of saying “in a culture dish”) model of organ-preservation, these stem cells significantly increased the survival of blood vessel (endothelial) cells. Endothelial cells are one of the main targets of ischemic injury, and the added cells bucked up these endothelial cells and rescued them from programmed cell death. In addition to these successes, Hauet and others showed that adding intact porcine afMSCs was not necessary, since addition of the culture medium used to grow the afMSCs (conditioned medium or CM) also rescued kidney endothelial cell death. The afMSC-treated kidneys survived because they had significantly larger numbers of blood vessels, and this seems to be the main factor that causes the extirpated kidney to survive intact.

While these experiments were successful, Hauet and others know that unless they were able to show that these cells improved kidney transplant outcomes in a living animal, their research would not be deemed clinically relevant. Therefore, Hauet and others injected afMSCs into the renal artery of pigs that had received a kidney transplant six days after the transplant. IR injuries following kidney transplants led to increased serum creatinine levels, but those pigs that had been infused with afMSCs showed reduced creatinine levels and lower protein levels in their urine (proteinuria). In fact, seven days after the stem cell infusion, the urine creatinine and protein levels had returned to pre-transplant levels. Three months after the transplant, the pigs were put down, and then the kidneys were subjected to tissue analyses. Microscopic examination of tissue slices from these kidneys showed that afMSC injection preserved the structural integrity of microscopic details of the kidneys and reduced the signs of inflammation. Control animals that were not treated with afMSCs showed disruption of the microscopic structures of the kidneys and extensive inflammation and scarring. Also, because the kidney controls blood chemistry, a comparison of the blood chemistries of these two groups of animals showed that the blood chemistries of the afMSC-treated animals were normal as opposed to the control animals.

Amniotic Fluid Stem Cells Aid Kidney Transplantation in Porcine Model

Molecular analyses also showed a whole host of pro-blood vessel molecules in the kidneys of the afMSC-treated pigs. VEGFA (pro-angiogenic growth factor), and Ang1 (capillary structure strengthening and maintenance of vessel stability), proteins were increased in the kidneys of afMSC animals compared to control animals. Thus the infused stem cells increased the expression of pro-blood vessel molecules, which led to the formation of larger quantities of blood vessels, reduced cell death and decreased inflammation.

These findings demonstrate the beneficial effects of infused afMSCs on kidney transplant. Since afMSCs are easy to isolate and grow in culture, secrete proangiogenic and growth factors, and can differentiate into many cell lineages, including renal cells (see Perin L, et al. Cell Prolif 2007; 40: 936-948; De Coppi P, et al. Nat Biotechnol 2007; 25: 100-106; and In ‘t Anker PS, et al. Stem Cells 2004;22:1338-1345). This makes these cells a viable candidate for clinical application. This study also highlights pigs as a preclinical model as a powerful tool in the assessment of stem cell-based therapies in organ transplantation.

Kidney Tubular Cells Formed from Stem Cells


A collaborative effort between several research teams has successfully directed stem cells to differentiate into kidney tubular cells. This is a significant advance that could hasten the day when stem cell-based treatments are used to treat kidney failure.

Chronic kidney disease is a major global public health problem. Unfortunately, once patients progress to kidney failure, their treatment options are limited to dialysis and kidney transplantation. Regenerative medicine, whose goal is to rebuild or repair tissues and organs, might offer a promising alternative.

A team of researchers from the Harvard Stem Cell Institute (Cambridge, Mass.), Brigham and Women’s Hospital (Boston) and Keio University School of Medicine (Tokyo) that included Albert Lam, M.D., Benjamin Freedman, Ph.D. and Ryuji Morizane, M.D., Ph.D., has been diligently developing strategies for the past five years to develop strategies to direct human pluripotent stem cells (human embryonic stem cells or hESCs and human induced pluripotent stem cells or iPSCs) to differentiate into kidney cells for the purposes of kidney regeneration.

“Our goal was to develop a simple, efficient and reproducible method of differentiating human pluripotent stem cells into cells of the intermediate mesoderm, the earliest tissue in the developing embryo that is fated to give rise to the kidneys,” said Dr. Lam. Lam also noted that these intermediate mesoderm cells would be the “starting blocks” for deriving more specific kidney cells.

Lam and his collaborators discovered a blend of chemicals which, when added to stem cells in a precise sequence, caused the stem cells to turn off their stem cell-specific genes and activate those genes found in kidney cells. Furthermore, the activation of the kidney-specific genes occurred in the same order that they turn on during embryonic kidney development.

At E10.5, the metanephric mesenchyme (red) comprises a unique subpopulation of the nephrogenic cord (yellow). Expression of the Glial-derived neurotrophic factor (Gdnf) is resticted to the metanephric mesenchyme by the actions of transcriptional activators, secreted factors, and inhibitors. GDNF binds the Ret receptor and promotes the formation of the ureteric bud, an outgrowth from the nephric duct (blue). Ret initially depends upon the Gata3 transcription factor for its expression in the nephric duct. Spry1 acts as an intracellular inhibitor of the Ret signal transduction pathway. BMP4 inhibits GDNF signaling and is in turn inhibited by the Grem1 binding protein. At 11.5, the ureteric bud has branched, forming a T-shaped structure. Each ureteric bud tip is surrounded by a cap of condensed metanephric mesenchyme. Reciprocal signaling between the cap mesenchyme and ureteric bud, as well as signals coming from stromal cells (red), maintain expression of Ret in the bud tips and Gdnf in the cap mesenchyme. Nephrons are derived from cap mesenchyme cells that form pretubular aggregates and then renal vesicles on either side of each ureteric bud tip. Wnt9b and Wnt4 induce nephron formation and are necessary for maintaining ureteric bud branching. The Six2 transcription factor prevents ectopic nephron formation. BMP7 promotes survival of the cap mesenchyme. Not all genes implicated in metanephros formation are shown for clarity (see text for further details). Green arrows indicate the ligand-receptor interaction between GDNF and Ret. Black arrows indicate the epistasis between genes but in most cases it is not known if the interactions are direct. T-shaped symbols indicate inhibitory interactions.
At E10.5, the metanephric mesenchyme (red) comprises a unique subpopulation of the nephrogenic cord (yellow). Expression of the Glial-derived neurotrophic factor (Gdnf) is resticted to the metanephric mesenchyme by the actions of transcriptional activators, secreted factors, and inhibitors. GDNF binds the Ret receptor and promotes the formation of the ureteric bud, an outgrowth from the nephric duct (blue). Ret initially depends upon the Gata3 transcription factor for its expression in the nephric duct. Spry1 acts as an intracellular inhibitor of the Ret signal transduction pathway. BMP4 inhibits GDNF signaling and is in turn inhibited by the Grem1 binding protein. At 11.5, the ureteric bud has branched, forming a T-shaped structure. Each ureteric bud tip is surrounded by a cap of condensed metanephric mesenchyme. Reciprocal signaling between the cap mesenchyme and ureteric bud, as well as signals coming from stromal cells (red), maintain expression of Ret in the bud tips and Gdnf in the cap mesenchyme. Nephrons are derived from cap mesenchyme cells that form pretubular aggregates and then renal vesicles on either side of each ureteric bud tip. Wnt9b and Wnt4 induce nephron formation and are necessary for maintaining ureteric bud branching. The Six2 transcription factor prevents ectopic nephron formation. BMP7 promotes survival of the cap mesenchyme. Not all genes implicated in metanephros formation are shown for clarity (see text for further details). Green arrows indicate the ligand-receptor interaction between GDNF and Ret. Black arrows indicate the epistasis between genes but in most cases it is not known if the interactions are direct. T-shaped symbols indicate inhibitory interactions.

The investigators were able to differentiate both hESCs and human iPSCs into cells that expressed the PAX2 and LHX1 genes, which are two key elements of the intermediate mesoderm; the developmental tissue from which the kidney develops. The iPSCs were derived by reprogramming fibroblasts obtained from adult skin biopsies into pluripotent cells. The differentiated cells expressed multiple genes found in intermediate mesoderm and spontaneously produced tubular structures that expressed those genes found in mature kidney tubules.

The researchers could then differentiate the intermediate mesoderm cells into kidney precursor cells that expressed the SIX2, SALL1 and WT1 genes. These three genes designate an embryonic tissue called the “metanephric cap mesenchyme.” Metanephric cap mesenchyme is a critical tissue for kidney differentiation. During kidney development, the metanephric cap mesenchyme contains a population of progenitor cells that give rise to nearly all of the epithelial cells of the kidney (epithelial cells or cells in a sheet, generate the lion’s share of the tubules of the kidney).

Metanephric cap mesenchyme is is red
Metanephric cap mesenchyme is is red

The cells also continued to behave like kidney cells when transplanted into adult or embryonic mouse kidneys. This gives further hope that these investigators might one day be able to create kidney tissues that could function in a patient and would be fully compatible with the patient’s immune system.

The findings are published online in Journal of the American Society of Nephrology.

Stem Cells Allow Kidney Transplant Recipients to Live Without Anti-Rejection Drugs


Researchers from Northwestern Medicine And University of Louisville are in the midst of a clinical trial to examine the use of stem cell infusions to re-educate the immune system of recipients of transplanted organs. Such re-education of the immune system might completely eliminate the need for anti-rejection medicines.

Organ transplant recipient must take several pills each day for the remainder of their lives. These medicines are drugs that suppress the immune system, and these drugs have many undesirable side effects. Prolonged use of these drugs can cause high blood pressure, diabetes, infections, heart disease, and cancer. Therefore a stem cell-based approach that obviates the need for drugs that inhibit the immune system would offer transplant recipients better quality of life and few health risks for transplant patients.

Joseph Leventhal, a transplant surgeon at Northwestern Memorial Hospital said, “The preliminary results are exciting and may have a major impact on organ transplantation in the future. With refinement, this approach may prove to be applicable to the majority of patients receiving the full spectrum of solid organ transplants.” Leventhal is the main author of this study in collaboration with Suzanne Ildstad, who is the director of the Institute of Cellular Therapeutics at the University of Louisville. The study is, in fact, one of the first of its kind, since it does not require that the organ donor and recipient do not have to be tissue matched.

For standard kidney transplants, the organ donor, who has agreed to donate a kidney, provides their kidney for transplantation to the recipient. In this study, the organ donor not only provides a kidney, but also a small quantity of blood cells. Approximately one month before the transplant, the organ donor gives some bone marrow by means of a procedure called “apheresis.”

Apheresis removes whole blood from a patient, and then uses a centrifuge-like instrument to separate blood components. These separated portions are removed and the remaining components used for retransfusion. The blood components are separated into fluids, otherwise known as plasma, platelets, and white blood cells. From the white cell fraction, a group of cells that the study cells “facilitating cells” are isolated. The organ recipient’s bone marrow is partially ablated with radiation.

The kidney is then transplanted into the recipient’s body, and one day later, the facilitating cells are given to the recipient. Because the organ recipient’s bone marrow has been semi-ablated, the facilitating cells have space to grow without competition from the recipient’s bone marrow. The goal of this is to make within the recipient two bone marrow stem cell systems that are completely functional in one person. The patient is given anti-immune system drugs, but he or she is slowly weaned off them, with the goal of all anti-rejection drugs being ended within one year of the transplant. To qualify for this study, patients must have compatible blood types

Ildstad provided this insight, “This is something I have worked for my entire life.”  Ildstad pioneered the discovery of the “facilitating cell.”  This trial is ongoing, but the initial results are immensely encouraging, since some transplant patients seem to not need their anti-rejection medicines anymore even though they now have a kidney inside them that was not tissue matched.  Specifically, five of eight people who underwent this treatment protocol were able to stop all immunosuppressive therapy within a year after their kidney and stem-cell transplants,. Note that four of these five patients received kidneys that came from unrelated donors. Notably, all of these patients maintained entirely donor-derived immune systems with no signs of Graft-versus-Host disease.  Ildstad and her team have since treated seven more people. “We continue to see good results,” she says. This could easily revolutionize solid organ transplantation.