Patient-Specific Stem Cells Made More Easily?


A Michigan State University research team uncovered the function of an already characterized gene that could be linchpin in the derivation of patient-specific stem cells that might be able to save millions of lives by differentiating into practically any cell in the body.

The gene is known as ASF1A, and even though it was not discovered by the team, ASF1A is one of the genes responsible for the mechanism of cellular reprogramming. Cellular reprogramming de-differentiates adult cells into less mature stem cells that have the capacity to differentiate into any cell type in the adult body.

This work was published in the journal Science. In this paper, the MSU team analyzed more than 5,000 genes from a human egg (oocyte) and determines that ASF1A in combination with another gene known as OCT4 and another molecule were primarily responsible for reprogramming.

Human oocytes
Human oocytes

“This has the potential to be a major breakthrough in the way we look at how stem cells are developed,” said Elena Gonzalez-Munoz, a former MSU post-doctoral researcher and first author of the paper. “Researchers are just now figuring out how adult somatic cells such as skin cells can be turned into embryonic stem cells. Hopefully this will be the way to understand more about how that mechanism works.”

An MSU team identified the thousands of genes expressed in oocytes in 2006. From this list of genes, the genes responsible for cellular reprogramming were then identified.

In 2007, a Japanese research team led by Shinya Yamanaka found that by introducing four other genes into adult cells, they could derive embryonic-like stem cells without the use of a human egg. These cells are called induced pluripotent stem cells, or iPSCs. “This is important because the iPSCs are derived directly from adult tissue and can be a perfect genetic match for a patient,” said Jose Cibelli, an MSU professor of animal science and a member of the team.

Apparently, ASF1A and OCT4 work in together in combination with a hormone-like substance that also is produced in the oocyte called GDF9 to facilitate the reprogramming process. “We believe that ASF1A and GDF9 are two players among many others that remain to be discovered which are part of the cellular-reprogramming process,” Cibelli said.

“We hope that in the near future, with what we have learned here, we will be able to test new hypotheses that will reveal more secrets the oocyte is hiding from us,” he said. “In turn, we will be able to develop new and safer cell-therapy strategies.”

Potential Marker Found for Stem Cell Population in the Inner Ear


Hearing loss is common as we get older. Presbycusis or age-related hearing loss results from the progressive death of sensory cells in the cochlea. Wouldn’t it be great if a stem cell population in the inner ear replaced these dying auditory sensory cells?

As it turns out, a stem cell population might exist in the mammalian inner ear and researchers from the UC Davis Comprehensive Cancer Center have identified a polysialylated glycoprotein that regulates neurodevelopment and is on the surface of cells in the adult inner ear. This glycoprotein acts as a marker of early cells in the inner ear and allows researchers to identify immature cells in the inner ear. This discovery was published in the journal Biochemical and Biophysical Research Communications and potentially opens the door to developing stem cell replacement treatments in the inner ear to treat certain hearing disorders.

“Hearing loss is a complex process and is usually regarded as irreversible,” said Frederic A. Troy II, principal investigator of the study from the UC Davis Comprehensive Cancer Center. “Finding this molecule in the inner ear that is known to be associated with early development may change that view.”

The existence of a marker for immature stem cells could make it possible to isolate neural stem cells from the adult inner ear in those people who suffer from hearing loss, culture and expand these cells in the laboratory, and then re-introduce them back into the inner ear as functioning neurons. These implanted cells might recolonize and establish themselves and improve hearing.

During development, certain glycoproteins (carbohydrate-protein linked molecules) are expressed on cell surfaces and serve critical functions essential to the normal growth and organization of the nervous system. One member of the class of cell-surface glycoproteins is an unusual molecule called polysialic acid or polySia. The large size of polySia allows it to fill spaces between cells and its strong electric charge repels other molecules. Therefore, polySia prevents cells from adhering or attaching to one another and thereby promotes cell movement to other areas.

Polysialic_acid_cell_interactions

Neural cell adhesion molecules (NCAMs) are also expressed on neuronal cell surfaces. As their name suggests, NCAMs help cells stick together and stay put. However, when NCAMs become modified with polysialic acid (or becomes “polysialylated”), the cells no longer adhere to other cells and are induced to migrate to new areas. Re-expression of the “anti-adhesive” polySia glycan on the surface of many adult human cancer cells facilitates their detachment, which enhances their metastatic spread.

Neural stem cells with these polySia-NCAMs on their cell surfaces play very important roles during embryonic development because these cells are able to travel throughout the body and differentiate into specialized cells. During adulthood, neural stem cells with polySia-NCAMs may migrate to injured areas and promote healing.

“The landscape of the cell surface of developing cells is decorated with a bewildering array of informational-rich sugar-protein molecules of which polysialylated NCAMs are of chief importance,” explained Troy. “During the life of a cell, these surface molecules are critical to cellular proliferation, self-renewal, differentiation and survival—essential processes for normal embryonic development and tissue regeneration in adults.”

It was already well-known that polySia-NCAM-expressing cells exist in the central nervous system, but Troy’s study is the first to document that they are also in the peripheral nervous system, and specifically in the spiral ganglia, those cluster of nerve cells in the inner ear that are essential to hearing.

Working with adult cells isolated from the inner ear spiral ganglia of guinea pigs, Troy and his team showed that these spiral ganglia cells expressed both polySia and NCAM. The polySia component was abundantly present on neural stem cells but was markedly reduced on mature cells. This implies that the polySia-NCAM complex is present on immature cells and can serve as a biomarker to identify these immature cells.

“Finding polySia-NCAM—a functional biomarker that modulates neuronal differentiation—on adult inner ear neural stem cells after differentiation gives researchers a ‘handle’ to identify and isolate these cells from among the many cells taken from a patient,” said Jan Nolta, director of the UC Davis Stem Cell Program and the university’s Institute for Regenerative Cures. “This discovery will enhance research into spiral ganglion neurons and may bring treatments closer to patients with hearing deficits.”

Lead author Park Kyoung Ho, a professor at Catholic University College of Medicine in Seoul, Korea, initiated the research for this article while on sabbatical leave in Troy’s laboratory at UC Davis. With his colleague and co-author, Yeo Sang Won, he is now planning clinical trials, based on the findings, in Korea with individuals who suffer from hearing disorders.

Stem Cells Aid Muscle Strengthing and Repair After Resistance Exercise


University of Illinois professor of Kinesiology and Community Health, Marni Boppart and her colleagues have published experiments that demonstrate that mesenchymal stem cells (MSCs) rejuvenate skeletal muscle after resistance exercise. These new findings, which were published in the journal Medicine and Science in Sports and Exercise, might be the impetus for new medical interventions to combat age-related declines in muscle structure and function.

Marni Boppart
Marni Boppart

Injecting MSCs into mouse leg muscles before several bouts of exercise that mimic resistance training in humans and result in mild muscle damage caused increases in the rate of muscle repair and enhanced the growth and strength of those muscles in exercising mice.

“We have an interest in understanding how muscle responds to exercise, and which cellular components contribute to the increase in repair and growth with exercise,” Boppart said. “But the primary goal of our lab really is to have some understanding of how we can rejuvenate the aged muscle to prevent the physical disability that occurs with age, and to increase quality of life in general as well.”

MSCs are found throughout the body, but several studies have established that MSCs from different tissue sources have distinct biological properties. Typically, MSCs can readily differentiate into bone, fat, and cartilage cells, but coaxing MSCs to form skeletal muscle has proven to be very difficult. MSCs usually form part of the stroma, which is the connective tissue that supports organs and other tissues.

Because of their inability to readily differentiate into skeletal muscle, MSCs probably potentiate muscle repair by “paracrine” mechanisms. Paracrine mechanisms refer to molecules secreted by cells that induce responses in nearby cells. Not surprisingly, MSCs excrete a wide variety of growth factors, cytokines, and other molecules that, according to this new study, stimulate the growth of muscle precursor cells, otherwise known as “satellite cells.” The growth of satellite cells expands muscle tissue and contributes to repair following muscle injury. Once activated, satellite cells fuse with damaged muscle fibers and form new fibers to reconstruct the muscle and enhance strength and restore muscle function.

“Satellite cells are a primary target for the rejuvenation of aged muscle, since activation becomes increasingly impaired and recovery from injury is delayed over the lifespan,” Boppart said. “MSC transplantation may provide a viable solution to reawaken the aged satellite cell.”

Unfortunately, satellite cells, even though they can be isolated from muscle biopsies and grown in culture, will probably not be used therapeutically to enhance repair or strength in young or aged muscle “because they cause an immune response and rejection within the tissue,” Boppart said. But MSCs are “immunoprivileged,” which simply means that they can be transplanted from one individual to another without sparking an immune response.

“Skeletal muscle is a very complex organ that is highly innervated and vascularized, and unfortunately all of these different tissues become dysfunctional with age,” Boppart said. “Therefore, development of an intervention that can heal multiple tissues is ideally required to reverse age-related declines in muscle mass and function. MSCs, because of their ability to repair a variety of different tissue types, are perfectly suited for this task.”

Mesenchymal Stem Cells Make Tendons on Fabricated Collagen


Ozan Akkus and his colleagues from the Department of Mechanical and Aerospace Engineering at Case Western Reserve University in Cleveland, Ohio has succeeded in making fibers made completely from the protein collagen. Why is this a big deal? Because it is so bloody hard to do.

In a paper published the journal Advanced Functional Materials, Akkus and others describe the generation of their three-dimensional collagen threads. This is the first time anyone has described the formation of such threads made purely from collagen.

Collagen is a very widely distributed protein in our bodies. It is the major structural component of tendons, and most connective tissues, and as a whole, collagen composes approximately one-third of all the protein in our bodies. There are almost 30 different types of collagen; some collagens for stiff fibers and others form flat networks that act as cushions upon which cells and other tissues can sit.

Collagen biosynthesis is very complicated and occurs in several steps. First, the collagen genes are transcribed into messenger RNAs that are translated by ribosomes into collagen protein. However, collagen proteins are made in a longer, inactive form that must undergo several types of modifications before it is usable.

Collagen synthesis begins in a compartment of the cell known as the endoplasmic reticulum, which is a series of folded membranes associated with the nuclear membrane. Within the endoplasmic reticulum, the end piece of the collagen protein, known as the signal peptide, is removed by enzymes called signal peptidases that clip such caps off proteins. Now particular amino acids within the collagen protein chains are chemically modified. The significance of these modifications will become clear later, but two amino acids, lysine and proline, and -OH or hydroxyl groups added to them. This process is called “hydroxylation,” and vitamin C is an important co-factor for this reaction. Some of the hydroxylysine residues have sugars attached to them, and three collagen protein chains now self-associate to form a “triple ɣ helical structure.” This “procollagen” as it is called, is shipped to another compartment in the cell known as the Golgi apparatus. Within the Golgi apparatus, the procollagen it is prepared to be secreted to the cell exterior. Once secreted, collagen modification continues. Other proteins of the collagen protein chains called “registration peptides” are clipped off by procollagen peptidase to form “tropocollagen.” Multiple tropocollagen molecules are then lashed together by means of the enzyme lysyl oxidase, which links hydroxylysine and lysine residues together in order to form the collagen fibrils. Multiple collagen fibrils form a proper collagen fiber. Variations on a theme are also available, since collagen can also, alternatively, attached to cell membranes by means of several types of proteins, including fibronectin and integrin.

collagen1

Now, if the cells has to go through all that just to make a collagen fiber, how tough do you think it is to make collagen fiber in a culture dish? Answer – way hard. In order to make collagen threads, Akkus and his team had to use a novel method for mature collagen production, and then they compacted the collagen molecules by means of the mobility of these molecules in an electrical field. This “electrophoretic compaction” method also served to properly align the collagen molecules until they formed proper collagen threads. Biomechanical analyses of these fabricated collagen threads showed that they had the mechanical properties of a genuine tendon. Akkus’ group when one step further and showed that a device they designed with movable electrodes could fabricate continuous collagen threads (). Thus, Akkus and his crew showed that they could make as many collagen threads as they needed and that these threads worked like tendons (see here for video). Are these guys good or what?

A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.
A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.

Nest, Akkus and his gang seeded collagen threads with mesenchymal stem cells (MSCs) from bone marrow. Remarkably, these collagen thread-grown mesenchymal stem cells differentiated into tenocytes, which are the cells that made tendons. Normally, MSCs do not readily form tenocytes in the laboratory, and they do not easily make tendons. However, in this case, the MSCs not only differentiated into tenocytes and made tenocyte-specific proteins and genes, but they do so without the addition of exogenous growth factors; the collagen threads were all the cells needed.

The seeded MSCs made Collagen I, which is the most abundant collagen of the human body, and is present in scar tissue, tendons, skin, artery walls, corneas, the endomysium surrounding muscle fibers, fibrocartilage, and the organic part of bones and teeth. Other tendon-specific proteins that were made included tenomodulin, and COMP (Cartilage oligomeric matrix protein). Furthermore, the electrically-aligned collagen does a better job of inducing the tenocyte fate in MSCs than collagen that is randomly oriented.

These remarkable and fascinating results demonstrate scaffolds made of densely compacted collagen threads stimulates tendon formation by Mesenchymal stem cells. Thus electrically aligned collagen as a very promising candidate for functional repair of injured tendons and ligaments. Now it is time to show that this can work in a living creature. Let the preclinical trials commence!!

Stem Cell Treatment Saves Man’s Leg From Amputation


Clive Randell loves motorcycles, but an unfortunate accident in 2011 seriously injured his leg and potentially prevented him from ever riding his beloved Harley-Davidson motorcycle again. His leg had several open fractures and one particular fracture that left some bone that protruded through his skin. He had extensive skin loss, and his doctors told me several times that his leg would have to be amputated. Things looked grim to say the least.

However, new stem cell procedure that repairs severely fractured bones has healed his bad leg and saved Clive from amputation. In fact, now Clive can ride his motorbike again. This new, pioneering stem cell procedure could give a new hope for victims of severe accidents who face limb amputation.

This new procedure uses stem cells extracted from the patient’s bone marrow from the patient’s pelvis and then mixes these cells with a specially created gel matrix to provide the cells with the right environment in order for them to form bone. This stem cell/matrix was then injected into the damaged bone with some hardware, such as a rod, which is inserted into the bone for support. Over time, the stem cells regenerate bone at the fracture site, traversing the fracture with new bone and completely healing the damaged bone.

Bone healing procedure

The intrepid physician who used this new procedure to heal Mr. Randall is Professor Anan Shetty, who serves as the Deputy Director of Minimally Invasive Surgery at Kent’s Canterbury Christ Church University. The motivation behind Dr. Shetty’s research is easy to understand. In the United Kingdom alone there are 350,000 serious fractures every year. Five to ten percent of these fractures are too extensive to heal and demand multiple surgeries, bone grafts, and other procedures that sometimes end in limb amputation if they fail to produce satisfactory results.

The fractured bone lacks an established blood supply, which means that it is very tough going for any implanted stem cells. Implanted stem cells have no way to receive signals to regenerate damaged cells. This new treatment circumvents this problem by using the bioengineered gel that contains the ingredients to that tells the stem cells what to do.

According to Professor Shetty, “Experiments have shown that collagen [gels] can trigger the transformation of stem cells into bone forming cells.” Dr. Shetty continued: “These “miracle” cells are abundant in bone marrow, so may be harvested, concentrated and applied with a collagen ‘scaffold’ into an area of poor healing.”

According to Clive Randall, “I may never dance the tango, but, thanks to Professor Shetty, I will be able to get as near to normal as possible.”

This bone-healing operation is performed under a general anaesthesia and only takes 30 minutes, after which the patient can walk out of the hospital and go home on the same day as the procedure. To date, six patients in the UK, four in India and 20 in South Korea have undergone this procedure.

Bone marrow for this procedure is drawn from the crest of the ilium of the patient’s pelvis by means of a stiff, hollow needle. Bone marrow contains a mixture of differ types of stem cells (including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells), and red blood cells. The bone marrow extract is then concentrated through centrifugation, but the red blood cells are usually removed. The concentrated bone marrow stem cell preparation is then mixed with collagen and this mixture is ready for implantation by injection.

For the surgical procedure, the surgeon stabilizes the fracture with a plate or long metal rod that is inserted through the central medullary canal or the bone. These stabilizing tools can be inserted with a small incision. The stem cell and collagen suspension is then injected into the fracture site and around the bone, guided by either live fluoroscopy or X-ray.

After the surgery, the patient is actually allowed put some weight on the affected limb, and is instructed to progressively increase the load he or she applies through his leg. Interestingly, Prof Shetty’s pioneering procedure cuts the healing time associated with these types of procedures in half, and at a cost of about $3,500 to $5,200, costs a fraction of the hundreds of thousands of dollars usually involved in amputation, rehabilitation and fitting the patient for prosthetics.

Professor Norimasa Nakamura, president elect of the International Cartilage Repair Society and one of the world’s leading authorities on stem cell treatment, has welcomed Prof Shetty’s work, saying: “It will revolutionize the whole field of bone fracture repairs. The patient has a more effective treatment and the health provider saves money. It’s a win-win situation.”

After his accident, Clive Randall, who worked as a high-altitude window cleaner who lived in Orpington, Kent, had a cage screwed to his damaged leg. He also underwent three bone grafts and several other procedures within 18 months after his accident. The accident also drastically changed his life. Even though the driver of the car was successfully prosecuted, he lost his job, girlfriend, and most of his money. He also had to take a deal of pain medication and became greatly depresses; at one time, understandably, he contemplated suicide. In a kind of “Hail Mary,” Clive turned to the internet and typed “I want to save my leg.”

He found Prof Shetty’s name, and the rest is history, but he is still in a state of disbelief over the reversal in his fortunes since having the operation in 2012. He said, “Six hours after the operation, Professor Shetty told me to get up and go for a walk. After being in and out of hospitals, I really couldn’t believe it. I’d suffered 15 months of being told there was a good chance I was going to lose my leg, yet eight weeks after the procedure I was told to start putting weight on it and to walk as much as I could. It still hurts to walk long distances, but that will improve. My foot is turned out a little bit to the side and I have a limp, but that’s a small price to pay to keep my leg. My hope is this procedure will eventually be available to everyone, since it can help so many people, particularly the military. The old way of mending broken bones is so painful and stops you getting on with your life. Professor Shetty’s stem cell surgery is quick and almost painless, so it’s important more people hear about it.”

There you have it from the patient himself.  Now only if the power-hungry, control-driven FDA would get off their duffs and look into bringing this procedure to the US?

Gene Therapy Creates a New Heart Pacemaker


When a patient’s heart beats too rapidly, too slowly or erratically, and if the usual heart medicines fail to properly regulate the heart rhythm, then the patient’s cardiologist may prescribe the implantation of an electronic pacemaker to regulate the heart rhythm. Even though implanted pacemakers are widely used, their installation requires an invasive surgery, they carry some risk of infection, and they also set off metal detectors during airport security checks. However, gene therapy might soon join the electronic pacemaker as a treatment for a poorly-regulated heart. It runs out that inserting a specific gene into heart-muscle cells can allow researchers to restore a normal heart rhythm in pigs, albeit temporarily.

Electronic pacemakers restore regular function to hearts by sending small electrical currents to the heart muscle in order to stimulate a heartbeat. This function is usually donned by the sinoatrial node, which is a cluster of a few thousand cardiac cells in the upper part of the right atrium that signals the heart to initiate a heartbeat and, therefore, sets the heart rate.

Heart Conducting System.  1) is the sinoatrial node or pacemaker and 2 is the atrioventricular node that receives the beat signal from the sinoatrial node and sends it to the ventricles.
Heart Conducting System. 1) is the sinoatrial node or pacemaker and 2 is the atrioventricular node that receives the beat signal from the sinoatrial node and sends it to the ventricles.

A research team led by Eduardo Marbán, who is a cardiologist at Cedars-Sinai Medical Center in Los Angeles, California, attempted to engineer heart cells outside the sinoatrial node to act as the pacemaker of the heart. The findings from Marbán’s laboratory were reported in the journal Science Translational Medicine.

Marbán and his colleagues used 12 laboratory pigs for their laboratory experiments. In these animals, Marbán and others induced a fatal heart condition in which electrical activity that originates from the sinoatrial node cannot spread through the heart. This forces other, less capable parts of the heart to take over and act as a pacemaker. Then, Marbán’s group used high-frequency radiowaves to destroy the sinoatrial nodes in the pigs’ hearts. This caused the animals’ average heart rate to slow to about 50 beats per minute (compared to the normal rate of 100 or more beats per minute). Such animals, if they were a human, would require an electronic pacemaker.

Next, Marbán and other injected the pigs’ hearts with a genetically modified virus that carried a pig gene called Tbx18, which is involved in heart development. Within one day, infected heart cells infected with the virus began to express those genes usually found in sinoatrial node cells. These cells acted as the pacemaker and began to direct the pumping the heart at a normal rate. The animals maintained this steady beating for the two-week study period, whether resting, moving or sleeping.

In an interview, Marbán said that his method is simpler than other biological approaches to restore a normal heart rhythm to hearts. These other approaches include inducing cardiac muscle cells to a pluripotent state, then coaxing them to differentiate into pacemaker cells. However, Marbán cautioned that the effects of gene therapy might be temporary. Over time, the body’s immune system would probably recognize the virus used to deliver Tbx18 to the heart and attack and destroy the infected cells. Marbán’s team is presently monitoring pigs that have received the gene-therapy treatment for several months to measure the persistence of this pacemaker effect.

However, even if the treatment’s effects are limited, it could still prove useful, according to Marbán. For example, if a pacemaker patient suffers from an infection as a result of the pacemaker, that pacemaker must be temporarily removed. This patient could then receive a biological pacemaker that could keep the heart pumping steadily until the infection clears and a new device is implanted. The gene-therapy approach could also help unborn children with heart defects, or even children who quickly outgrow implanted pacemakers or people for whom surgery is simply too risky.

“I think it’s a truly creative idea,” says Ira Cohen, a cardiac electrophysiologist at Stony Brook University Medical Center in New York. He would like to see the therapy tested in dogs, whose average heart rate is 60-100 beats per minute, which is more similar to that of a human.

Marbán is presently in talks with the US Food and Drug Administration about developing a human trial, which he says could be just two to three years away.

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.