Manipulation of a Master Molecular Switch Called 190RhoGAP May Improve Stem Cell Treatment Of Heart Attacks

New research findings have provided vitals clues as to why heart-based stem cells differentiate into muscle or blood vessels. Such a discovery might hold the key to better treatments for heart attacks in the future.

Human heart tissue lacks the capacity to heal after a heart attack and instead of reforming heart muscle; it tends to form a non-contracting heart scar. Stem cells in the heart can augment the healing process and direct the heart to make heart muscle and blood vessels rather than scars, but why this does not normally occur is unclear.

Particular physicians and their colleagues have shown that introducing heart stem cells into the heart can reduce the formation of heart scar tissue and increase the regeneration of heart muscle. However, uncovering the molecular switch that directs the fate of these cells could result in even more effective treatments for heart patients.

A recent report has shown that scientists who have manipulated a protein called “p190RhoGAP” managed to direct the differentiation of cardiac stem cells to become either blood vessels or heart muscle. Members of this research group even said that altering levels of this protein can affect the activities of these stem cells.

Andre Levchenko, a biomedical engineering professor who supervised the research effort said: “In biology, finding a central regulator like this is like finding a pot of gold.” The lead author of this paper, Kshitiz, said, “Our findings greatly enhance our understanding of stem cell biology and suggest innovative new ways to control the behavior of cardiac stem cells before and after they are transplanted into a patient. This discovery could significantly change the way stem cell therapy is administered in heart patients.”

Earlier in 2012, a medical team at Cedars-Sinai Medical Center in Los Angeles, CA reported reductions of scar tissue in heart attack patients after harvesting some of the patient’s own cardiac stem cells, growing more of these cells in the lab and then transfusing them back into the patient. Using the stem cells from the patient’s own heart prevented the rejection problems that often occur when tissue is transplanted from another person.

The goal of Levchenko’s research is to determine what directs the stem cells, at the molecular level, to change into helpful heart tissue. Answering this question could improve the results from experiments like the one done at Cedars-Sinai and boost regeneration in the heart after a heart attack to an even greater degree.
Levchenko’s team (from Johns Hopkins) tried to change the surface upon which they grew the harvested cardiac stem cells. Surprisingly, growing the cells on a surface that had a similar rigidity to that of heart tissue caused the stem cells to grow faster and to form blood vessels. The increase in growth was substantially greater than that observed with any other protocol with regard to these stem cells. The increased population growth on such a medium also gave prominent hints as to why the formation of a cardiac scar (a structure with very different rigidity), can inhibit stem cells that reside there from regenerating the heart.

By digging further into this phenomenon, the Johns Hopkins group found that the increased cell growth under these conditions was due to decreases in the levels of a protein called p190RhoGAP. This same molecule, when absent, could also direct stem cells to form blood vessels.
Levchenko explained: “It was the kind of master regulator of this process. And an even bigger surprise was that if we directly forced this molecule to disappear, we no longer needed the special heart-matched surfaces. When the master regulator was missing, the stem cells started to form blood vessels, even on glass.”

When Levchenko’s group artificially increased levels of 190RhoGAP, the stem cells formed heart muscle. According to Levchenko, “The stem cells started to turn into cardiac muscle tissue, instead of blood vessels. This told us that this amazing molecule was the master regulator not only of the blood vessel development, but that it also determined whether cardiac muscles and blood vessels would develop from the same cells, even though these types of tissue are quite different.”

Can such findings make a difference in the treatment of living beings? To get a handle on the clinical consequences of this finding, Levchenko’s group limited the production of p190RhoGAP in cardiac stem cells not within a culture dish, but inside the heart of a living animals. The cells with less 190RhoGAP integrated more smoothly into an animal’s blood vessel networks in the aftermath of a heart attack. Also, more of these transplanted heart cells survived, compared to what had occurred in earlier cell-growing procedures.

Kshitiz said that the special heart-like surface on which the cardiac stem cells were grown triggers regulation of the master molecule, and this then guides the next steps in differentiation.

“This single protein can control the cells’ shape, how fast they divide, how they become blood vessel cells and how they start to form a blood vessel network,” he said. “How it performed all of these myriad tasks that require hundreds of other proteins to act in a complex interplay was an interesting mystery to address, and one that rarely occurs in biology. It was like a molecular symphony being played in time, with each beat placed right at the moment before another melody has to start.”
See Matrix Rigidity Controls Endothelial Differentiation and Morphogenesis of Cardiac Precursors;” Kshitiz et al; Science Signaling, 2012; 5 (227): ra41 DOI: 10.1126/scisignal.2003002.

Stem Cells Derived from Fat Show Promise for Regenerative Medicine

A detailed review article in the June issue of Plastic and Reconstructive Surgery, the official medical journal of the American Society of Plastic Surgeons, has examined the safety and clinical efficacy of fat-derived stem cells. Stem cells from fat, also known as ACSs, are a promising source of cells for use in plastic surgery and regenerative medicine, according to this review, but there are still many questions that remain about them. Much more research is needed in order to completely establish the safety and effectiveness of ASC-based therapies in human patients. The review article was written by ASPS Member Surgeon Rod Rohrich, MD of University of Texas Southwestern Medical Center, Dallas, and his colleagues (Dr. Rohrich is Editor-in-Chief of Plastic and Reconstructive Surgery).

ASCs are very easily procured from humans, since simple procedures such as liposuction can provide more than enough material for therapies. On the average, one gram of fat yields about 5,000 stem cells, whereas the yield from the same quantity of bone marrow is about 1,000 cells (B. M. Strem, K. C. Hicok, M. Zhu et al., “Multipotential differentiation of adipose tissue-derived stem cells,” Keio Journal of Medicine, vol. 54, no. 3, pp. 132–141, 2005.). Once isolated from the fat, ASCs have the capacity to form fat cells, but also bone, cartilage and muscle cells.

From a therapeutic standpoint, ASCs promote the development of new blood vessels (angiogenesis). ASCs are also not recognized by the immune system and they seem to staunch inflammation. According the Dr. Rohrich and is co-authors, “Clinicians and patients have high expectations that ASCs may well be the answer to curing many recalcitrant diseases or to reconstruct anatomical defects.”

Fortunately, there is great interest in ASCs, and this means that the number of studies that examine ASCs or utilize them for experimental treatments have soared. Unfortunately, there is continued concern about the “true clinical potential” of ASCs. In the words of this new article, “For example, there are questions related to isolation and purification of ASCs, their effect on tumor growth, and the enforcement of FDA regulations.”

Rohrich and others conducted a rather in-depth review of all known clinical trials of ASCs. Thus far, most studies have been performed in Europe and Korea, and only three in the United States, to date. This reflects the stringency of FDA regulations.

Most ASC clinical trials to date have been examined the use of ASCs in plastic surgery. In this case, plastic surgeon-researchers have used ASCs for several types of soft tissue augmentation (breast augmentation, especially after implant removal and regeneration of fat in patients with abnormal fat loss or lipodystrophy). Studies exploring the use of ASCs to promote healing of difficult wounds have been reported as well. ASCs have also been used as in so-called soft tissue engineering or tissue regeneration. In these cases, the results have been inconclusive.

Other medical specialties have also made use of ASCs as treatments for other types of medical conditions. For example, ASCs have been studied for used to treat certain blood and immunologic disorders, heart and vascular problems, and fistulas (abnormal connection between an organ, vessel, or intestine and another structure). There are some other studies that have examined the use of ASCs for generating new bone for use in reconstructive surgery. A few studies have reported promising preliminary results in the treatment of diabetes, multiple sclerosis, and spinal cord injury. Perhaps one of the most encouraging results was the complete absence of serious adverse events related to ASCs in any of these studies.

These results are encouraging, but all of these applications are in their infancy. Globally speaking, less than 300 patients have been treated with ASCs, and no standardized protocol exists for the preparation or clinical applications of ASCs. Additionally, there is no consensus as to the number of ASCs required per treatment, or how many treatments are required for the patient to show clinical improvement. Thus Rohrich and his colleagues have taken a “proceed with caution approach.” They conclude that “further basic science experimental studies with standardized protocols and larger randomized controlled trials need to be performed to ensure safety and efficacy of ASCs in accordance with FDA guidelines.”

Fat-Based Mesenchymal Stem Cells Form Bone Easily Inside Lab Animals

Bone production by stem cells is usually the domain of bone marrow-derived stem cells. However, fat-derived stem cells can make bone in culture, although they do not seem to be as efficient at making bone as bone marrow stem cells. Nevertheless, some enterprising stem cells scientists have designed protocols for using purified adipose-derived stem cells to make high quality bone tissue faster than the currently-used methods. The new technology may someday eliminate the need for painful bone grafts that use material taken from the patient during invasive procedures.

Fat is thought to be an ideal source of MSCs, since they are plentiful and easily acquired by liposuction.

Traditionally, cells taken from fat had to be grown in culture for weeks in order to isolate the bone-making stem cells. Unfortunately, the expansion of these cells increases the risk of infection and genetic instability. However, a fresh, non-cultured cell composition called stromal vascular fraction (SVF) is also used to grow bone, but SVF cells extracted from fat constitute a highly heterogeneous population that includes cells that do not have the ability to make bone.

To solve this problem, researchers used a cell sorting machine to isolate and purify human perivascular stem cells (hPSC) from adipose tissue and demonstrated that these purified cells worked far better than SVF cells in bone-making assays. In addition, they also showed that a growth factor called NELL-1 enhanced bone formation in their animal model.

According to Dr. Chia Soo, vice chairman for research at UCLA Plastic and Reconstructive Surgery, who was involved with this study: “People have shown that culture-derived cells could grow bone, but these are a fresh cell population and we didn’t have to go through the culture process, which can take weeks. The best bone graft is still your own bone, but that is in limited supply and sometimes not of good quality. What we show here is a faster and better way to create bone that could have clinical applications.”

In the animal model, Soo and colleague Bruno Pйault placed hPSCs with NELL-1 in a muscle pouch (a location where bone normally does not grow). They then used X-rays to show that the cells did indeed become bone.
“The purified human hPSCs formed significantly more bone in comparison to the SVF by all parameters,” Soo said. “And these cells are plentiful enough that patients with not much excess body fat can donate their own fat tissue.”

Conceivably, patients may one day have rapid access to high-quality bone graft material by which doctors extract some of the patient’s fat tissue, purify the hPSCs from it, and then replace their own stem cells with NELL-1 back into the area where bone is required. The hPSCs cultured with NELL-1 could grow into bone inside the patient, which eliminates the need for painful bone graft harvestings. The goal is to isolate the hPSCs and add the NELL-1 with a matrix or scaffold in order to aid cell adhesion, all in less than an hour.

“Recent studies have already demonstrated the utility of perivascular stem cells for regeneration of disparate tissue types, including skeletal muscle, lung and even myocardium,” said Pйault, a professor of orthopedic surgery at UCLA. “Further studies will extend our findings and apply the robust osteogenic potential of hPSCs to the healing of bone defects.”

Human-Eye Precursors are Grown from Embryonic Stem Cells

Yoshiki Sasai of the RIKEN Center for Developmental Biology (CBD) in Kobe, Japan has managed to grow eye precursors in the laboratory from embryonic stem cells.  Such an achievement provides a remarkable opportunity to investigate early eye development and the pathology of eye abnormalities.

Eye development is a complex process, since mammalian eyes develop as an extension of the central nervous system.  The development of the central nervous system begins at about 18 days after fertilization with the formation of a thickened layer of cells on the surface of the embryo called the neural plate.  The neural plate is induced by a cluster of cells that clumps together to form a hollow tube called the “notochordal plate.”  The neural plate rolls into a tube called the neural tube and this neural tube is the beginnings of the central nervous system.  The front of the neural tube will inflate to form the brain and the portion of the tube behind the brain forms the spinal cord.  The neural tube forms as a result of high points that form in the neural plate called the neural folds.  These neural folds fuse to form a tube that is below the outermost layer of the embryo (ectoderm).

About 22 days after fertilization, inflations on either side of the developing brain extend from the brain, and these are the beginnings of the eye.  These “optic vesicles” as they are called continue to grow until their connection to the brain becomes narrower and narrower.  The narrow connections between the optic vesicles and the brain are called the optic stalks and they will become the rudiments of the optic nerves.

The optic vesicles make contact with the surface of the embryo and this does two things.  The vesicle collapses into a two-layered structure called the optic cup and the embryonic ectoderm pinches in and forms a vesicle that will form the lens of the eye (lens vesicle).  The optic cup is about 550 micrometers in diameter and initially contains two layers of cells.  These cells divide quickly to form an inner neural retina and an outer retinal pigment epithelium.  The neural retina divides to form multiple layers of cells, including photoreceptor cells, which respond to light.  Austin Smith, director of the Centre for Stem Cell Research at the University of Cambridge, UK said of the developing eye: “The morphology is the truly extraordinary thing.”

Previously, stem-cell biologists were able to grow embryonic stem-cells into two-dimensional sheets, but over the past four years, Sasai and his colleagues have used mouse embryonic stem cells to grow well-organized, three-dimensional cerebral-cortex tissue (Eiraku, M., et al. Cell Stem Cell 3, 519–532, 2008)., pituitary-gland (Suga, H., et al. Nature 480, 57–62, 2011)., and optic-cup tissue (Eiraku, M., et al. Nature 472, 51–56, 2011).  His present successes represent the first time that anyone has managed a similar feat using human cells.

The fact that Sasai’s laboratory was able to grow the optic cup in the laboratory, and that it recapitulated the same developmental events in the same order shows that the cues for this the formation of the eye rely, primarily, on cue from inside the cell, rather than relying on external triggers.  “This resolves a long debate,” says Sasai, over whether the development of the optic cup is driven by internal or external cues.

This achievement could make a big difference in the clinic.  There have been increasing successes cell transplantations in the last few years.  For example, a last month, a group at University College London showed that a transplantation of stem-cell derived photoreceptors could rescue vision in mice (Pearson, R. A., et al. Nature 485, 99–103, 2012).  The transplant involved only rod-shaped receptors, not cone-shaped ones, which would leave the recipient seeing fuzzy images. Sasai’s organically-layered structure provides hope that integrated photoreceptor tissue might be transplantable someday.   The developmental process could also be adapted to treat a particular disease, and stocks of tissue could be created for transplant and frozen.

Sasai emphasized that the cells in the optic cup are differentiating and there are no embryonic stem cells in them.  This reduces concerns that transplants of these optic cups or structures derived from them might develop cancerous growths or fragments of unrelated tissues. “It’s like pulling an apple from a tree. You wouldn’t expect iron to be growing inside,” says Sasai. “You’d have no more reason to expect bone to be growing in these eyes.”

Masayo Takahashi, an ophthalmologist at the CBD, has already started transferring sheets of the retina from such optic cups into mice, and she would like to do the same with monkeys sometime this year.  The big question is whether transplanted tissue will integrate into native tissue.

The big question is whether or not clinicians and stem-cell biologists can easily repeat Sasai’s results?  Some, in fact, have already tried and failed to reproduce Sasai’s mouse experiment using human cells. “We need to know how robust, how reproducible it is,” says Smith.

Bone Marrow Stem Cells Rejuvenate Tissue Mitochondria

Within almost every cell in your body are small vesicles called mitochondria that act as the powerhouses of the cell. Chemical energy is synthesized in the mitochondria, and without these vital structures, the ability of the cell to do all the incredible things that it does goes away. Cells are chemical and biological marvels but all the things they do require energy.

Cells make their energy from energy-rich food molecules such as sugars, amino acids, and fats. Cells degrade these energy-rich food molecules to smaller molecules, and harvest the released energy in the form of high-energy electrons and am energy storage molecule called “ATP.”  ATP stands for “adenosine triphosphate,” and this molecule as a series of high-energy phosphate anhydride bonds that store energy. The high-energy electrons are carried by electron carriers and these carriers give their high-energy electrons to electron transport chains that are embedded in the membranes of mitochondria and the electrons are passed through the electron transport chains, ultimately to molecular oxygen, which makes water. Passing the electrons down the electron transport chain to make water releases a whole lot of energy that is captured and stored in the form of ATP. Thus mitochondria are the “powerhouses” of the cell.

Mitochondria, however, pay a price for their assiduous energy making. All their exposure to oxygen (a toxic molecule under various conditions) and versions of oxygen with extra electrons (known as reactive oxygen species or ROS) tends to damage the mitochondria over time. With time, the mitochondria become to damaged that they are unable to make energy for the cell. Cells that cannot make energy are of no value to the body. How do you fix what has been broken?

Cells have ways to destroy damaged mitochondria (autophagy) and working mitochondria also have the capacity to divide. However, what if the mitochondria become collectively so damaged that the cell cannot make its own energy?

New work shows that stem cells can come to the rescue. A paper in the journal Nature Medicine (18(5)) by Mohammad Naimul Islam and colleagues from the laboratory of Jahar Bhattacharya at the College of Physicians and Surgeons of Columbia University, New York, has discovered something potentially ground breaking. The paper, entitled “Mitochondrial transfer from bone-marrow–derived stromal cells to pulmonary alveoli protects against acute lung injury” used a mouse model to study lung damage. They sprayed a molecule from bacteria into the respiratory systems of mice. This molecule, lipopolysaccharide (LPS) is found in the outer-most membrane of certain types of bacteria, and production of this molecule and its distribution throughout the body causes tissue damage and inflammation. After spraying LPS into the lungs, they added bone marrow-derived stem cells from mouse or human bone marrow to help mitigate the damage induced by exposure to LPS.

They used live optical studies to examine the nature of the interaction of bone marrow MSCs with respiratory cells. Optical viewing of the respiratory tract showed that intimate connections were made between the stem cells and the respiratory cells. These connections consisted of special channels that are normally found between cells that are in the process of transferring materials between each other.

The astounding result was that the connections between the stem cells and respiratory cells showed the transfer of mitochondria from the stem cells to the respiratory cells. The transfer of mitochondria resulted in increased concentrations of ATP in respiratory cells and also decreased the signs and symptoms of inflammation within the respiratory tract. Mutant stem cells that had defective mitochondria were not able to abrogate the damage to the respiratory system.

This is a significant finding, since as well as being the powerhouse of the cell, mitochondria also control the onset of programmed cell death. Fresh, new mitochondria probably rejuvenate the cell by preventing the onset of programmed cell death, and refurbishing the energy-production machinery of the cell. This significant finding elucidates what might be a major mechanism of how stem cells heal damaged tissue.

FGF Proteins Maintain Stem Cells in Developing Kidney

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Olfactory Stem Cells Show Potential As Therapy

A new study has examined the ability of olfactory neural stem cells to differentiate into different cell types and the efficacy of this stem cell population in regenerative treatments. Olfactory neural stem cells are highly proliferative (they grow fast) and they are easily harvested by means of a biopsy procedure that is minimally invasive. Because these cells are constantly replaced throughout someone’s lifetime, they are quickly replaced, and can even be grown in tissue culture.

Dr. Andrew Wetzig of the King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia, who was the leading author of this study said: “There is worldwide enthusiasm for cell transplantation therapy to repair failing organs. The olfactory mucosa of a patient’s nose can provide cells that are potentially significant candidates for human tissue repair.”

These studies utilized rats as the preferred model system. The olfactory neural stem cells in rats seem to have rather similar characteristics to those in humans. According the Wetzig, “Previously, we found that they (i.e., olfactory neural stem cells) have performed well in preclinical models of disease and transplantation and seem to emulate a wound healing process where the cells acquire the appropriate phenotype in an apparently orderly fashion over time.”

When grown in culture, the olfactory stem cells form clusters of cells that grow as hollow balls of cells called “neurospheres.” These neurospheres contain stem cells that have the ability to differentiation into several different cell types. The specific cell type formed by the stem cells in the neurospheres is determined by specific signals from the immediate environment. Wetzig noted that olfactory stem cells grew well in culture, but when they were transplanted, the olfactory neural stem cells differentiated into the cell types that surrounded them. Therefore, the olfactory stem cells seemed to be able to sense their environment, and then assume the same cellular identity as their immediate neighbors.

While such work must be replicated with human olfactory stem cell before they can be considered for human clinical trials, such results are certainly encouraging.

Mature Liver Cells Seem to be Better Than Stem Cells for Liver Therapy

Japanese research team has compared the ability of liver progenitor cells (liver stem cells) and mature liver cells to effectively repopulate a damaged liver. They have concluded that mature liver cells (hepatocytes) are better than stem cells for liver repopulation.

Workers in the laboratory of Toshihiro Mitaka of the Cancer Research Institute of the Sapporo Medical University School of Medicine, Sapporo, Japan used a rat model to test these hypotheses. They injured the livers of these rats with surgery and chemicals and then used transplanted cells to repopulate these livers. Up to two weeks after transplantation, the growth rate of the stem cells was significantly higher than that of the mature liver cells, but after two weeks the majority of the stem cells died before they could confer any significant benefit on the liver. The mature cells, however, grew slower, but survived much better.

Toshihiro Mitaka made this comment: “Cell-based therapies as an alternative to liver transplantation to treat liver disease have shown promise. However, the repopulation efficiency of hepatic progenitor/stem cells and mature hepatocytes (liver cells) had not been comprehensively assessed and questions concerning the efficiency of each needed to be resolved.”

Mitaka’s team noticed that the shortage of liver cell sources and the difficulties of preserving the available liver cell sources by freezing (cryopreservation) have limited the available material, and therefore, the clinical applications of cell-based therapies for liver disease. Liver stem cells (liver progenitor cells) have been considered to be the best option to treat liver disease, since they can be expanded in culture and preserved by freezing for long periods of time.

However, once the liver progenitor cells were transplanted into the damaged livers of rats, the stem cells failed to survive terribly well. After two months, the vast majority of the transplanted stem cells had disappeared. In contrast, the mature liver cells gradually repopulated the rat livers and the even continued growing and repopulating the damaged livers for one year after transplantation.

Transplanted liver cells did not make uniform cells. Mitaka noted that “the small hepatocytes repopulated significantly less well than the larger ones. We also found that serial transplantations did not enhance nor diminish the repopulation capacity of the cells to any significant degree.”

In this paper, Mitaka and his colleagues argue that because the stem cells died much earlier than the mature hepatocytes, the stem cells were eliminated from the host livers and this reduced their potential regenerative capacity. They conclude in the paper that further “experiments are required to clarify the mechanism by which this might occur.”

My take on this is that damaged livers probably contain a respectable amount of inflammation. Therefore, they are probably a rather hostile environment for any transplanted cells. We have seen in previous posts that stem cells have a mechanism to resist stressful conditions, but also, that this pathway for resisting stress must be activated in the stem cells before they are capable of resisting stress. Therefore, I would suggest that the next experiment Mitaka and his co-workers should do, is to either precondition his stem cells with oxygen and glucose deprivation or pre-treat them with insulin-like growth factor-1 (IGF-1). Both of these treatments have been shown to activate the stress resistant pathway in stem cells transplanted into the heart. Therefore, if this pathway could be induced in liver progenitor cells, then perhaps the stem cells can be stress-adapted and tolerate the stressful conditions in the damaged livers.

Stem Cell Dormancy Enables Them to Remain Viable Days After Death

A collaboration of several researchers from French Institutions has demonstrated that humans and mouse stem cells have the ability to become dormant when their environment becomes hostile, including several days after the death of the organism. This ability to significantly reduce metabolic activity enables them to preserve their potential for cellular division, even a long time after death. Once isolated from the cadaver, the stem cells retain their healing abilities. This discovery could be the beginning of new therapeutic avenues for treating numerous diseases.

Skeletal muscle stem cells have the ability to survive for seventeen days in humans and sixteen days in mice, after death. This discovery was made by researchers from the Institut Pasteur, the Université de Versailles Saint-Quentin-en-Yvelines, the Paris Public Hospital Network (AP-HP), and the CNRS under the direction of Professor Fabrice Chrétien, in collaboration with a team led by Professor Shahragim Tajbakhsh. These laboratories showed that once the stem cells from the cadavers were grown in culture, they retained their capacity to differentiate into perfectly functioning muscle cells.

Once they made this surprising discovery, the next step was to determine precisely how these cells survive such adverse conditions. As it turns out, the stem cells enter a deeper state of sleep (quiescence), and this drastically lowers their metabolism. This so-called “dormant” state results from stripping the functional structures of the cell to their bare minimum. For example, these cells have fewer mitochondria (cellular power plants using oxygen to produce energy in cells) and diminished stores of energy.

Fabrice Chrétien explained it this way: “We can compare this to pathological conditions where cells are severely deficient in resources, before regaining a normal cell cycle for regenerating damaged tissues and organs. When muscle is in the acute phase of a lesion, the distribution of oxygen is highly disrupted. We have even observed that muscle stem cells in anoxia (totally deprived of oxygen) at 4°C have a better survival rate than those regularly exposed to ambient levels of oxygen.”

Chrétien’s team wondered if other cell types showed similar capacities. Once again, the results were surprising. Stem cells from bone marrow where blood remained viable for four days after death in mice. More importantly, they retained their ability to reconstitute tissue after a bone marrow transplant.

This discovery could form the basis of a new source, and more importantly new methods of conservation, for those stem cells used to treat different conditions. For example, leukemia treatments require a bone marrow transplant to restore those blood and immune cells that were destroyed by chemotherapy and radiation. By harvesting stem cells from the bone marrow of consenting donors after death, doctors could address to some extent, the shortage of tissues and cells. Although highly promising, this approach in the realm of cellular therapy still requires more testing and validation before it can be used in clinical applications. However, it paves the way to investigate the viability of stem cells from all tissues and organs post-mortem.

Reprogramming Heart Fibroblasts into Heart Muscle Cells Goes to Human Trials

Last month, this blog reported on the conversion of heart-based fibroblasts into heart muscle cells after a heart attack in living, laboratory animals by means of gene therapy. Another researcher has utilized a different strategy to achieve the same result. This work has also provided the means for biotechnology companies to begin clinical trials using this very strategy.

Scar formation (fibrosis), prevents the regeneration of heart muscle and creates a scar that does not contract. The loss of contractile function leads to heart failure and death. Therapeutic goals for these conditions include limiting scar formation.

To that end, Eric C. Olson and his colleagues from UT Southwestern were able to introduce four genes (GATA4, HAND2, MEFC2, and TBX5) into heart-based fibroblasts and convert them into beating heart muscle cells. To do this, Olson and his army of graduate students, technicians, and postdoctoral research fellows made genetically engineered viruses that encoded the four genes (collective known as GHMT).  When the GHMT-viruses were injected into mouse hearts after a heart attack, the four genes reprogrammed the fibroblasts into heart muscle cells in tissue culture and inside living animals.  Furthermore, when GHMT is introduced into fibroblasts after a heart attack, the fibroblasts do not make scar tissue, but heart muscle.

Olson and his team also used techniques that allowed them to trace cells and their descendents.  These techniques showed that the heart muscle that formed after the heart attack were the result of cells that had been infected by the engineered viruses (that is, they contained viral DNA).  Thus the new heart muscle came about because the virally-infected fibroblasts turned into heart muscle that began to beat.  Also, heart imaging also showed that infection of the heart with GHMT viruses greatly boosted heart function after a heart attack in comparison to control heart that were infected by the viruses that did not contain GHMT.

Can such a technology make it way to clinical trials?  Fortunately, Eric Olson is not only chairman of the Molecular Biology department as UT Southwestern, but he is also co-founder of a medical technology company known as LoneStar Heart Inc.  Olson’s company hopes to extend his findings in laboratory animals and eventually gain approval to begin human clinical trials.  Olson noted, “These studies establish proof-of-concept for in vivo cellular reprogramming as a new approach for heart repair. However, much work remains to be done to determine if this strategy might eventually be effective in humans. We are working hard toward that goal.”

LoneStar Heart is capitalizing on previous work by Olson and others in his laboratory that have established that the delivery of the four previously mentioned genes increases heart regeneration in laboratory animals and in cultured human heart cells.  LoneStar Heart is currently trying to complete the animal studies required before the Food and Drug Administration will consider permitting a human clinical trial

Lonestar Heart, however, has other products that might play a role in treating the hearts of patients whose hearts have started to enlarge. Heart enlargement results when the heart is overworked and it reacts to this overwork by enlarging. The enlargement stretches the heart and makes the walls of the heart thinner. The result is that the heart does not beat in a coordinated fashion, and patients with enlarged hearts are at risk for irregular heart beats or sudden cardiac death.

To address enlargement of the heart, LoneStar Heart has made a product called Algisyl-LVR that is a biopolymer that stiffens when it is injected into the heart. Injection of Algisyl-LVR into the walls of a heart that has enlarged thickens the heart wall without interfering with heart function. The artificial thickening of the heart walls decreases the stress on the heart and helps reverse heart enlargement. Algisyl-LVR is presently being tested in Europe in clinical trials under the product name AUGMENT-HF.  These remarkable products will hopefully be on the market before long.

British Medical Association (Again) Urges Doctors To Abdicate Their Professional Duty

Wesley Smith blogs on the British Medical Journal article that argues that British physicians should end their opposition to physician-assisted suicide. Instead, the Journal argues, this is a question for society and doctors should be neutral about it.

Smith rightly notes that this is preposterous, since doctors are a part of society and have a sworn duty to the well-being of their patients. Shame on the British Medical Association for pulling a stunt like this and shame on the Journal of being complicit in the promotion of doctor-based murder.

Read Smith’s post here.

Transplantation Of Lung Stem Cells Improves Emphysema

In an animal model of emphysema, transplantations of their own lung-derived mesenchymal stem cells (MSCs) increased blood flow, oxygen transport and the synthesis of extracellular matrix. This approach could offer a potential alternative to conventional stem cell-based therapies for the treatment of emphysema.

Emphysema results from destruction of the tiny little sacs in the lung called alveoli. The alveoli surfaces are densely-packed with a network of delicate blood vessels. These blood vessels are the site of oxygen exchange. When a patient contracts emphysema, the walls of the alveoli break down and the tiny air sacs are transformed into a giant air sac. This provides far less surface area for the exchange of oxygen, and the patient has shortness of breath and difficulty catching their breath.

Edward P. Ingenito of Brigham and Women’s Hospital, who was part of this study, gave this perspective: “Mesenchymal stem cells are considered for transplantation because they are readily available, highly proliferative and display multi-lineage potential. Although MSCs have been isolated from various adult tissues, including fat, liver and lung tissues, cells derived from bone marrow (BM) have therapeutic utility and may be useful in treating advanced lung diseases, such as emphysema.”

According to the authors, previous transplantation studies that used bone marrow-derived MSCs and delivered them via an intravenous method have shown that such a treatment only marginally improves the condition of the lung. Also, therapeutic responses in those studies were limited to animal models of inflammatory lung diseases, such as asthma and acute lung injury. For this study, however, researchers isolated highly proliferative mesenchymal cells from adult lung tissue, and delivered them by means of an endoscopic delivery system that included the MSCs and a scaffold composed of natural extracellular matrix components.

According to Ingenito, “LMSCs display efficient retention in the lung when delivered endobronchially and have regenerative capacity through expression of basement membrane proteins and growth factors,”

Despite the use of autologous cells, only a fraction of the LMSCs delivered to the lungs alveolar compartment appeared to engraft. The lost likely reason for the low engraftment rates is due to the rates of cell death. Just as in the heart after a heart attack, diseased lungs represent a hostile environment, and this stressed the cells, which induced programmed cell death. The inability of the stressed cells to attach to their proper niches prevented them from surviving in the lung.

Even though the rates of engraftment were quite low, the findings of this study did suggest that LMSCs could contribute to lung remodeling and functional improvement 28 days after transplantation in 13 female sheep.  “Although the data is from a small number of animals, results show that autologous LMSC therapy using endoscopic delivery and a biocompatible scaffold to promote engraftment is associated with tissue remodeling and increased perfusion, without scarring or inflammation,” Ingenito said. “However, questions concerning mechanism of action and pattern of physiological response remain topics for future investigation.”
For the abstract of this study, see here.

University of Wisconsin Scientists Find a New, Better Way to Turn Stem Cells into Heart Muscle Cells

Stem cell researchers and cardiologists from the University of Wisconsin-Madison have designed a new and improved protocol to turn embryonic and induced pluripotent stem cells into heart muscle cells.

The study leader, Sean Palecek, who is also professor of chemical and biological engineering at the University of Wisconsin-Madison, and his colleagues Timothy Kamp, professor of cardiology at UW School of Medicine and Public Health, and Xiaojun Lian, a UW graduate student, have developed a technique for efficient and abundant production of heart muscle cells. This technique will provide scientists a better and more abundant source of material for drug studies and a better model system to study diseases and heart pathologies.

Heart muscle cells (also known as cardiomyocytes) are essential cells that compose the beating heart. However, it is rather difficult to make large quantities of them. Typically, cultured heart muscle cells only survive or a short period time, which greatly complicates using them for any experiments or drug tests. Now, however, these UW researchers have devised an inexpensive method for developing an abundance of heart muscle cells in the laboratory.

Cardiologist Timothy Kamp explained: “Many forms of heart disease are due to the loss or death of functioning cardiomyocytes, so strategies to replace heart cells in the diseased heart continue to be of interest. For example, in a large heart attack up to 1 billion cardiomyocytes die. The heart has a limited ability to repair itself, so being able to supply large numbers of potentially patient-matched cardiomyocytes could help.”

Why is their method so much more efficient? The UW research team discovered that by changing a signaling pathway called Wnt pathway, they could guide the stem cells to differentiate into heart muscle cells. All they had to do was turn the Wnt pathway on and off at different times by using two small molecules.

The Wnt signaling pathway is an extremely common signaling pathway that exists in virtually all multicellular organisms and is used multiple times during development.  Wnt signaling begins with the secretion of a small protein can a Wnt protein.  Wnt proteins are produced by cells to send signals to nearby cells.  When the cells receiving the signal are bound by the Wnt protein, a series of events are set into motion inside the cell.  The receptor that binds the Wnt protein consists of a protein that is a member of the Frizzled gene family.  Frizzled receptors bind the Wnt protein in combination with another protein called LRP.  The binding of Wnt, LRP and Frizzled brings an internal protein called Disheveled to the membrane.  Once Disheveled come to the membrane, it becomes activated.  How this activation occurs in still unclear, but Disheveled inhibits GSK-3 (glycogen synthase kinase-3).  GSK-3 normally attaches phosphate groups to a protein called beta-catenin.  This phosphate group attachment marks beta-catenin for destruction, but once GSK-3 is inhibited by activated Disheveled, beta-catenin is no longer destroyed and when the levels of beta-catenin build up in the cytoplasm, it goes to the nucleus where it combines with another protein called TCF and regulates gene expression.  Once again we see that a signal transduction pathway begins at the cell surface and results in changes in gene expression.

“Our protocol is more efficient and robust,” said Palecek. “We have been able to reliably generate greater than 80 percent cardiomyocytes in the final population while other methods produce about 30 percent cardiomyocytes with high batch-to-batch variability.”

Palacek continued: “The biggest advantage of our method is that it uses small molecule chemicals to regulate biological signals. It is completely defined, and therefore more reproducible. And the small molecules are much less expensive than protein growth factors.”

Kamp noted that the “fact that turning on and off one master signaling pathway in the cells can orchestrate the complex developmental dance completely is a remarkable finding as there are many other signaling pathways and molecules involved.”

This protocol has the capacity to revolutionized the use of heart muscle cells for drug testing.  Also, because the Wnt signaling pathway is required during heart development, this protocol also has the ability to clarify the exact role of this pathway during heart differentiation.  Finally, if stem cells are eventually used for therapeutic purposes, this protocol or one like it will certainly be employed to convert stem cells into heart muscle cells.

Infanticide Advocate Peter Singer is Awarded Australis’s Highest Civic Award

Princeton University’s Professor of Ethics Peter Singer has been appointment as a Companion of the Order of Australia (he is a native Australian). I will not mince words on this one. This is a new low for the government of Australia. Here are some of the things Singer has advocated:

He is best known for ethically endorsing infanticide. According to Singer, people are not human persons unless they can do certain things. This is called functionalism, and it leads people to regard certain human beings as being in a class of “human non-persons.” For example, Singer does not think humans reach “full moral status” until after the age of two. He supports non-voluntary euthanasia of human “non-persons” fo0r any reason. Not liking the color of their eyes, they cry too much. they pooped on your carpet, they threw up on your nice clothes, they are a girl and not a boy. Mind you, this is the same chap who gets all choked up about the use of animals in research because is multiples animal suffering. Instead of appealing to the more noble aspects of human nature, where we exercise those properties that make us truly human (compassion, defending the weak and defenseless), Singer would have us eat our young the way brute beasts do. Furthermore, he would commend us for it. We used to demarcate between barbaric societies and civilizations that did such things. Now we have become the barbarians, but according to Singer, that’s just fine.

In keeping with this disgusting, misanthropic philosophy, Singer supports using cognitively disabled human beings in medical experiments instead of animals. The laboratory animals, you see, have a higher “quality of life” according to Singer. How does he know that? Well they can do more. They can walk, groom themselves, feed themselves, and defecate without anyone’s help. The mentally disabled person it still essentially a person, but Singer doesn’t let that get in the way. People who cannot do are not people any more. They might even be trapped inside a body that no longer works, but Singer does not let that get in the way either. As far as he is concerned, person is as person does. He forgets that must BE something to eventually DO something. He has gotten the cart before the horse and we have abortion on demand, euthanasia in Holland and Brazil as the result of it.

Singer has also defended bestiality. These are, according the Singer, “mutually satisfying activities” between humans and animals should not be opposed. Now, pray tell, how does Singer know that the animal is enjoying it? Is he also Dr. Doolittle and can talk to the animals? This is disgusting. We used to think such people were sick in the head (not to mention to horrific sexually transmitted diseases you can get from such activities), but Singer thinks they are just alright.

Singer started the “Great Ape Project.” This project would establish a “community of equals” among humans, gorillas, bonobos, chimpanzees, and orangutans. The day one of those creatures asks me for admission to such a project, I will think about it, but for now, they are too busy killing each other in the wild and spreading their feces all over each other to care about it.

Singer has also questioned whether “the continuance of our species is justifiable.” Do we need any more evidence of his own self-loathing?

Finally, Singer believes “speciesism” — viewing humans as having greater value than animals — is akin to racism. Oh, just between you and me, racism is a HUMAN concept. Bringing animals into it is a category mistake of the first degree. Humans are exceptional among the creatures of the earth. We and we alone are the stewards of the earth and its resources. The animals don’t give a rip about such things and it is not even on their cognitive radar. Human exceptionalism is the basis of human law, human rights, and everything from property values, antislavery movements, anti-genocide activities and so on. Without human exceptionalism, we become no better than the animals.

Singer’s philosophy is perverted. It takes what is profane, disgusting and devilish, and calls it morally upright. It is the result of misanthropy and self-loathing and he wants use to hate ourselves as much as he hates himself. His philosophy produces a society that is unworkable and objectionable in every way. He should not be rewarded, but derided.

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.”

When Scientists Mislead the Public

Wesley Smith has an interesting post about the ability of fat-based stem cells to differentiate into bone-making cells that make good bone. Apparently, human clinical trials are in the works. An Israeli biotechnology company used a bioreactor to grow the cells, and then seeded the stem cells into a three-dimensional scaffold. This scaffold directed the bone-making cells to form bone that resembled living human bone. These bones have been implanted into living animals and seem to be ready for human clinical trials.

Nevertheless, Smith uses these reports to reminisce back to Amendment 2 in the state of Missouri in 2006, when scientists testified before the state legislature than adult stem cells were “unipotent,” which means that they are only able to form one kind of adult cell type. This was a lie in 2006 and is even more a lie in 2012. This goes to show that scientists are funded by public money and it is not beneath them to shade the facts to get more public money. We should always view what scientist say with some degree of skepticism and criticism. Bowing to them as “experts” is an insult to the scientific method, which does not recognize authority, only the quality of evidence. Check out Smith’s blog entry here and the 2006 one here.

Reprogramming Skin Cells into Neural Stem Cells By Introducing One Gene

Transforming skin cells into nerve cells that interconnect and send nerve impulses to each other requires an extensive amount of reprogramming. The production of induced pluripotent stem cells is rather labor-intensive and introduces some risks. However, a new procedure designed by Yadong Huang at the Gladstone Institutes has shown that the introduction of a single gene into skin cell can generate nerve cells from skin cells.

This single gene, Sox2, transforms skin cells within days into early-stage brain stem cells known as induced neural stem cells or iNSCs. In culture, iNSCs self-renew and mature into neurons that can connect with each other and then transmit electro-chemical signals between each other. When the iNSCs were cultured for one month, they had already formed a completely new neural network.

An excited Huang made these points: “Many drug candidates, especially those developed for neurodegenerative diseases, fail in clinical trials because current models don’t accurately predict the drug’s effects on the human brain. Human neurons derived from reengineered skin cells could help assess the efficacy and safety of these drugs, thereby reducing risks and resources associated with human trials.”

Huang’s findings build on the work of Japanese research Shinya Yamanaka, who was the first scientist to publish the production of induced pluripotent stem cells. Since that time, other researchers have used genetic engineering techniques to directly reprogram adult cells into other types of adult cells without passing through the embryonic-stem-cell stage. Last year, Sheng Ding managed to use a combination of small molecules and genes to transform skin cells directly into neural stem cells. Huang’s technique now simplifies this technique even more so that only one gene is required to reprogram skin cells into neural stem cells. By avoiding the induced pluripotent stem cell stage, Huang and Ding hope to avoid the risk of tumor formation and the mutations induced by the production of induced pluripotent stem cells.

Karen Ring, a graduate student in Biomedical Sciences at the University of California, San Francisco, who was the lead author on this paper vouched for the safety of the iNSCs: “We wanted to see whether these newly generated neurons could result in tumor growth after transplanting them into mouse brains. Instead, we saw the reprogrammed cells integrate into the mouse’s brain, and not a single tumor developed.”

Huang’s paper also addresses the function Sox2 in the reprogramming of the skin cells. Huang and his research team also want to identify similar regulators that direct the development of specific types of neurons in the brain that tend to degenerate in the case of particular types of neurodegenerative diseases. Huang noted: “If we can pinpoint which genes control the development of each neuron type, we can generate them in the Petri dish from a single sample of human skin cells. We could then test drugs that affect different neuron types, such as those involved in Parkinson’s disease.” Huang added that such a discovery would help drug developers design treatments for neurodegenerative diseases that are much more specific, and the drug design would probably occur much faster.

Alzheimer’s disease still afflicts 5.4 million people in the US alone and this number is thought to triple by 2050. There are still no medications that can reverse the devastation wrought by this disease. Huang’s data might provide the means to test such new drugs.

Adult Stem Cell Possess Internal Machinery to Resist Ischemic Injury

Adult stem cells that have been transplantation into a sick patient are often faced with harsh conditions that lead to their untimely death before they can help the patient. Several different strategies have been applied to stem cells to “toughen them up” so that they can resist these conditions. Treating them with particular growth factors have proven effective in some experiments, as has oxygen or glucose deprivation. A recent paper in the journal ANTIOXIDANTS & REDOX SIGNALING by Gang Lu, Muhammad Ashraf, and Khawaja Husnain Haider from the Department of Pathology at the University of Cincinnati, Ohio has shown that treatment of stem cells with insulin-like growth factor-1 (IGF-1) and glucose and oxygen deprivation activate a biochemical pathway that seems to be common to many different types of stem cells that help cell resist ischemic (oxygen-poor) conditions.

This paper examined bone marrow stem cells. When the bone marrow stem cells were deprived of oxygen and glucose for 12 hours, they discovered that a well-known signaling molecule called ERK1/2 was activated. ERK1/2 stands for “extracellular signal-related kinases-1/2. These molecules are “kinases,” which simply means that they are enzymes that attach phosphate groups to other proteins. These phosphate groups change the 3-D structure of proteins can induce them to change they function. ERK1/2 are activated whenever the cell binds particular types of growth factors. Growth factor-binding sets a series of steps in motion that leads to the activation of ERK1/2. ERK1/2 phosphorylates its target, which sets a phosphorylation cascade into motion, and this changes the behavior of the cell.

There are ways to inhibit ERK1/2 activation, and when the University of Ohio team did just that (with a drug called PD98059), the bone marrow stem cells did not activate ERK1/2 when derived of oxygen and glucose and the cells died. This shows that activation of ERK1/2 occurs as a result of oxygen and glucose deprivation, and the activation of ERK1/2 is required for the cells to survive the harsh conditions.

Next, they discovered that ERK1/2 is also activated by treating the cells with IGF-1. Since IGF-1 treatment also helps stem cells adapt to harsh conditions, it is possible that these two treatments use the same internal mechanism to help stem cells adapt to harsh conditions.

What is a downstream target of ERK1/2 that throws the switch that allows cells to adapt the harsh conditions? The researchers received two clues when they discovered that drugs that prevent the release of calcium ion stores into the cell interior also prevent cells from adapting to harsh conditions. This tipped them off that a target of calcium-ion signaling was probably the downstream target of ERK1/2. That target is protein kinase C (PKC).

To shore up their hypothesis, they treated stem cells with a chemical that is known to activate PKC (phorbol esters). These chemicals completely acclimatized the cells to harsh conditions and when the bone marrow stem cells were grown after they had been engineered with a permanently active form of PKC, the stem cells did not require any preconditioning in order to resist harsh conditions.

These data are remarkable and since calcium signaling is a pathway that we know a great deal about and there are lots of chemicals available to manipulate it, it should be possible to precondition a whole host of stem cells to resist harsh conditions before they are ever used. These types of treatments should improve adult stem cell treatments for a variety of conditions.

Huntington’s Disease Model System Derived from Patient-Specific Induced Pluripotent Stem Cells

Huntington’s Chorea or Huntington’s disease is an inherited condition that results from the progressive and relentless degeneration of nerve cells in the central nervous system.  Huntington’s disease (HD) broadly affects the patient’s functional abilities and decreases his or her ability to move, think or behave properly.  Most of the time, patients develop the signs or symptoms of HD when they are 40 or 50 years old or slightly older.  In the case of Juvenile HD, symptoms begin before the age of 20.

Mutations in the HTT gene, which encodes the Huntingtin protein cause HD, and typically, the mutations in the gene that are associated with HD are so-called “triplet expansions.”  To understand triplet expansions, we must understand how genes encode proteins.  Genes are stretches of a DNA molecule that are transcribed into RNA copies.  The enzyme that synthesizes the RNA copy is called RNA polymerase, and a gene has a set of sequences that tell the RNA polymerase where to start making RNA copies and where to stop.  Once the RNA copy of the stretch of DNA is made, the RNA either has a function of its own, or the RNA is translated into protein.  Translation is the process by with RNA-protein complexes called “ribosomes” bind to the front of the RNA and use the nucleotide sequence to synthesize a protein that has a specific sequence of amino acids.  Amino acids are encoded in genes by a three-nucleotide sequence or codon, and ribosomes read the RNA molecules three nucleotides at a time.

The nucleotide sequence CAG (cytosine, adenine, guanine) encodes the amino acid glutamine.  The HTT gene has a stretch of these nucleotides, and they code for the amino acid glutamine.  Normal copies of the HTT gene will have anyways from nine to thirty-five glutamines in these stretches.  However, these CAG stretches have a tendency to expand because the enzymes that replicate DNA (DNA polymerases) have a tendency to slip when they get to CAG stretches, and this causes the CAG stretches to increase in size, or, occasionally, decrease in size.  The glutamine stretches can reach large numbers, and if the number of glutamines in the glutamine stretch exceeds 35, people will usually start showing symptoms.  The larger the number of glutamines in the glutamine stretches, the earlier the symptoms will appear (Juvenile HD usually occurs in patients with 60 or more glutamines in the glutamine stretch), and the more aggressive the disease.

How does the abnormal Huntingtin protein kill nerve cells?  This is unclear, but it is clear that Huntingtin proteins with abnormally large numbers of glutamines in their glutamine stretches are poisonous to cells, and the nerve cells that die tend to dump their neurotransmitters, which kills other nearby cells, which then cause them to dump their neurotransmitters, and the cascade of cell death begins.

Cell transplantation experiments in animals have produced a variety of positive results, but these results are probably not representative of the situation in human patients.  HD in animals, you see, is induced by the injection of chemicals into the brains of laboratory animals, and these chemicals kill off particular groups of nerve cells that cause the symptoms of the disease.  The rest of the brain is essentially normal.  Human patients have a brain that has been transformed into a toxic waste dump, and transplanted cells do not survive well in them.  I have other blogs on this site that speak about this here, here, and here.

To address this problem, a South Korean group has developed a model system for HD based on induced pluripotent stem cells made from an actual HD patient.  This paper was published in the journal Stem Cells on May 24, 2012 (doi: 10.1002/stem.1135), and is entitled:  Neuronal Properties, In Vivo Effects and Pathology of a Huntington’s Disease Patient-Derived Induced Pluripotent Stem Cells.  The lead author is I. Jeon from the CHA Stem Cell Institute, CHA University, Seoul, Korea.

In this paper, Jeon and colleagues took skin cells from HD patients and used them to make induced pluripotent stem cells (iPSCs).  By carefully manipulating the cells in culture, the South Korean scientists were able to convert the iPSCs into nerve cells.  The particular patient whose IPSCs were used in this experiment had a HTT gene that encoded a Huntington protein with 72 glutamines and the patient had a juvenile form of HD.

The specific nerve cells that degenerate in the brains of HD patients are neuron that produce the neurotransmitter GABA (gamma-amino butyric acid).  Therefore, Jeon and his coworkers had GABA-specific neurons from the iPSCs.  WHile the initial induction rate for nerve cell production from the iPSCs from the HD patient was low, they were able to produce a respectable quantity of GABA-neurons from the HD iPSCs.

Nest, they took rats and generated the types of lesions necessary to cause HD, but they transplanted the GABA-neurons that were made from the HD-iPSCs into the brains of the lesioned rats.  Interestingly, the rats recovered from the lesions and their behavior returned to normal.  At 12 weeks after the transplantations, the brains of the rats still looked normal.

However, once the rats were treated with a chemical that prevents cells from getting rid of excessive amounts of junk proteins, now the rats started to show the symptoms of HD and their brains showed pathologies that greatly resembled those found in HD patients.  Also, if the GABA neurons made from the HD iPSCs were implanted into the brains of neonatal rats, which grow very quickly, they produce HD-like pathology 33 weeks after transplantation.

What does this mean?  Even though these rats carried GABA neurons that contained a severe version of the HTT gene, the neurons still were able to work and give rise to normal neurons inside the body of the animals.  However, those animals were extremely susceptible to any sort of perturbations that caused junk proteins to build up.  If the levels of junk protein built up, they eventually killed the cells.  What are those triggers in human patients that cause cells to clog up with junk proteins?  Clearly this HD model will help neuroscience researchers answer some very vital questions about the cause and pathology of HD.  Answers that might lead to efficacious treatments that will reduce the extreme suffering of some patients.

Mifepristone – Not as Safe a Drug as you Might Think

According to data released by the US Food and Drug Administration (FDA) on the abortion pill, mifepristone, more than 1.2 million unborn children have lost their lives because of it, but even more stunningly, thousands of women have been injured and this includes more than a dozen who have died in the United States alone.

Just after the approval of mifepristone during the Clinton administration, the FDA released a report in 2006 that showed that more than 1,100 women had been subjected to “adverse effects” after taking mifepristone.  Pro-life advocates have waited five years for the FDA to come out with a new report of the adverse effects associated with this drug.  This drug seems to continue to kill and injure women all across the globe.

Mifespristone, which is marketed under the trade names Mifeprex and Korlyn, is still known by the name given to it when it was an experimental drug, RU486.  Mifepristone is a synthetic steroid drug that binds to the progesterone receptors in cells in the endometrium and prevents the progesterone receptor from receiving signals from progesterone.  Because the endometrium requires constant progesterone signaling to maintain itself, mifepristone causes the endometrium to breakdown.  It also causes the cervix to soften and induces the release of mo9lecules called prostaglandins.  These prostaglandins causes the smooth muscle of the uterus to contract, but mifepristone, also increases the sensitivity of the smooth muscle of the uterus to prostaglandins.  The breakdown of the endometrium and the contractions of the uterine smooth muscle cause the embryo to detach.  This eventually kills of all sources of progesterone production in the mother’s body, and the embryo dies.  Typically, mifepristone is followed by an oral prostaglandin (misoprostol) to increase uterine smooth muscle contraction and expulsion of the dead embryo.  Mifepristone is used to terminate pregnancies that are not older than 49 days.  The approval of mifepristone did include a Black Box Warning, as required under Subsection H.

There are several excellent articles about this FDA data.  Read about it here, here and here.  Mifepristone also has caused problems in women all around the world.  These data in this report is limited to adverse effects in the United States only.