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.

Amniotic Fluid Stem Cells Treat Mice With Spinal Muscular Atrophy

A multi-center study that included labs from the United Kingdom, Italy and France has culminated in a publication that describes the invention of a novel strategy to regenerate muscle in laboratory animals using human amniotic fluid stem cells.

Amniotic fluid fills a sac that surrounds the developing baby known as the amnion.  The amnion forms after about 12 days after the onset of fertilization, and amniotic fluid cushions and protects the baby, helps maintain a steady temperature around the baby, helps the baby’s lungs grow and develop since the baby breathes in the fluid, helps the baby’s digestive system develop since the baby swallows the fluid, provides a medium through which the baby swims and moves and therefore helps the bones and muscle develop, and prevents the umbilical cord from being squeezed.  Amniotic fluid stem cells are sloughed from the amniotic membranes and other surfaces as well, and have the ability to develop into several different cell types, including skin cells, muscle cells, neurons, cardiac tissues, kidney, liver, cartilage, bone, tendon, and others.  These cells are potentially useful for a broad range of future uses and therapeutic applications.

Particular muscular diseases result from the progressive degeneration of skeletal muscles. Stem cell treatments that use a patient’s own stem cells are problematic in such cases because the patient’s own stem cells have the same abnormalities as the degenerating muscles. Therefore, such “autologous” treatments would only add more dying cells to the muscle. New stem cells that can form new muscle that will not degenerate is required to effectively treat these diseases.

To this end, one such muscular degenerative disease, spinal muscular atrophy is a name given to a group of inherited diseases that cause progressive muscle damage and weakness that gets worse over time and eventually leads to death. Spinal muscular atrophy (SMA) patients possess mutations in SMN1 gene. SMN stands for “survival of motor neurons,” which indicates what this gene does; it encodes a protein that is absolutely essential for the continued survival of motor neurons. Motor neurons are spinal nerve cells that extend long processes called “axons” to skeletal muscles. Activation of the motor neurons causes the skeletal muscle to contract. Without motor neuron activation, the muscle is unable to contract. When the motor neurons die, the muscle is paralyzed and is unable to move.

Humans have two copies of the SMN gene on chromosome 5. SMN1 is found at the tip of chromosome 5 and SMN2 is found towards the middle of chromosome 5. SMA2 is expressed at very low levels in motor neurons. People with SMA have received two mutant copies of SMA1, one from each parent. Approximately, 4 of every 100,000 people have SMA.

There are four  forms of SMA.  SMA type I (Werdnig-Hoffman disease), which is the most severe, SMA type 1 results from mutations in SMN1 that prevent the production of any functional SMN1 protein. Even though SMN2 is available, not enough SMN protein is produced to prevent the neurons from dying. Symptoms appear in the first months of life, and there is rapid motor neuron death. The body organs operate inefficiently and the respiratory system operates poorly,which leads to a high risk of pneumonia-induced respiratory failure. Babies diagnosed with SMA type I do not usually live past two years of age and death can occur as early as within weeks in the most severe cases

SMA type II or Dubowitz disease affects children. Children with SMA type II are never able to stand and walk. However, they can maintain a sitting position at some time in their life. Weaknesses manifests some time between 6 – 18 months. The progression of this disease varies greatly. Body muscles are weakened, and the respiratory system is a major concern. Life expectancy is somewhat reduced but most SMA II patients live well into adulthood. SMA type II patients have at least three copies of SMA2, since the copies of SMN1 cause reduced production of SMN1 protein to levels similar to those observed as SMN2.

SMA type III, which is the least severe, results from at least three copies of SMN2, and in rare cases, SMA type IV, patients have four copies of SMN2, since both copies of SMN1 have undergone mutations that reduce their levels of expression to those of SMN2. The symptoms of SMA type IV begin in adulthood, In almost all cases, there is a family history of SMA.

Mouse models of SMA have been particularly useful in the study of this disease, and Dr. Paolo de Coppi, who, which his colleagues at the UCL institute of Child Health showed that intravenous administration of human amniotic fluid stem cells could increase the strength of SMA animals and improve their survival. This study demonstrated the integration of amniotic stem cells into skeletal muscle.

According to Dr. Coppi, “SMA is a genetic disease affecting one in 6,000 births. It is currently incurable and in its most severe form children with the condition may not survive long into childhood. Children with a less severe form face the prospect of progressive muscle wasting, loss of mobility, and motor function. There is an urgent need for improved treatments. We now need to perform more in-depth studies with human AFS (amniotic fluid stem cells) in mouse models to see if it is viable to use cells derived from the amniotic fluid to treat diseases affecting skeletal muscle tissue.”