Positive Results from Mesoblast’s Phase 2 Trial of Cell Therapy in Diabetic Kidney Disease


Mesoblast Limited has announced results from its Phase 2 clinical Trial that evaluated their Mesenchymal Precursor Cell (MPC) product, known as MPC-300-IV, in patients who suffer from diabetic kidney disease. In short, their cell product was shown to be both safe and effective. The results of their trial were published in the peer-reviewed journal EBioMedicine.  Researchers from the University of Melbourne, Epworth Medical Centre and Monash Medical Centre in Australia participated in this study.

The paper describes a randomized, placebo-controlled, and dose-escalation study that administered to patients with type 2 diabetic nephropathy either a single intravenous infusion of MPC-300-IV or a placebo.

All patients suffered from moderate to severe renal impairment (stage 3b-4 chronic kidney disease for those who are interested).  All patients were taking standard pharmacological agents that are typically prescribed to patients with diabetic nephropathy.  Such drugs include angiotensin-converting enzyme inhibitors (e.g., lisinopril, captopril, ramipril, enalapril, fosinopril, ect.) or angiotensin II receptor blockers (e.g., irbesartan, telmisartan, losartan, valsartan, candesartan, etc.).  A total of 30 patients were randomized to receive either a single infusion of 150 million MPCs, or 300 million MPCs, or saline control in addition to maximal therapy.

Since this was a phase 2 clinical trial, the objectives of the study were to evaluate the safety of this treatment and to examine the efficacy of MPC-300-IV treatment on renal function.  For kidney function, a physiological parameter called the “glomerular filtration rate” or GFR is a crucial indicator of kidney health.  The GFR essentially indicates how well the individual functional units within the kidney, known as “nephrons,” are working.  The GFR indicates how well the blood is filtered by the kidneys, which is one way to measure remaining kidney function.  The decline or change in glomerular filtration rate (GFR) is thought to be an adequate indicator of kidney function, according to the 2012 joint workshop held by the United States Food and Drug Administration and the National Kidney Foundation.

nephronanatomy

Diabetic nephropathy is an important disease for global health, since it is the single leading cause of end-stage kidney disease.  Diabetic nephropathy accounts for almost half of all end-stage kidney disease cases in the United States and over 40% of new patients entering dialysis treatment.  For example, there are almost 2 million cases of moderate to severe diabetic nephropathy in 2013.

Diabetic nephropathy can even occur in patients whose diabetes is well controlled – those patients who manage to keep their blood glucose levels at a reasonable level.  In the case of diabetic nephropathy, chronic infiltration of the kidneys by inflammatory monocytes that secrete pro-inflammatory cytokines causes endothelial dysfunction and fibrosis in the kidney.

Staging of chronic kidney disease (CKD) is based on GFR levels.  GFR decline typically defines the progression to kidney failure (for example, stage 5, GFR<15ml/min/1.73m2).  The current standard of care (renin-angiotensin system inhibition with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers) only delays the progression to kidney failure by 16-25%, which leaves a large residual risk for end-stage kidney disease.  For patients with end-stage kidney disease, the only treatment option is renal replacement (dialysis or kidney transplantation), which incurs high medical costs and substantial disruptions to a normal lifestyle.  Due to a severe shortage of kidneys, in 2012 approximately 92,000 persons in the United States died while on the transplant list.  For those on dialysis, the mortality rate is high with an approximately 40% fatality rate within two years.

The main results of this clinical trial were that the safety profile for MPC-300-IV treatment was similar to placebo.  There were no treatment-related adverse events.  Secondly, patients who received a single MPC infusion at either dose had improved renal function compared to placebo, as defined by preservation or improvement in GFR 12 weeks after treatment.  Third, the rate of decline in estimated GFR at 12 weeks was significantly reduced in those patients who received a single dose of 150 million MPCs relative to the placebo group (p=0.05).  Finally, there was a trend toward more pronounced treatment effects relative to placebo in a pre-specified subgroup of patients whose GFRs were lower than 30 ml/min/1.73m2 at baseline (p=0.07).  In other words, the worse the patients were at the start of the trial, the better they responded to the treatment.

The lead author of this publication, Dr David Packham, Associate Professor in the Department of Medicine at the University of Melbourne and Director of the Melbourne Renal Research Group, said: “The efficacy signal observed with respect to preservation or improvement in GFR is exciting, especially given that this trial was not powered to show statistical significance. Patients receiving a single infusion of MPC-300-IV showed no evidence of developing an immune response to the administered cells, suggesting that repeat administration is feasible and may in the longer term be able to halt or even reverse progressive chronic kidney disease. I hope that this very promising investigational therapy will be advanced to rigorous Phase 3 clinical trials to test this hypothesis as soon as possible.”

Patients who received s single IV infusion of MPC-300-IV cells showed no evidence of developing an immune response to the administered cells.  This suggests that repeated administration of MPCs is feasible and might even have the ability to halt, or even reverse progressive chronic kidney disease.

Packham and his colleagues hope that this cell-based therapy can be advanced to a rigorous Phase 3 clinical trial to further test this treatment.

Stem Cells from Abdominal Fat Helps Fight Kidney Disease


Researchers from Chicago, Illinois have shown that a fatty fold of tissue within the abdomen contains a rich source of stem cells that can help heal diseased kidneys.

Scientists from the laboratory of Ashok K. Singh at Hospital of Cook County used a rat model of chronic kidney disease to examined the efficacy of these cells.

In past experiments, transplanted stem cells have failed to live very long in the body of the recipient. To solve this problem, Singh and his co-workers connected the a fatty fold of tissue located close to the kidney called the “omentum” to the kidney. The omentum is a wonderfully rich source of stem cells and by connecting the kidney to the omentum, Singh and his colleagues subjected the diseased kidney to a constant supply of stem cells.

Omentum

After 12 weeks of being connected to the kidney, the kidney showed significant signs of improvement.

The progression of chronic kidney disease was slowed due to this continuous migration of stem cells from the omentum to the diseased kidney. The influx of these stem cells seemed to direct healing of the kidney.

This experiment is significant in that it suggests that resident stem cells that facilitate healing of the kidney, but only when they are in contact with the tissue over a long period of time. Also, it implies that a supposedly useless organ that lies close to the kidney can be fused with the kidney to heal it with a patient’s own stem cells. This therapeutic strategy seems to be ideal for kidney patients.

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.

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.

A New Way to Treat Kidney Disease and Heart Failure


St. Michael’s Hospital in Toronto, Ontario is the site of new research that uses bone marrow stem cells to treat chronic kidney disease and heart failure in rats.

Darren Yuen and Richard Gilbert of St. Michael’s Hospital were the first to show in 2010 that enriched stem cells improved heart and kidney functions in rats afflicted with both diseases. Their work generated concerns about the side effects of returning such stem cells to the body.

Since 2010, Yuen and Gilbert have found that enriched bone marrow stem cells secrete stromal cell–derived factor-1α (SDF-1α), a chemokine that is made by ischemic tissue but is rapidly degraded by dipeptidyl peptidase-4 (DPP-4), in culture dishes.  Injection of SDF-1α into rats has many of the same positive effects as when the stem cells themselves are injected into rats.  Even more remarkably, if a drug that inhibits the enzyme DPP-4 is given (sitagliptin) produced many improvements as well.

“We’ve shown that we can use these ‘hormones’ to replicate the beneficial effects of the stem cells in treating animals with chronic kidney disease and heart failure,” said Yuen, who practices as a nephrologist. “In our view, this is a significant advance for stem cell therapies because it gets around having to inject stem cells.”

Yuen said that they do not yet know what kind of hormone the cells are secreting, but identifying the hormone would be the first step toward the goal of developing a synthetic drug.

Chronic kidney disease (CKD) is much more prevalent than was once believed, with recent estimates suggesting that up to five percent of the Canadian population may be affected with this condition.

The number of people with CKD and end-stage renal failure is expected to rise as the population ages and more people develop Type 2 diabetes. People with kidney disease often develop heart disease, and many of them die from heart failure rather than kidney failure.

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.