DC Court Says that Stem Cells are Drugs

On the 23rd of July, 2012, the US District Court in Washington DC acknowledged the right of the Food and Drug Administration (FDA) to regulate clinical therapies that are made from the patient’s own processed stem cells. This case answered the question, “Does the court agree with the FDA that stem cells are drugs?”

According to the judge, the FDA is right and stem cells cultured outside the body are drugs. This ruling upholds the injunction brought by the FDA against Regenerative Sciences, the Broomfield, Colorado-based clinic that offers the Regenexx stem cell treatment procedure.

The Regenexx procedure uses mesenchymal stem cells that are isolated from patients’ bone marrow. These stem cells are then processed and injected back into the patients to treat joint pain. The FDA has labeled this procedure the “manufacturing, holding for sale, and distribution of an unapproved biological drug product.” In August 2010, the FDA ordered Regenerative Sciences to stop offering the treatment, since they were offering a drug without FDA approval

According Nature magazine science reporter, David Cyanoski, investigations by the FDA that led to the injunction showed that there were flaws in the cell processing protocol that violated the FDA’s regulations that refer to “adulteration.” These regulations are meant to ensure the safety of patients who receive the therapy.

Not surprisingly, academics are praising the decision and a shot across the bow of any enterprising physician who wants to offer stem cell treatments. For example, Jeanne Loring, a regenerative-medicine scientist at the Scripps Research Institute in La Jolla, California, has said that this court decision will send a warning to others who want to offer unapproved stem-cell treatments. In her words: “So many people want to start these companies. They say, ‘FDA? What FDA?’.”

Chris Centeno, the medical director of Regenerative Sciences and one of two majority shareholders, told Nature that he plans to appeal against the ruling. Centeno has replied to the ruling in an internet book entitled “The Stem Cells They Do Not Want You To Have.” Centeno’s main objection during the trial was that the ‘Regenexx’ procedure does not significantly modify the mesenchymal stem cells before they are reinjected into the patient. Therefore, the procedure should be considered a routine medical procedure. The company also argued that because all the processing work is done in Colorado, the procedure should be subject to state law, rather than to regulation by the FDA.

Unfortunately for Centeno, the Ninth Circuit court disagreed with both arguments. According to the court: “the biological characteristics of the cells change during the process.” This and other considerations mean that the cells are more than “minimally manipulated,” which makes them a drug a subject to regulation by the FDA. .

University of Minnesota bioethicist Leigh Turner, agrees with the court on this one. Turner noted: “It is much too simplistic to think that stem cells are removed from the body and then returned to the body without a ‘manufacturing process’ that includes risk of transmission of communicable diseases,” he says. “Maintaining the FDA’s role as watchdog and regulatory authority is imperative.”

The FDA injunction only applies to one of the Regenexx stem-cell products; the Regenexx-C procedure. In this procedure, the bone marrow mesenchymal stem cells are processed for 4–6 weeks. The Regenexx-C procedure will still be available, since after the 2010 injunction, the company moved its treatment location to an affiliated Cayman Island clinic.

Centeno plans to continue providing the other three procedures; Regenexx-SD, Regenexx-AD, and Regenexx-SCP, for joint pain, in the United States. In those treatments, the cells are reinjected within one-two days. Centeno claims that those cells are “minimally manipulated”, and that the FDA sees them as the “practice of medicine” and “has no issues” with them.

According the Nature’s David Cyanoski, until July 25th of this year, a graphic on the Regenerative Sciences website claimed that these three other procedures were “FDA approved.” However, the FDA has not approved these three procedures, and Centeno was not able to provide documentation to support his claims that the agency views the three treatments as outside its purview. This graphic was removed from the Regenexx website after Nature’s enquiries.

Stem-cell ethics and regulation expert, Doug Sipp, who is at the RIKEN Centre for Developmental Biology in Kobe, Japan, is concerned that this ruling will simply drive entrepreneurs to move their stem cell clinics outside the United States to avoid regulation. Indeed, Regenerative Sciences has done just that by setting up their Regenexx-C procedure in the Cayman Islands. According to Sipp, “Other US stem-cell outfits have close ties with partner clinics in Mexico and other neighboring countries, which are traditionally regulatory havens for other forms of fringe medicine as well. I suppose it will be business as usual in such places,”
We will have more to say about this in the days to come, but for now, this is it.

Scientists Identify the Stem Cells From Which Sweat Glands Grow

Sweat glands control our body temperatures, but little is known about how they develop and the cells that give rise to them. However, researchers from Rockefeller University have now identified in mice the stem cell population from which sweat glands originally develop and the stem cells that regenerate adult sweat glands.

In this study, scientists from the laboratory of Elaine Fuchs invented a strategy to purify and molecularly characterize the different kinds of stem cell populations that make up the complex sweat duct and glands found in mammalian skin. Afterwards, they examined how these different stem cell populations respond to normal tissue homeostasis and to different types of skin injuries. They also found how sweat glands differ from their close cousins, the mammary glands.

Elaine Fuchs, who is an investigator at the Howard Hughes Medical Institute, said: “Mammary gland stem cells respond to hormonal induction by greatly expanding glandular tissue to increase milk production. In contrast, during a marathon race, sweat gland stem cells remain largely dormant,and glandular output rather than tissue expansion accounts for the 3 liters of sweat our body needs. These fascinating differences in stem cell activity and tissue production are likely at the root why breast cancers are so frequent, while sweat gland cancers are rare.”

These findings from Fuchs’ lab might someday help improve treatment strategies for burn patients and to develop topical treatments for people who sweat too much, or too little.

“For now, the study represents a baby step towards these clinical goals, but a giant leap forward in our understanding of sweat glands,” said the study’s lead author, Catherine P. Lu, a postdoctoral researcher in Fuchs’s laboratory.

Each one of us has millions of sweat glands but they have rarely been extensively studied, and much of this has to do with the difficulty of gathering enough of the tiny organs to research in a lab. Mice are traditionally used as a model for human sweat gland studies. Therefore, for this study, Lu and colleagues laboriously extracted sweat glands from the tiny paw pads of mice, the only place they are found in these and most other mammals. The goal was to determine if the different cells that make up the sweat gland and duct contained stem (progenitor) cells that can repair damaged adult glands.

According to Lu, “We didn’t know if sweat stem cells exist at all, and if they do, where they are and how they behave.” The last major studies on the proliferative potential within sweat glands and sweat ducts were conducted in the early 1950s before modern biomedical techniques were used to understand fundamental biomedical science.

Fuchs’ team determined that just before birth, the nascent sweat duct forms as a down-growth from progenitor cells in the epidermis, the same master cells that at different body sites give rise to mammary glands, hair follicles and many other epithelial appendages. As each duct grows deeper into the skin, a sweat gland emerges from its base.

Lu then led the effort to look for stem cells in the adult sweat gland. Sweat glands are composed of two layers: an inner layer of luminal cells that produce the sweat and an outer layer of myoepithelial cells that squeeze the duct to discharge the sweat.

Lu devised a strategy to fluorescently tag and sort the different populations of ductal and glandular cells. The Fuchs team then injected each population of purified cells into different body areas of female host recipient mice to see what the cells would do.

When introduced into the mammary fat pads, the sweat gland myoepithelial cells generated fluorescent sweat gland-like structures. “Each fluorescent gland had the proper polarized distribution of myoepithelial and luminal cells, and they also produced sodium-potassium channel proteins that are normally expressed in adult sweat glands but not mammary glands,” Lu said.

When the host mice became pregnant, some of the fluorescent sweat glands began to express milk, even though they still retained some sweat gland features as well. Sweat gland myoepithelial cells produced epidermis when engrafted to the back skin of the mice.

“Taken together, these findings tell us that adult glandular stem cells have certain intrinsic features that enable them to remember who they are in some environments, but adopt new identities in other environments,” Fuchs said. “To test the possible clinical implications of our findings, we would need to determine how long these foreign tissues made by the stem cells will last, unless it is long-term, a short-term “fix” might only be useful as a temporary bandage for regenerative medicine purposes,” Fuchs said.

The findings can now be used to explore the roots of some genetic disorders that affect sweat glands, as well as ways to potential ways to treat them.

“We have just laid down some critical fundamentals of sweat gland and sweat duct biology,” Lu said. “Our study not only illustrates how sweat glands develop and how their cells respond to injury, but also identifies the stem cells within the sweat glands and sweat ducts and begins to explore their potential for making tissues for the first time.”

Umbilical Mesenchymal Stem Cells Improve the Symptoms of Patients With Decompensated Liver Cirrhosis

One of the most central organs for the body’s metabolism is the liver. When the gastrointestinal tract absorbs food molecules, the first stop for most of these molecules is the liver. The liver makes many blood-specific proteins, detoxifies foreign molecules to make them more water-soluble so that the body can excrete them, and stores energy reserves in the form of glycogen. Consequently, damage to the liver from chronic liver infections (e.g., hepatitis B & C, bilharzia or schistosomiasis, illegal drug use, etc.), alcoholism, or exposure to liver-damaging chemicals (carbon tetrachloride, chloroform, etc.) seriously compromises the capacity of the body to store energy, process food molecules, make blood specific proteins (which include clotting factors), and process and synthesize metabolic wastes. Repeated damage to the liver causes extensive scarring and deposition of fatty tissues, and such a condition is called “cirrhosis.”

Cirrhosis ultimately leads to liver failure, and tough scar tissue with nodules replaces once healthy liver tissue. There are two main types of cirrhosis. Compensated cirrhosis of the liver refers to early liver damage in which the body functions well despite the damaged liver tissue. Even though liver function is decreases, the body still operates within normal parameters, and the patient often shows no symptoms of disease. Even though people with compensated liver cirrhosis are often asymptomatic, they may display symptoms of weakness, fatigue, loss of appetite, vomiting, weight loss and easy bruising. Liver function tests may reveal increased levels of certain liver enzymes. Liver damage is not reversible, but treating the underlying cause can prevent further damage. Additionally, constant monitoring is required for the early detection of loss of liver function that leads to life-threatening complications.

Decompensated liver cirrhosis is a life-threatening complication of chronic liver disease, and it is also one of the major indications for liver transplantation. The symptoms of decompensated cirrhosis are internal bleeding from the esophagus (bleeding varices), fluid in the belly (ascites), confusion (encephalopathy), yellowing of the eyes and skin (jaundice). When someone becomes this sick, there is little to be done, but receive a liver transplant.

Can stem cells help patients with decompensated liver cirrhosis? Perhaps they can. A paper from the Journal of Gastroenterology and Hepatology (2012; 27 Suppl 2:112-20) has examined the ability of human umbilical cord mesenchymal stem cells to improve symptoms in patients with decompensated liver cirrhosis (DLC). The paper’s first author is Z. Zhang and the title of the paper is “Human Umbilical Cord Mesenchymal Stem Cells Improve Liver Function and Ascites in Decompensated Liver Cirrhosis Patients.” These authors are from the Research Center for Biological Therapy at the Beijing 302 Hospital, in Beijing, China.

In this study, the safety and efficacy of umbilical cord-derived MSCs (UC-MSC) were infused into in patients with DCL. They used a total of 45 chronic hepatitis B patients, all of whom were diagnosed with DCL. 30 patients received transfusions of UC-MSCs, and another 15 patients were given saline as the control. After transfusions, all 45 patients were followed for a 1-year follow-up period.

In none of the 45 patients who were infused, were any significant side-effects observed. Also, there were no significant complications were observed in either group. As to the symptoms suffered by the patients, those who had received the UC-MSC transfusion showed a significant reduction in the volume of ascites in comparison to those patients who had received the control saline transfusions. When liver function parameters were examined, UC-MSC therapy also significantly increased of serum albumin levels (albumin is made by the liver), decreased in total serum bilirubin levels (bilirubin is a waste that is processed by the liver), and stabilized the sodium levels for patients (patients with cirrhosis have low blood sodium levels).

Further follow-up of these patients is clearly warranted, but for the year follow-up. It seems clear that UC-MSC transfusions are clinically safe. Furthermore, when compared to controls, they also seem to improve liver function and reduce the volume of belly fluid in patients with DCL. UC-MSC transfusions might represent a novel therapeutic approach for patients with DCL.

What is the Best Way to Deliver Stem Cells to the Heart?

Three main techniques have been used to deliver stem cells to the heart. The simplest technique to introduce stem cells intravenously and hope that they will home to the heart and stay there. A more technically demanding way to introduce stem cells to the heart is to inject them into the wall of the heart. This method is called transendocardial cell injection, and it requires electromechanical mapping guidance (NOGA) in order to direct the surgeon to the site where the stem cells should be injected. Finally, stem cells are introduced to hearts by means of intracoronary delivery. Intracoronary delivery takes advantage of angioplasty technology to deliver stem cells through the coronary arteries where they move across the blood vessels and enter the heart.

Stem cell delivery by means of intravenous introduction has been shown in several studies to be extremely inefficient. The vast majority of the stem cells end up in the lungs, liver and the spleen and only a tiny, insignificant fraction goes to the heart.

The transendocardial injection method is the most difficult of the three methods, but it is also the most direct, since it deposits the stem cells directly into the cardiac muscle . This method requires special equipment and also increases the risk of rupture of the heart wall.

The intracoronary method was adapted from the same procedure that cardiologists use to implant stents. This procedure, known as percutaneous infusion (PCI), uses an over-the-wire technique to inflate the coronary artery nearest the damage, and then deposits the stem cells into the artery (see this video here). Because PCI has been used so much recently and because the technique has been greatly refined, stem cell delivery with this technology is second nature to many cardiologists. However, it does have some risk of causing narrowing of the artery (stenosis).

Which technique is better, transendocardial injection or intracoronary delivery? While some papers have compared the two procedures, there has been no randomized comparison with blinded endpoint analysis of the two techniques; until now.

In a paper published in the Journal of Cellular and Molecular Medicine by a Dutch lab at the University of Utrecht in the Netherlands, these two techniques were used to deliver stem cells to the hearts of pigs that had suffered heart attacks. The number of stem cells delivered in both cases were exactly the same, and the outcomes were compared and statistically compared. The stem cells were also labeled with a low-energy radioactive isotope so that they could be easily visualized in imaging experiments.

The results showed that both sets of pigs, which were given the stem cell treatments four weeks after the heart attack, improved about the same. Also the retention of stem cells in the heart was the same for both groups. The only difference was that the cells delivered by transendocardial injection tended to be clustered near the border of the infarct, but in the case of intracoronary delivery, the cells were spread throughout the heart muscle.

Also the safety profiles of both techniques were about the same, with the exception that the intracoronary delivery technique was easier and did not show the variation of the transendocardial technique, which is a much more difficult technique.

The authors conclude that both of these delivery techniques are feasible and safe. Furthermore, the conditions and cost of the techniques should determine which is used, since the safety and efficacy of the two is essentially the same.

Slow Adhering Skeletal Muscle Cells Improve Function of Sick Hearts

Skeletal muscles consist of cells that have fused together to form a so-called “myotube.” Myotubes, upon closer examination, are filled with contractile proteins that help them contract. These rows of contractile proteins are organized into stripes, and for this reason, skeletal muscle is often called “striated muscle” because of its stripped appearance under the microscope. Skeletal muscles are collections of these myotubes all bound together, and attached to bones by means of tendons.

Skeletal muscles also contain a stem cell population called muscle satellite cells. Muscle satellite cells divide and form new muscle in response to increase demand on the muscles. Muscle satellite cells are responsible for the increase in muscle size when you lift heavy weights.

Because muscle satellite cells are easily isolated from patients, and they resist the hostile conditions of a heart that has had a heart attack, they were one of the first stem cells used to treat heart attack patients. Preclinical work on laboratory animals produced hopeful results. The implanted satellite cells did not become heart muscle cells (Reinecke H, Poppa V, Murry CE (2002) J Mol Cell Cardiol. 34(2):241-9). However, the hearts that had received satellite transplants after a heart attack showed functional improvements and no deterioration in comparison to the control animals (rats – CE Murray, et al., J. Clin. Invest. 98(11): 2512–2523; rabbits – Blatt A,, et al., Eur J Heart Fail. 2003 Dec;5(6):751-7). These positive results were the impetus for the first human clinical trials that used a patient’s own satellite muscle stem cells as a treatment for acute heart attacks.

Early trials were quite small but the implanted patients seemed to improve. Unfortunately, these early studies were not controlled terribly well, and the results somewhat hopeful, but not completely conclusive (Siminiak T., et al., Am Heart J. 2004;148(3):531-7). More controlled clinical trials, the MAGIC and MYSTAR trials, however, revealed a problem with satellite cells. They had a tendency to not connect with the resident heart muscle cells, and were, therefore, functionally isolated from the rest of the heart (Léobon B, et al., Proc Natl Acad Sci USA. 2003;100:7808–7811). Such isolation had a tendency to cause implanted hearts to beat irregularly, and for this reason, muscle satellite clinical trials have been tabled for the time being (Menasché P, et al., Circulation. 2008;117(9):1189-200).

However, skeletal muscles possess several distinct cell types and some of these are probably better candidates for heart treatments (Winitsky SO, et al., PLoS Biol. 2005;3:e87). To that end, Johnny Huard’s laboratory at the University of Pittsburgh has characterized a population of cells that show superior therapeutic possibilities from skeletal muscle.

Masaho Okada was the lead author of this paper, and he and his colleagues observed that most of the cells in skeletal muscle adhere very readily to the culture flasks after the muscle tissue was pulled apart. They designated these fast adhering cells as RACs or rapidly-adhering cells. A minority population of cells were slow-adhering cells or SACs.

Comparisons of SACs and RACs showed that the SACs were more resistant to cellular stresses than their RAC counterparts. SACs also more readily formed myotubes that RACs. The gene expression profiles of the two cell populations were also sufficiently different to confirm that even though these two cell populations were clearly derived from skeletal muscle, they were distinct populations.

Finally, transplantation of SACs into the heart of laboratory animals that had suffered heart attacks showed definitively, that SACs improved cardiac function better than RACs. Also, SACs decreased the quantity of scar tissue in the heart and increased the number of blood vessels that had formed since the heart attack. There was also less cell death in the SAC-implanted hearts as opposed to the RAC-implanted hearts.

From these data, it seems clear that the SAC population more effectively improves heart function than the RAC population. If such a population exists in the skeletal muscles of adult humans, then such cells might prove more effective for cardiac treatments than muscle satellite cells. The only caveat is that such cells may not exist in humans, since searches for such cells have not turned up anything useful to date (see Susanne Proksch, et al., Mol Ther. 2009; 17(4): 733–741).

Stem Cells From Burnt Tissue May Augment Burn Treatment

Researchers from the Netherlands have discovered that cells from the non-viable tissue that remains after burn injuries, which are normally removed by debridement, to prevent infection, are a potential source of mesenchymal cells that can be used for tissue engineering. In this study, the research team of Magda Ulrich compared cells isolated from burn eschars (dry scabs or sloughs formed on the skin as a result of a burn or by the action of a corrosive or caustic substance) with fat-derived stem cells and dermal fibroblasts, and determined how well they conform to those criteria established for multipotent mesenchymal stromal cells.

According the Dr. Ulrich, who is member of the Association of Dutch Burn Centers in the Netherlands: “In this study we used mouse models to investigate whether eschar-derived cells fulfill all the criteria for multipotent mesenchymal stromal cells as formulated by the International Society for Cellular Therapy (ISCT). The study also assessed the differentiation potential of MSCs isolated from normal skin tissue and adipose tissue and compared them to cells derived from burn eschar.”

Burn treatment advances have increased the percentage of patients who survive severe burn injuries. This growing survival rate has also increased the number of people who are left with burn scars, and these scars cause skin problems, such as contracture (shortening and hardening of muscles, tendons, or other tissues that leads to deformity and rigidity of joints), and the social and psychological aspects of disfigurement.

Tissue engineering attempts to rebuild the skin are some of the most promising approaches to addressing these problems. Unfortunately, two shortcomings with this approach include finding a viable source of stem cells for the therapy and designing the scaffold that produces a suitable microenvironment to guide the stem cells toward those behaviors that engender tissue regeneration.

“The choice of cells for skin tissue engineering is vital to the outcome of the healing process,” Ulrich said. “This study used mouse models and eschar tissues excised between 11 and 26 days after burn injury. The delay allowed time for partial thickness burns to heal, a process that is a regular treatment option in the Netherlands and rest of Europe.”

Since elevated levels of MSCs have been detected in the blood of burn victims, Ulrich and her co-workers suspected that shortly after being burned, the severely damaged tissues attract stem cells from the surrounding tissues,.

“MSCs can only be beneficial to tissue regeneration if they differentiate into types locally required in the wound environment,” Ulrich said. “We concluded that eschar-derived MSCs represent a population of multipotent stem cells. The origin of the cells remains unclear, but their resemblance to adipose-derived stem cells could be cause for speculation that in deep burns the subcutaneous adipose tissue might be an important stem cell source for wound healing.”

Further work is needed to properly identify the origins of the stem cells found in the burn eschar, and how their function is influenced by the wound environment.

Nanoscale Scaffolds and Stem Cells Show Promise For Cartilage Repair

Tissue engineers have designed scaffolds for stem cells made from nanotubes that induce them to form cartilage. These nanofibers are tiny, artificial fiber scaffolds that are thousands of times smaller than a human hair. Cartilage formation has succeeded in laboratory and animal model systems.

Much more work is necessary before these scaffolds can be used inside a human body; the results of this present study hold promise for designing new techniques to help millions who endure joint pain.

Jennifer Elisseeff, Ph.D., Jules Stein Professor of Ophthalmology and director of the Translational Tissue Engineering Center at the Johns Hopkins University School of Medicine, said: “Joint pain affects the quality of life of millions of people. Rather than just patching the problem with short-term fixes, like surgical procedures such as microfracture, we’re building a temporary template that mimics the cartilage cell’s natural environment, and taking advantage of nature’s signals to biologically repair cartilage damage,”

Unfortunately, cartilage does not repair itself when damaged. For the last decade, Elisseeff’s research team has been trying to better understand the development and growth of cartilage cells (chondrocytes). Part of these studies has involved building scaffolds that mimics the environment inside the body that produces new cartilage tissue. The cartilage-making environment consists of a three-dimensional mix of protein fibers and gel. This matrix provides support to connective tissue throughout the body, and physical and biological cues for cells to grow and differentiate.

In the laboratory, the Elisseeff team created a nanofiber-based network that utilized a process called electrospinning. Electrospinning shoots a polymer stream onto a charged platform, to which a compound called chondroitin sulfate is added. Chondroitin sulfate is commonly found in many joint supplements. After characterizing the manufactured fibers, they made several different scaffolds from spun polymer or spun polymer plus chondroitin sulfate. Elisseeff and her colleagues then seeded the scaffolds with bone marrow stem cells from goats in order to test how well the scaffolds supported the growth of the stem cells.

The results showed that the scaffold-supported stem cells formed more voluminous, cartilage-like tissues that those grown without the manufactured scaffolds.

“The nanofibers provided a platform where a larger volume of tissue could be produced,” said Elisseeff, adding that 3-dimensional nanofiber scaffolds were more useful than the more common nanofiber sheets for studying cartilage defects in humans.

Next, Elisseeff and her group tested the ability of these nanotube scaffolds and the cartilage they produce in an animal model. They implanted the nanofiber scaffolds into damaged cartilage in the knees of rats. They compared the quality of the knee repair to damaged cartilage in knees that were not treated with any stem cells. The nanofiber scaffolds improved tissue development and repair. The nanofiber-implanted knees produced far more cartilage as measured by the production of collagen, which is a component of cartilage. The nanofiber scaffolds induced the production of larger quantities of a more durable type of collagen, which is typically absent in surgically repaired cartilage tissue. In rats, the limbs with damaged cartilage treated with nanofiber scaffolds generated a higher percentage of the more durable collagen (type 2) than those damaged areas that were left untreated.

“Whereas scaffolds are generally pretty good at regenerating cartilage protein components in cartilage repair, there is often a lot of scar tissue-related type 1 collagen produced, which isn’t as strong,” said Elisseeff. “We found that our system generated more type 2 collagen, which ensures that cartilage lasts longer. Creating a nanofiber network that enables us to more equally distribute cells and more closely mirror the actual cartilage extracellular environment are important advances in our work and in the field. These results are very promising.”

Embryonic Stem Cell Cultures Fluctuate Between Pluripotence and Totipotence

In the journal Nature, a fascinating paper appeared from the laboratory of Samuel Pfaff at the Howard Hughes Medical Institute at the Salk Institute for Biological Studies in La Jolla, California near San Diego. In this paper, Todd Macfarlan and his colleagues show that embryonic stem cells cycle between a very primitive developmental stage and a later stage. This cycling is also due to gene expression that is linked to transposable DNA elements.

First, we need some background. The term “totipotent” means that a cell can form any structure in the embryonic or adult body. For example, when the egg undergoes fertilization, it becomes a zygote, which has the capacity to grow into the embryo and all the extraembryonic membranes (amnion, chorion, allantois, placenta, and so on). Another example is a sponge. When a small piece of the sponge is cut from it, the cells in that small piece can de-differentiate and grow into an entire new sponge. Sponge cells are, therefore, totipotent.

Secondly, there is a term “pluripotent,” which means that the cells can form all the adult cell types. Embryonic stem cells are generally thought to be pluripotent and not totipotent. Once the embryo forms the two-cell stage, these two blastomeres are totipotent. However, when the blastocyst stage forms, the inner cell mass cells become pluripotent and lose the ability to form the placenta.

Many years ago, Beddington and Robertson, (1989) implanted mouse embryonic stem cells into the outer layer of cells (trophoblast) to determine if the inner cell mass cells could form the placenta (Development 105, 733–737). The embryonic stem cells were incorporated into the placenta at a very low rate. These data led to an intriguing question: Was the low incorporation due to contamination with trophoblast cells, or could a small proportion of the embryonic stem cells actually become placenta? When gene expression studies examined embryonic stem cells, gene expression was stable in the majority of the cells, but unstable in a small minority of cells (a condition called metastable). It was not surprising that embryonic stem cells were a mixed bag of different cells, but some cells expressed genes that were normally found at earlier developmental stages or were normally expressed in cells that make placenta:
1. Niakan, K. K. et al. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev. 24, 312–326 (2010).
2. Hayashi, K., Lopes, S. M., Tang, F. & Surani, M. A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3, 391–401 (2008).
3. Singh, A. M., Hamazaki, T., Hankowski, K. E. & Terada, N. A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25, 2534–2542 (2007).
4. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007)
5. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

Weird, huh?

Into the fray comes Macfarlan and company to save (or explain) the day. It turns out that our genomes are loaded with DNA from transposable elements. These DNA elements either have or had at one time, the ability to jump from one location in the genome to another. There are large numbers of these transposable elements in our genomes and almost 50% of the human genome is composed of the remains of these elements.  Current transposable elements include Long INterspersed Elements (LINEs), Short INterspersed Elements (SINEs) and SVA (SINE/VNTR/Alu) elements.  Others include elements such as Mariner, MIR, HERV-K, and others.  The significance of all this is that during development, when the embryo gets to the two-cell stages, in the mouse, particular transposable elements are expressed at very high levels (they produce 3% of the transcribed messenger RNAs, see Peaston, A. E. et al., Dev. Cell 7, 597–606 (2004); Evsikov, A. V. et al., Cytogenet. Genome Res. 105, 240–250 (2004); Kigami, D., et al., Biol. Reprod. 68, 651–654 (2003)), and after two-cell stage, the expression of these transposable elements is silenced (Svoboda, P. et al. Dev. Biol. 269, 276–285 (2004); Ribet, D. et al. J. Virol. 82, 1622–1625 (2008)).

Since these transposons are characteristic of gene expression at the two-cell stage, they can be used as a marker for embryonic stem cells that have reverted back to the two-cell stage.  MacFarlan and his co-workers made a reporter gene and placed it into embryonic stem cells that was controlled by the same sequences as the transposons that are activated at the two-cell stage.  After growing these engineered embryonic stem cells in culture, they discovered that a small minority of cells expressed this reporter gene.

Did these reporter-expressing cells have characteristics like unto those of the two-cell stage embryos?  Yes they did.  When Macfarlan and his buddies examined the genes expressed in the cells that expressed the reporter, they found that the traditional genes that are so characteristic of inner cell mass cells (Oct4, Nanog, Sox2, etc.) were not expressed and other genes normally expressed in two-cell stage embryos, such as Zscan4, were expressed.  Other features that are found in two-cell-stage embryos were also found in these cells that expressed the reporter gene. (methylation of histone 3 lysine 4 (H3K4) and acetylation of H3 and H4 for those who are interested).

Finally, the reporter-expressing cells were able to contribute to the formation of the placenta when transplanted into a mouse embryo.  This shows that these cells not only express the genes of the totipotent stage of development, but they also are totipotent.

These experiments show that most, maybe all embryonic stem cells pass through a short-lived state during which they display features that are characteristic of the totipotent two-cell stage: unlike the vast majority of the ES cells in the culture.  During this transition, they lack expression of the pluripotency-promoting proteins Oct4, Sox2 and Nanog, and have the ability to form cells of both the placenta and the fetus.

There is also a moral implication of these experiments.  In his book Challenging Nature and on the book’s web site, Lee Silver argues that embryonic stem cells are essentially embryos, and if we don’t object to using and discarding embryonic stem cells, then we should not have any problem with using and discarding embryos.  His reasons for asserting that embryonic stem cells are embryos is that in the mouse, embryonic stem cells can be inserted into the inner cell mass of an embryo that has four copies of each chromosome.  The tetraploid embryos can form the placenta, but they cannot form the embryo that is attached to the embryo.  Inserting embryonic stem cells into the inner cell mass of these embryos will rescue them from dying because the embryonic stem cells with make the embryo and the tetraploid embryos will form the placenta.  This experiment is called “tetraploid rescue” and Silver uses it to argue that embryonic cells are essentially embryos.

I find this argument unconvincing for several reasons.  First of all, these embryonic stem cells are being manipulated by being inserted into an embryo.  Granted this embryo is abnormal, but it is an embryo all the same and it provides a vital function that the embryonic stem cells cannot supply – the making of the placenta.  This manipulation helps the embryonic stem cells make the embryo, but not everything else.  In this case the embryonic stem cells are only doing part of the job and they are also receiving the structure and inductive signals from the tetraploid embryo to form the embryo proper.  This is something that embryonic stem cells do not do in culture.

Secondly, embryos undergo development, a process that we understand rather well.  This process of development has a goal toward which the embryo proceeds during development.   Embryonic stem cells are not in the process of development.  They can be induced to form particular cell types or even tissues, but this is part of the embryo or fetus and forming part of the fetus does not constitute embryonic development but only a small part of it.  Embryos do not go backwards during development.  Cells that do go backwards are usually cancer cells that grow uncontrollably and cannot move to a more differentiated state that puts the brakes on cell division.  The fact that embryonic stem cells do move developmentally backward is another indication that they are not embryos.  They do something that embryos do not do and this disqualifies them from being embryos.

Thus another argument against the humanity of the early embryo falls into the pit of very bad arguments.

Sox2 Marks Incisor Stem Cells

Finnish stem cell researchers have discovered a gene that serves as a marker for front teeth. Researchers in the group of Professor Irma Thesleff at the Institute of Biotechnology in Helsinki, Finland have developed a method to record the division, movement, and specification of these dental stem cells. Apparently, building a tooth requires a detailed recipe to instruct cells to differentiate towards proper lineages and form dental cells.

Building a tooth from stem cells is a very difficult talk. However the development of new bioengineering protocols might make this possible in a few years. There is definitely a demand for tissue engineered teeth, since tooth loss affects oral health, quality of life, and your appearance. To build a tooth, a detailed recipe to instruct cells to direct cells to differentiate towards proper cell lineages and form dental cells is needed. However, in order to study of stem cells, scientists need a specific protein that only those cells make (a marker), that allows the isolation of and purification of dental stem cells. Unfortunately, the lack of an identifiable marker has hindered such studies so far.

The mouse system is an excellent system for such studies, since mouse incisors grow continuously throughout life and this growth is fueled by stem cells located at the base of the tooth. In Professor Thesleff’s lab, her students traced the descendants of genetically labeled cells, and showed that a gene called Sox2 labels stem cells that give rise to enamel-forming ameloblasts as well as other cell lineages of the tooth.

Even though human teeth don’t grow continuously, the mechanisms that control and regulate their growth are similar to those in mouse teeth. Therefore, the discovery of Sox2 as a marker for dental stem cells is an important step toward developing a complete bioengineered tooth.

In the future, it may be possible to grow new teeth from stem cells to replace lost ones, said researcher Emma Juuri, a co-author of the study.

Letter from a Nurse With MS – FDA’s Cells = Drugs Hurts People

At the Regenexx Blog site is a letter from a Registered Nurse who has Multiple Sclerosis. The drugs for MS do little to stop the progression of the disease and they have remarkably bad side effect. On top of that they are very expensive. Despite some exceedingly robust results with animals models with a form of MS and stem cell treatments, and human clinical trials with a patient’s own mesenchymal stem cells, the FDA has yet to budge because according to your FDA cells = drugs and they get to regulate them into the ground.

This letter from the nurse really nails it on the head and shows how the FDA’s policy is a) crap, and b) actively hurting sick people. Read about it here, and then write your Congressperson.

Getting Genes into Stem Cells Without Viruses

Genetic engineering of cells and, in particular, of stem cells has the ability to adjust the functional capacities of cells. Unfortunately, genetically engineering cells requires the use of viruses that introduce genes into cells and, by doing so, produce mutations in cells.

However, there are new ways to put genes into cells without the use of viruses. By surrounding DNA that encodes the genes you want to put into cells with positively-charged lipids, you have made a structure called a liposome. Liposomes can fuse with the membranes of cells and deliver the genes to cells without viruses that can cause mutations.

A paper that has appeared in the journal Stem Cells and Development examined the use of liposomes to introduce genes into blood cell-making stem cells (HSCs). They used commercially-available systems to transfer genes into these stem cells, but they found that their own lab-designed system did a better job than the commercially-available systems.

The lead author of this paper is Hilal Gul-Uludag and the senior author is Jie Chen from the University of Alberta in Edmonton, Alberta, Canada. In this paper, Chen’s research group isolated blood cell-making stem cells from umbilical cord blood. Then they used liposomes to insert the CXCR4 gene. The CXCR4 gene encodes a receptor for “stromal cell-derived factor-1alpha” (SDF-1alpha). When cells bind to SDF-1alpha, they move towards the source of SDF-1alpha.

Interestingly, one of the best sources of SDF-1alpha is the bone marrow. If HSCs could be engineered to make CXCR4, then they would readily move into the bone marrow. This means that implanted HSCs would only need to be introduced into the peripheral blood and not into the bone. This would increase the efficiency of bone marrow or umbilical cord transplants.

Chen’s group showed the feasibility of such experiments, and that these treatments are not toxic in any way to the HSCs. Thus, such a strategy could potentially increase the efficiency of bone marrow and umbilical cord blood transplantation.

Neurons Derived from Cord Blood Cells

A research group at the Salk Institute in San Diego has discovered a new protocol for converting umbilical cord blood cells into neuron-like cells. These new cells could prove valuable for the treatment of a wide variety of neurological conditions, including stroke, traumatic brain injury and spinal cord injury.

Physicians have used umbilical cord blood for more than 20 years to treat many different types of illnesses, including cancer, immune disorders, and blood and metabolic diseases. However, these Salk Institute researchers demonstrated that cord blood (CB) cells can be differentiated into cell types from which brain, spinal and nerve cells arise.

Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory, who led the research team, said: “This study shows for the first time the direct conversion of a pure population of human cord blood cells into cells of neuronal lineage by the forced expression of a single transcription factor.”

Izpisua Belmonte’s group used an engineered retrovirus to introduce a gene called Sox2, a transcription factor that acts as a switch inside cells that converts them into neurons. Therefore, by introducing Sox2 into CB cells, and culturing them in the lab, the cells formed colonies that expressed genes normally found in neurons.

Were these cells actual neurons or faux neurons? Cells might make neuron-specific genes, but they do not assemble those gene products into neuron-specific machinery, then they are not neurons. To if such cells are neurons, they should be able to manipulate the electrical charges across their cell membranes. But subjecting cells to electrophysiological tests, they determined that these new cells, which they called induced neuronal-like cells or iNCs, could transmit electrical impulses. This shows that the iNCs were mature and functional neurons. Next, they implanted these Sox2-transformed CB cells to a mouse brain and found that they integrated into the existing mouse neuronal network and were capable of transmitting electrical signals like mature functional neurons.

Mo Li, a scientist in Belmonte’s lab and a co-first author on the paper, said: “We also show that the CB-derived neuronal cells can be expanded under certain conditions and still retain the ability to differentiate into more mature neurons both in the lab and in a mouse brain. Although the cells we developed were not for a specific lineage-for example, motor neurons or mid-brain neurons-we hope to generate clinically relevant neuronal subtypes in the future.”

Scientists can use these cells in the future to model neurological diseases such as autism, schizophrenia, Parkinson’s or Alzheimer’s disease.

CB cells offer several advantages over other types of stem cells. First, they are not embryonic stem cells and are not controversial. They are more plastic, or flexible, than adult stem cells from sources like bone marrow, which may make them easier to convert into specific cell lineages. The collection of CB cells is safe and painless and poses no risk to the donor, and they can be stored in blood banks for later use.

“If our protocol is developed into a clinical application, it could aid in future cell-replacement therapies,” said Li. “You could search all the cord blood banks in the country to look for a suitable match.”

X (Chromosome) Marks the Plot

In female mammalian embryos, the X chromosome represents a problem. Since mammalian females have two X chromosomes, the embryo contains twice as much of the gene products of the X chromosome as opposed to male mammalian embryos, which only have one copy of the X chromosome. How is this problem solved? X chromosome inactivation (XCI). XCI occurs very early during female mammalian development, and it occurs on a cell-by-cell basis, and occurs randomly. The embryo has some cells that have one copy of the X chromosome inactivated and all the other cells have the other copy of the X chromosome inactivated. This is the reason the bodies of mammalian females are mosaics in which some cells have one copy of the X chromosome inactivated and yet other cells in which the other copy of the X chromosome is inactivated. Thus genetic diseases that map to the X chromosome will affect the entire body of the mammalian male but only a portion of the mammalian female’s body.

What does this mean for stem cells? Quite a bit. Embryonic stem cells are derived from the inner cell mass of the blastocyst-stage embryo. This is precisely the time when the cells of the embryo begin to randomly select a copy of the X chromosome to inactivate. The timing of XCI differs slightly from one species to another. In mice, for example, both copies of the X chromosome are active in mouse embryonic stem cells (ESCs) (Fan and Tran, Hum Genet 130 (2011):217-22; Chaumeil, et al., Cytogenet Genome Res 99 (2002):75-84), and XCI occurs when the cells differentiate (Murakami, et al., Development 138 (2011):197-202). Human ESCs, however, vary tremendously (Dvash and Fan, Epigenetics 4 (2009):19-22), with a few hESC lines showing activation of both copies of the X chromosome and many others showing inactivation of one or the other copy of the X chromosome. Human induced pluripotent stem cells (iPSCs) are derived from adult cells that already have one copy of the X chromosome inactivated. Therefore, de-differentiation of adult cells into iPSCs undoes XCI and activates both copies of the X chromosome (Maherali, et al., Cell Stem Cell 1 (2007):55-70 & Hanna, et al., PNAS 107 (2010):9222-7).

XCI is a process that is linked to pluripotency. The genes necessary for the maintenance of pluripotency (OCT4, Sox2, Nanog) all repress genes necessary for XCI (Xist) and activate genes that repress XCI (Tsix). Therefore, XCI seems to be a factor in the down-regulation of pluripotency in early embryonic cells.

There is a new study that underscores this link between XCI and pluripotency. Researchers at the Gladstone Institutes at the University of California, San Francisco have expanded upon the so-called Kyoto method for making iPSCs. The Kyoto method uses an animal cell line that grows in the culture dish and makes a protein called LIF (leukemia inhibitory factor). LIF activates the growth of cultured iPSCs and allows them to grow and establish an iPSC line.

According to Kiichiro Tomoda from the Gladstone Institute, iPSC derivation on LIF-making feeder cells always produces IPSCs that have two active copies of the X chromosome. However, if iPSCs are derived on feeder cells that do not make LIF, the result is very poor iPSCs derivation and the resultant iPSCs only have one active copy of the X chromosome. Furthermore, by passaging iPSCs that were derived from non-LIF-making feeder cells on LIF-making feeder cells, the inactivated X chromosome became active. This shows that iPSC derivation is highly sensitive to the environment in which the cells are derived. If also shows how to make iPSCs that more closely resemble early embryonic cells.

Beta-Blocker Enhances the Survival of Implanted Mesenchymal Stem Cells After a Heart Attack

Transplantation of adult stem cells into the heart after a heart attack has shown remarkable promise as a treatment for heart patients. The implanted stem cells improve heart function, reinforce heart structure, improve blood circulation in the heart, and reduce the size of the heart scar. Such treatments. however, are hampered by the lack of persistence of implanted stem cells. Only a vast minority of the implanted stem cells survive in the inhospitable environment of the infarcted heart, and the massive cell die-off limits the efficacy of stem cell transplants in the heart.

Fortunately, there are ways to allay this problem. Genetically engineering stem cells to express proteins known to enhance cell survival is one way to ensure that implanted cells survive when implanted. However, getting FDA approval for a clinical trial with genetically-engineered cells will prove to be immensely difficult. A more promising approach is to pretreat the cells with various growth factors, growth conditions or drugs to precondition them to survive in the heart. To that end, scientists at the Davis Heart and Lung Research Institute at Ohio State University have used a commonly prescribed heart drug called “carvedilol” to enhance the survive of bone marrow mesenchymal stem cells in the heart.

Faternat Hassan and his colleagues in the laboratory of Mahmood Khan treated mesenchytmal stem cells (MSCs) from rats with carvedilol and a related drug called “atenolol.” These drugs are members of a drug category called “beta-blockers.”

Beta-blockers are given to lower blood pressure, or to protect the heart after a heart attack from undergoing further deterioration. They bind to the receptors for epinephrine and norepinephrine and block them, which slows the heart down and reduces blood pressure. After a heart attack, however, Beth Haebecker at Oregon Health and Science University has shown that the sympathetic nerves to the heart make very large amounts pf norepinephrine and this is responsible for the remodeling and eventual deterioration of the heart. Beta-blockers can prevent this norepinephrine-based deterioration of the heart.

Over ten years ago, Yue et al. (1992) and Feuerstein (1998) showed that carvedilol has the ability to quench the deleterious effects of damaging molecules. Therefore, carvedilol might protect stem cells from dying in the heart after transplantation.

To begin, Khan’s group cultured MSCs with carvedilol and atenolol for one hour and then subjected the cells to chemical stress by treating them with hydrogen peroxide. The carvedilol-treated cells survived the hydrogen peroxide treatment much better than either the atenolol-treated MSCs or the negative controls that were not pretreated with anything.

For their next experiment, they divided into five groups of six animals each. The first group was operated on but were not give heart attacks. The second group was given heart attacks and no further treatments. The third group was given carvedilol (5 mg/kg body weight) after the heart attack. The fourth group, was MSC treatments, and the fifth group received MSC transplantations plus carvedilol at the previously mentioned dosages. The results showed that the MSC + carvedilol group fared substantially better than all the rest (except for the sham operated group). The heart structure and heart physiology were far superior in the MSC + carvedilol group.

Finally, Khan’ group made a remarkable discovery. Carvedilol prevented the heart from undergoing extensive cell death and decreased the formation of scar tissue. When combined with MSCs, carvedilol’s effect on cell death was amplified. Further investigation demonstrated that carvedilol prevented activation of a protein called “caspase-3.”

Caspases are proteins that degrade other proteins, but they are activated when the cell is damaged beyond all reasonable expectations of repair and the only fitting response for the cell is to die. This process of programmed cell death is called “apoptosis.” The induction of apoptosis is, as you might guess, very tightly controlled, and one of the main regulators of the initiation of apoptosis are the caspases. Caspases exist as inactive enzymes in the cell, but they are activated if the cells is exposed to drugs,conditions, or chemicals that induce cell death. There are three caspases that activate the rest of them and they are caspase 3, 8, & 9, and of these, caspases 3 and 9 are the most important.

Carvedilol treatment caused a significant down-regulation of caspase-3 in heart muscle cells after a heart attack. Furthermore, it prevented the expression of caspase-3 in implanted MSCs, thus increasing MSC survival. Additionally, genes that are known to improve cell survival were also activated in heart muscle cells after carvedilol and MSC treatment.

Thus carvedilol did double duty. It helped the ailing heart, but it also helped the heart help itself by preventing the untimely death of transplanted MSCs. This allowed the MSCs to work their healing processes for a much longer time. The final result was that the carvedilol + MSC-implanted rats showed hearts that were in much better shape than the those in the other groups with the exception of the sham-operated group.

This also suggests that carvedilol should be used with transplanted MSCs in the next clinical trial that utilizes transplanted MSCs.

Unique Drug Responses of Stem Cells from Parkinson’s Patients

Induced pluripotent stem cells (iPSCs) are made from adult cells by means of genetic engineering techniques that introduce specific genes into the adult cell and force it to de-differentiate into an embryonic-like cell. This procedure might provide cells for therapeutic uses some day, but this technology must overcome the mutations introduced into these cells by this procedure and the tumors they can cause. Until then, iPSCs will remain off-limits as therapeutic tools.

That does not disqualify iPSCs as tools for research and even therapeutic investigation. This present paper that comes from a collaborative effort led by Ole Isacson, professor of neurology at McLean Hospital and Harvard Medical School in Boston, uses this very strategy to examine the response of patients with particular forms of Parkinson’s disease to various drugs.

Parkinson’s disease is a progressive, insidious disease that affects a portion of the brain called the midbrain. Within the midbrain is a black body called the substantia nigra, which is Latin for “black stuff.” The substantia nigra is rich in neurons that release a neurotransmitter called “dopamine.”

First of all, to review, neurotransmitters are chemicals that neurons (the cells that make and transmit nerve impulses to other neurons in the brain) use to talk to each other. Neurotransmitters bind to the surfaces of nearby neurons and initiate the production of a nerve impulse. If the neuron receives enough neurotransmitter, it will generate a nerve impulse. Neurons typically can only respond to particular neurotransmitters. The neurotransmitters to which they respond elicit particular responses from them.

Parkinson’s disease results from the death of dopamine-releasing neurons in the midbrain. These neurons connect to cells of the “striatum.” The striatum is responsible for balance, movement control, and walking. Dopamine, produced in the substantia nigra, passes messages between the striatum and the substantia nigra, and when the cells of the substantia nigra deteriorate, which is the case of Parkinson’s disease, there is a corresponding decrease in the amount of dopamine produced between these cells. The decreased levels of dopamine cause the neurons of the striatum to fire uncontrollably, and this prevents the patient from properly controlling their direct motor functions.

Most of the cases of Parkinson’s disease are spontaneous and have no apparent cause. However, there are several types of inherited forms of Parkinson’s and mutations in approximately 17 different genes are associated with inherited forms of Parkinson’s disease.  Of these, only nine have been studied in any detail.  Nevertheless, two genes in particular are important in this paper.

Isacson found two Parkinson’s patients with inherited forms of the disease.  One of then had a mutation in the LRRK2 (Leucine-rich repeat kinase-2) gene, which encodes the Dardarin protein and is intimately involved in the onset of Parkinson’s disease.  The other had a mutation in the PINK1 gene (PTEN induced putative kinase 1), which encodes a protein known to enter mitochondria (the powerhouses of the cell).  Isacson used cells from each patient to make iPSCs.  He also used additional patients, and he had a total of 3 patients with mutations in LRRK2 and two with mutations in PINK1.

Because mutations in LRRK2 and PINK1 are thought to interfere with the function of mitochondria in neurons, Isacson examined the mitochondria of these patient-specific iPSCs.  When compared to mitochondria from volunteers without Parkinson’s disease, Isacson found that the Parkinson’s patient-specific iPSCs were much more susceptible to damage after exposure to toxins.  Thus, the mitochondria of these patient-specific iPSCs were certainly much more fragile than normal mitochondria.

Could this mitochondrial fragility be ameliorated with medicines?  Isacson tested the ability of particular substance to mitigate this condition in the patient-specific iPSCs.  A supplement called Q10, which is known to aid mitochondrial function was administered the to Parkinson’s patient-specific iPSCs, was given to the cells, and all cells were prevented from experiencing mitochondrial damage after exposure to toxins.  However, when a different drug called rapamycin was administered, the results were very different.  Rapamycin diminishes the immune response of an organism, and therefore, it can spare weak cells from being cleared by the immune system.  Rapamycin prevented damage in the cells with mutations in LRRK2, but not those with mutations in PINK1.

This paper shows how iPSC-based research can lead to information that can fashion personal treatments for each patient.  Even though this work focused on Parkinson’s disease, there are many other diseases that could benefit from iPSC-based research.

Kobe Bryant’s Knee Treatment (Orthokine) Versus Platelet-Rich Plasma

Chris Centeno at his Regenexx blog has a really good article on the Orthokine knee treatment that Kobe Bryant traveled to Germany to receive. Because Dr. Centeno has done quite a few Platelet-Rich Plasma (PRP) injections and has a fairly wide patient database to tap, his opinion should not be treated lightly.

In a word, the data in hand does not support the efficacy of the Orthokine treatment. The WOMAC Knee Function Study that was published in the journal Osteoarthritis and Cartilage in 2008 showed that Orthokine was only slightly more effective than placebos.

Thus, it is expensive, and is only a little better than placebo treatments. PRP, on the other hand, is cheaper, you do not have to go to Germany for it, and has a much better chance of working. Therefore, if you want to try one of the two, Centeno strongly argues that you should pick the PRP treatment rather than the Orthokine treatment.

See Centeno’s excellent post here.

How Stem Cells Make New Skin Cells Throughout Life

Beneath the upper epidermal layers of our skin lies a layer of stem cells and their progeny (human epidermal progenitor cells) that continually make new skin throughout our lifetime. How these stem cells manage to form skin and not some other structure is still poorly understood, but a new study from the University of San Diego School of Medicine in the laboratory of George L. Sen has pulled back the curtain on this vital process.

Sen and his colleagues have examined a component of the machinery of the cell known as the “exosome.” The term exosome is confusing because it refers to two different entities. Exosomes are vesicles secreted by cells that are loaded with proteins and RNA molecules that the cell wants to dump (Kooijmans, et al., Int J Nanomedicine. 2012; 7: 1525–41). Exosomes are used by cells to export materials to other others cells. Cells also use exosomes to regulate processes, since by ridding themselves of proteins and RNAs that direct particular processes, effectively shuts those processes down. However, exosome also refers to a complex of proteins that are involved in 3′–5′ exonucleolytic degradation. This exosome consists of ~11 proteins that degrade RNAs and regulate processes.

In skin-based stem cells, the exosome (RNA degradation machinery) functions in skin stem cells and provides one of the main mechanisms by which stem cells stay stem cells and skin cells stay skin cells. Exosomes and their targets may help point the way to new drugs or therapies for not just skin diseases, but other disorders in which stem and progenitor cell populations are affected.

Stem cells can divide throughout their lifetime, and their progeny can differentiate to become any required cell type. The progeny of stem cells, progenitor cells, have more limited developmental capabilities, and are only able to divide only a fixed number of times and form a few distinct cell types. When it comes to skin, progenitor and stem cells deep in the epidermis constantly produce new skin cells called keratinocytes that gradually rise to the surface where they will mature, die, and be sloughed off.

Exosomes degrade and recycle different RNA molecules, such as messenger RNAs that wear out or that contain errors. Such errors would cause the production of junk protein, and this would be deleterious to the cell.

According to Sen: “In short, the exosome functions as a surveillance system in cells to regulate the normal turnover of RNAs as well as to destroy RNAs with errors in them.” Sen and his colleagues discovered that in the epidermis the exosome functions to target and destroy mRNAs that encode for transcription factors that induce differentiation. One of the targets of the exosome in epidermal progenitor cells is a transcription factor called GRHL3. GRHL3 promotes the expression of genes necessary for skin cell differentiation. Routine destruction of GRHL3 keeps epidermal progenitor cells undifferentiated. When the epidermal progenitor cells receive signals to differentiate, the progenitor cells down-regulate the expression of certain subunits of the exosome, and this leads to higher levels of GRHL3 protein. The increase in GRHL3 levels promotes the differentiation of the progenitor cells to skin cells.

“Without a functioning exosome in progenitor cells,” said Sen, “the progenitor cells prematurely differentiate due to increased levels of GRHL3 resulting in loss of epidermal tissue over time.” Sen also noted that these findings could have particular relevance if future research determines that mutations in exosome genes are linked to skin disorders or other diseases.

“Recently there was a study showing that recessive mutations in a subunit of the exosome complex can lead to pontocerebellar hypoplasia, a rare neurological disorder characterized by impaired development or atrophy of parts of the brain,” said Sen. “This may potentially be due to loss of progenitor cells. Once mutations in exosome complex genes are identified in either skin diseases or other diseases like pontocerebellar hypoplasia, it may be possible to design drugs targeting these defects.”

Some Induced Pluripotent Stem Cell Lines Cause Tumors When Transplanted into Mouse Cochleas

Japanese researchers have been carefully evaluating the safety of different stem cell lines to determine the tendency of these cells to form tumors when transplanted into mice. Such studies have made it abundantly clear that the tendency for cell lines to form tumors depends upon the cell line and where it is transplanted (see Blum & Benvenisty, The Tumorigenicity of Human Embryonic Stem Cells. Advances in Cancer Research, Volume 100, 2008, Pages 133–158). However, little is known about the cochlea and the tendency of stem cells to cause tumors when transplanted into the cochlea. Therefore, Takayuki Nakagawa of Kyoto University and his group examined the results of stem cell transplantation into mouse cochlea.

Nakagawa made it clear that his motivation for this work is to achieve successful stem cell transplantation into the cochlea to treat hearing loss. He said: “Hearing loss affect millions of people world-wide. Recent studies have indicated the potential of stem cell-based approaches for the regeneration of hair cells and associated auditory primary neurons. These structures are essential for hearing and defects result in profound hearing loss and deafness.”

In this study, Nakagawa’s group transplanted embryonic stem cells and three distinct clones of mouse induced pluripotent stem cells into the cochlea of adult mice. According to Nakagawa; “Our study examined using induced-pluripotent stem cells generated from the patient source to determine if they offer a promising alternative to ES (embryonic stem) cells. In addition, the potential for tumor risk from iPS cells needed clarification.”

Upon transplantation into the cochlea, each cell line showed a distinct ability to form neural structures and integrate into the adult cochlea four weeks after transplantation. Some cells showed poor survival in the cochlea and one induced pluripotent stem cell line formed tumors in the cochlea. “To our knowledge, this is the first documentation of teratoma formation in cochleae after cell transplantation,” said Nakagawa.

These data demonstrate the necessity of screen individual iPS cell lines before their use, since some lines have greater tumor-causing potential than others.  Furthermore, it essential for researchers to design and develop screens to eliminate tumorigenic iPS cell lines.

John Sladek from the University of Colorado School of Medicine said: “While this study do not look at the ability of the transplanted cells to repair hearing loss, it does provide insight into the survival and fate of transplanted cells.  It highlights the importance of factors such as knowing the original source of the cells and their degree of differentiation to enable the cells to be ranked in order of their likelihood of forming tumors.”

Using Stem Cells to Model the Blood-Brain Barrier

Our central nervous system include the brain and spinal cord. The central nervous system (CNS) is surrounded by a series of tough coverings called meninges that protect it and is bathed and fed by a circulating fluid called cerebrospinal fluid (CSF). The blood vessels that circulate blood through the CNS are composed of specialized cells that are very tightly apposed. These specialized blood vessels prevent molecules from spreading from the body to the CNS. In order for something to enter the CNS, the blood vessel-making cells (endothelia) must possess specific receptors that bind the desired molecule and allow it to pass into the CNS. Over 100 years ago, scientists found that if dye was injected into the bloodstream of a laboratory animal, the dye would enter everywhere except the CNS. This shows that there is a barrier that prevents the passage of all but a select set of molecules into the CNS and this barrier is called the blood-brain barrier (BBB).

The BBB is selectively permeable, which means that it allows some materials to cross into the CNS, but prevents others from doing so. In most parts of the body, the smallest blood vessels, called capillaries, are lined with endothelial cells. Endothelial tissue has small spaces between each individual cell so substances can move readily between the inside and the outside of the vessel. However, in the brain, the endothelial cells fit tightly together and substances cannot pass out of the bloodstream into the CNS. Some molecules, such as glucose, are transported out of the blood by special mechanisms.

Generally speaking. the BBB does not allow passage of large molecules, and non-fat soluble molecules also do not enter the brain. However, fat soluble molecules, such as barbituate drugs, can rapidly enter the brain. Also molecules that have a high electrical charge, if they enter the CNS at all, only do so rather slowly.

The BBB also prevents cancer drugs from entering the CNS, and this is one of the main reasons cancers of the CNS are difficult to treat. Designing cancer drugs that can enter the CNS and bypass the BBB is also challenging.

Fortunately, a new study from the University of Wisconsin, Madison, has shown that embryonic stem cells can be coaxed into differentiating into structures that greatly resemble the BBB. This might provide drug companies with a new model system to study the movement of experimental drugs into the CNS.

Eric Shusta is professor of chemical and biological engineering, is one of he senior authors of this new study. Since his laboratory has succeeded in differentiating embryonic stem cells into endothelial cells with BBB characteristics, he thinks that this “has the potential to streamline drug discovery for neurological disease. You can look at tens of thousands of drug candidates and just ask the question if they have a chance to get into the brain. There is a broad interest from the pharmaceutical industry.”

The endothelial cells generated in Shusta’s lab exhibit the active and passive regulatory characteristics of endothelial cells from the brains of a living animal.

Shusta and his team were able to induce embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into forming BBB-like structures. The ability to drive iPSCs to form BBB-like structures is significant, since scientists could use cells from patients with particular neurological diseases to make BBB-like structures and then tailor drug treatments that will take into account the capacity of the patient’s BBB to admit or exclude particular drugs.

From an industrial standpoint, because these cells can be grown in culture and mass-produced, the can be used for diverse high-throughput screens for molecules that may have therapeutic value for neurological conditions or to identify neurotoxic properties of existing drugs.

According to Shusta: “The nice thing about deriving endothelial cells from induced pluripotent stem cells is that you can make disease-specific models of brain disease that incorporate the BBB. The cells you create will carry the genetic information of the condition you want to study.”

The BBB is also complete at birth but fragile. Former experiments led scientist to believe that the BBB was immature at birth, but these experiments used conditions that were destructive for the BBB. Therefore, high levels of particular molecules that are not a problem for an adult, such as bilirubin, can cause profound problems in a baby. Kernicterus is a form of mental retardation that results from high blood bilirubin levels in a newborn. The bilirubin accumulates in the brain and causes brain damage. This is a phenomenon that results from the BBB in neonates being overloaded with bilirubin. Certain medical conditions increase the risk of kernicterus. For example, premature birth, Rh incompatibility, polycythemia (too many red blood cells), certain drugs such as sulphonamides, which displace bilirubin from serum albumin, Crigler-Najjar syndrome, Gilbert’s syndrome or G6PD deficiency all predispose babies to kernicterus. A model system in which BBB cells from patients with these diseases are cultured and grown in the lab to provide profound and potentially life-saving insights into these diseases and how they affect the BBBs of newborn babies.

Making the BBB in culture also led to another important insight, The formation of the BBB requires the activity of brain-specific cells such as neurons. Shusta explained that neurons develop at the same time as the endothelial cells. Therefore, the developing neurons seem to secrete chemical cues that help determine functional specificity to the growing endothelial cells. Presently, Shusta and his group do not know what those chemicals are, but with this in the dish model, it will be relatively easy to go back and look.

Finally, in quoting from the abstract of this paper, “The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.” In other words their model BBB looks like a BBB and functionally acts like one. The possibilities for this model system are truly tremendous.