Making Pancreatic Beta Cells from Embryonic Stem Cells


Type 1 diabetes results from an inability to produce sufficient quantities of the hormone insulin. Without insulin, the body does not receive the signal to take up sugar from the blood, and the result is high blood sugar levels, which are damaging to tissues, and a general wasting of tissues because they cannot take up enough sugar to feed them.

Pancreatic beta cells

The cells in the pancreas that produce insulin are the beta cells, and animal studies have shown that transplantation of new beta cells into diabetic animals can reverse and even in some cases cure the diabetic animals. Therefore researchers have tried to make beta cells from pluripotent stem cells in order to make a source of beta cells for transplantation.

Unfortunately, beta cell production in the laboratory has been fraught with problems. The cells produced by differentiation of embryonic stem cells do not have the characteristics of mature beta cells and they produce little insulin and are not glucose responsive (D’Amour, et al., (2006) Nat Biotechnol 24, 1392-1401).

A different strategy, however, works much better. Instead of differentiating stem cells into beta cells, differentiate them into those cells that will form beta cells and other types of pancreatic cells in the embryo – immature endocrine cell precursors – and then transplant those into the pancreas of diabetic mice. In this case, the endocrine cell precursors differentiate in the bodies of the mice into pancreatic beta cells that greatly resemble normal beta cells.

Why don’t embryonic stem cells for beta cells in culture? This question was pursued by a collaboration between research team led by Maike Sander at UC San Diego and a company called ViaCyte, Inc.

When it comes to endocrine precursors transplanted into mice, Dr. Sander noted that, “We found that the endocrine cells retrieved from transplanted mice are remarkably similar to primary human endocrine cells.” He continued, “This shows that hESCs (human embryonic stem cells) can differentiate into endocrine cells that are almost indistinguishable from their primary human counterparts.”

Well, ESCs can make perfectly fine beta cells in the mouse body, but not in culture. What’s up with that?

Sander and her colleagues examined the gene expression patterns of embryonic stem cells as they were differentiated and compared them with the gene expression patterns in those cells that were transplanted into mice and allowed to differentiate inside the body of the mouse.

What Sander and her team found was astounding. As cells progress through their developmental program, particular genes are brought on-line and expressed, and then turned off as the cells passed through each stage of endocrine cell differentiation. The cellular machinery that shuts off genes after they have been activated consists of a family of proteins that remodel chromatin known as the Polycomb group (PcG). PcG-mediated repression of genes silenced those genes that were only turned on temporarily once they were no longer required.

Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .
Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .

In the transplanted endocrine precursors, Sanders and his team noted an orderly progression of genes that were turned on and then turned off once as needed. However, in the embryonic stem cells that were differentiated into beta cells in culture, they discovered that these cells failed to express the majority of genes critical for endocrine cell function. The main reason for this appeared to be that the PcG-mediated repression of genes was not fully eliminated when particular genes had to be expressed at specific developmental stages. Thus these cultured cells failed to fully eliminate PcG-mediated repression on endocrine-specific genes, which contributes to the abnormality of the culture-derived beta cells.

Sander commented: “This information will help devise protocols to generate functional insulin-producing beta cells in vitro. This will be important not only for cell therapies, but also for identifying disease mechanisms that underlie the pathogenesis of diabetes.”

When To Use Umbilical Cord Blood Stem Cells


Umbilical cord blood stem cells (UCB-SCs) have been used in a variety of clinical trials and treatments. Their use in treatment bone marrow-based conditions is very well-known, but they have also been used in other experimental treatments as well.

Treatments with UCB-SCs suffer from inconsistent results that stem from a variable number of viable cells in UCB-SC samples. Establishing high numbers of viable cells in UCB-SC samples is not easy, and there is a great interest in being able to grow UCB-SCs in culture and expand them. However, even though UCB-SCs can be grown in culture, the effects of culturing UCB-SCs is presently unclear.

To address this question in a rigorous fashion, Miguel Alaminos at the University of Granada and his colleagues grew UCB-SCs in culture and analyzed cell viability and gene expression at every passage.

What they discovered was astounding. When UCB-SCs were passaged two or three times, the cells showed signs of cells death, and gene expression studies revealed that many of the cells expressed genes associated with programmed cell death. Cells passaged eight, nine, or ten times also showed extensive cell death. However, cells passaged five or six times showed the highest viability.

This suggests that different studied have used cells that were grown for different periods of time and probably had different viabilities. This explains why UCB-SCs have performed so variably in experiments and clinical trials. This suggests that therapies that utilize UCB-SCs should use them after they are passaged for the fifth or sixth time in order to ensue the highest levels of viability.

Specificity Protein 2 Required for Neuron Formation


In mammals, cells contain a group of proteins known as “specificity factors” that acting during gene expression. Specificity proteins, or Sps, bind to DNA at specific sequences and activate gene expression at those genes that possess the binding target for the Sps. Sps control the expression of a many genes, including house keeping, tissue-specific, development-specific and cell-cycle-regulated genes. There are nine different Sps that have been discovered in mammals, and they are numbered Sp1 to Sp9. For a good article on Sps, see Suske G, Bruford E, Philipsen S (2005) Mammalian SP/KLF transcription factors: bring in the family. Genomics 85: 551–556.

Several of the Sps have been shown to play rather central roles during development. For example, mouse embryos that lack Sp1, die very early (around embryonic day 10.5). Mice that do not have functional Sp3, develop until the end of pregnancy but die immediately after birth due to respiratory failure. Such embryos also show impaired skeletal bone ossification, tooth development, heart development, and abnormal organization of the placenta. Newborn Sp4-deficient mice either die within the first month of life or are severely growth-retarded, and males do not breed and females show delayed in sexual maturation. Sp4 is also required for the development of the conduction system in the heart.

Of the Sps, Sp2 has been very poorly characterized. For this reason, Troy Ghashghaei at North Carolina State University has investigated the role of Sp2 in neural stem cells.

In collaboration with Jon Horowitz, a colleague at the Center for Comparative Medicine and Translational Research, they made mouse neural stem cells that lacked functional Sp2. The neural stem cells without Sp2 were able to divide, but the progeny were unable to differentiate into neurons. Instead the neural stem cells simply divided over and over without ever forming neurons.

This result was unexpected, since Horowitz had shown in an earlier publication that overproducing Sp2 did something similar in skin stem cells. Instead of dividing and forming new skin cells, skin stem cells that expressed excessive amounts of Sp2 continued to divide and without forming new skin cells. Instead they formed tumors (Kim TH, et al., Cancer Res. 2010 Nov 1;70(21):8507-16. doi: 10.1158/0008-5472.CAN-10-1213).

In Horowitz’s view: “We believe that Sp2 must play a fundamental role in the lives of normal stem cells. Trouble ensues when the mechanisms that regulate its activity are overwhelmed due to its excess abundance.”

However, this recent work shows that in a different system, the absence of Sp2 has much the same effect – prevent of stem cells from producing progeny that differentiates into mature cell types and continued, uncontrolled proliferation.

Of this Ghashghaei said: “It’s interesting that both an overabundance of this protein and a total lack of it result in similar disruptions in how stem cells divide. So while this work confirms that Sp2 is absolutely necessary for stem cell function, a lot of questions still remain about what exactly it is regulating, and whether it is present in all stem cells or just a few. We also need to find out if Sp2 deletion or overabundance can produce brain tumors in our mice as in the skin.”

Ghashghaei continued: “Lastly, we are very interested in understanding how Sp2 regulates a very important decision a stem cells has to make: whether to produce more of itself or to produce offspring that can become neurons or skin cells. We hope to address these questions in our future research, because these cellular mechanisms have implications for cancer research, neurodevelopmental diseases and regenerative medicine.”

See Liang H, Xiao G, Yin H, Hippenmeyer S, Horowitz JM, Ghashghaei HT. Neural development is dependent on the function of specificity protein 2 in cell cycle progression.  Development. 2013 Feb;140(3):552-61. doi: 10.1242/dev.085621.

Local Anesthesia Inhibits Mesenchymal Stem Cells


Anyone who has had dental work or particular out-patient procedures has had local anesthesia. Local anesthesia inhibits local sensory nerve function and induces numbness. Several studies have shown that when used at high concentrations, local anesthesia can cause particular cells to die. Therefore, some physicians worry that local anesthesia might affect stem cells, but the effects of local anesthesia on mesenchymal stem cells is largely unknown.

To this end, Michael Zaugg from the University of Alberta and his talented co-workers examined the effects of local anesthesia on mesenchymal stem cells from bone marrow. Their results were from experiments on cultured mesenchymal stem cells.

When mouse bone marrow mesenchymal stem cells were isolated and grown in culture and exposed to 100 micromolar concentrations of three different local anesthetics, lidcocaine, ropivacaine, and bupivacaine, they discovered that the mesenchymal stem cells grew much more slowly. In fact, the stem cells seemed to divide and then give up the ghost. Therefore, local anesthetics seemed to inhibit mesenchymal stem cell proliferation.

Upon further investigation, the stem cells stopped dividing at the point when they were supposed to start making new DNA. This phase of the life of the cell is called the S phase for synthesis phase, and the molecule made by the cell at this time is DNA. However, the mesenchymal stem cells exposed to local anesthetics failed to initiate DNA synthesis.

The next question Zaugg and his team asked was whether or not the stem cells had trouble making energy, which is a common feature of cell exposed to too much local anesthetic. Indeed, the mesenchymal stem cells exposed to local anesthetics showed reduced energy production.

A more sophisticated analysis called “microarray analysis,” which examines the gene expression patterns in a cell by a gene-by-gene basis, showed that those genes necessary for cell membrane synthesis were greatly decreased when the cells were exposed to local anesthetics. Furthermore, the mesenchymal stem cells exposed to local anesthetics differentiated quite poorly, and the microarray analysis confirmed this observation, since those genes necessary for differentiation in mesenchymal stem cells were down regulated in the presence of local anesthetics.

Before conclusions can be drawn about what local anesthetics do to a living creature during wound healing, more work must be done, First of all, these results from cultured cells may not hold true in a living organism. Also, the concentration of anesthetic used in this study is well above what are acknowledged to be toxic levels for these drugs. Therefore, while these results are informative and interesting, the must be interpreted with some caution.

Leprosy Bacterium Reprograms Adult Cells into Stem Cells


Hansen’s disease is another name for the modern known as leprosy. Leprosy is known from old documents, for example, the Bible, but what is described in the Old Testament as leprosy seems to be a combination of various conditions. Plague psoriasis, for example, could fit the biblical description of leprosy. Also, in the Old Testament, a house or fabrics could get leprosy (Leviticus 13-14, which suggests that mildew, or something like it, was regarded as leprosy. Thus leprosy in the Old Testament seems to refer to a broad category of diseases. However, in the New Testament, when leprosy is described, it might be a variant of the modern Hansen’s disease.

Hansen's disease

Hansen’s disease is caused by a microorganism called Mycobacterium leprae. It causes skin lesions, loss of sensation, muscle weakness, and numbness in the hands, arms, feet and legs. The skin lesions are lighter than normal skin color, which have decreased sensation to touch, heat, or pain. These lesions do not heal after several weeks to months.

Leprosy is not very contagious and it has a long incubation period (time before symptoms appear). This makes it rather difficult to know where or when someone caught the disease. Children are more likely than adults to get leprosy.

There are two common forms of leprosy, tuberculoid and lepromatous leprosy. Both forms produce sores on the skin, but the lepromatous form is most severe. It causes large lumps and bumps. Leprosy is common in many countries worldwide, but it is also found in temperate, tropical, and subtropical climates. There are about 100 cases diagnosed per year in the United States, and most are in the South, California, and Hawaii.

Mycobacterium leprae (M. leprae) attacks, among other things, the peripheral nerves. The organism causes the insulating myelin sheath that surrounds the nerve to unravel, thus leaving the nerves without their insulating layer, which causes nerve malfunction. However, recent work has shown that M. leprae unravels the myelin sheath by reprogramming the cells that make the myelin sheath. These myelin-making cells are known as Schwann cells, and M. leprae, reprograms Schwann cells to revert to a stem-cell-like state, which causes them to leave the nerves, leaving the nerves in the lurch.

schwann cells

These finding were published in the prestigious international journal Cell.

Scientists from the laboratory of Anura Rambukkana, who holds a dual appointment at the Rockefeller University in New York and The MRC Centre for Regenerative Medicine in Edinburgh, Scotland, discovered this remarkable finding while examining how leprosy spreads around the body.

The initial target of M. leprae is Schwann cells. To understand how the organism affects Schwann cells, Rambukkana and co-workers isolated Schwann cells from mice and infected them with M. leprae. Once infected with M. leprae, the infecting bacteria reprogrammed the cells into a stem-like state. It turned off genes associated with mature Schwann cells and turned on genes associated with embryonic stages or other developmental stages.

M. leprae seemed to trigger Schwann cells’ plasticity. Plasticity refers to the ability of cells to revert to an immature state and differentiate into new types of cells. In fact, healthy Schwann cells do exactly that in order to help nerves recover and regenerate after an injury.

Rambukkana notes that however the bacteria are reprogramming the Schwann cells, they seem to be employing a “very sophisticated mechanism — it seems that the bacterium knows the mechanistic interaction of the Schwann cell better than we do.”

Upon being reprogrammed, the stem cells can migrate to different locations in the body with the bacterium housed inside then. Once the infected cells reach another tissue, such as skeletal muscle, the stem cells integrate with that tissue’s cells, thus spreading the bacteria. The infected stem cells also attract immune cells by secreting summoning proteins called chemokines. This brings more potential carriers to the bacteria’s doorstep.

What do the bacteria do to trigger a reprogramming event? At this point these researchers do not know, but they suspect that the mechanism could exist in other infectious diseases.

According to Sheng Ding, a stem cell biologist at the Gladstone Institute of Cardiovascular Disease in San Francisco, CA, “Cellular plasticity may represent an underlying mechanism of disease, as other cellular reprogramming events have been shown in cancers and metabolic diseases.”

By understanding these precise mechanisms, scientists could devise precise ways to improve treatment and earlier diagnosis of leprosy itself. These latest findings also provide vital clues about how leprosy spreads throughout the body, and how to catch the disease before it spreads rapidly.

In the future, bacteria or products made by the bacteria could be used to change adult tissue cells into stem cells in the laboratory, thus potentially leading to new regenerative treatments for diseases such as diabetes and Alzheimer’s.

See Masaki, T. et al. Cell 152, 51–67 (2013).

Stem Cells Heal Damaged Baboon Arteries in the Lab


A research group at the Texas Biomedical Research Institute in San Antonio, Texas has reprogrammed embryonic stem cells derived from baboon embryos to completely restore a severely damaged artery. Such results lay the ground work for what might be a new way to completely heal large blood vessels that have been damaged by congenital diseases, the ravages of disease, or simply old age.

John L. VandeBerg, chief scientific officer at the Texas Biomedical Research Institute, said: “We first cultured the stem cells in Petri dishes (culture dishes) under special conditions to make them differentiate into cells that are the precursors of blood vessels, and we saw that we could get them to form tubular and branching structures, similar to blood vessels.”

Since VandeBerg and his colleagues were able to differentiate baboon embryonic stem cells into blood vessel precursors, they wanted to try a much more difficult experiment and try to use these blood vessels precursor cells to repair a damaged blood vessel in a simulated environment.

By removing the cells that line the inside of a baboon artery, VandeBerg and co-workers replaced the lining with the blood vessel precursors derived from baboon embryonic stem cells. Then they connected this artery segment to a plastic tubing inside a device known as a “bioreactor.” Bioreactors are designed to grow cells and tissues under conditions that closely mimic those inside the human body. In this case, the bioreactor also pumped fluid through it as though it were inside a real, living baboon.

The artery was bathed in culture medium, and by three days, the complex inner layer of the artery showed signs of regenerating, and by 14 days, it was perfectly restored to its complex, natural state. In two weeks, the artery had gone from stripped to a fully functioning artery.

VandeBerg said of these experiments: “Just think of what this kind of treatment would mean to a patient who had just suffered a heart attack as a consequence of a damaged coronary artery. And this is the real potential of stem cells regenerative medicine – that is, a treatment with stem cells that regenerates a damaged or destroyed tissue or organ.”

A control experiment also showed that the arteries could not regenerate without the added cell stems, they used an artery that can been internally stripped and hooked it up to the bioreactor without seeding it with stem cells. Under these conditions, no healing occurred.

When the arteries were stained for those proteins normally found in a properly functioning artery, the healed artery showed all the same staining characteristics as arteries that had not been internally stripped. Of this result, VandeBerg noted: “This is evidence that we can harness stem cells to treat the gravest of arterial injuries.”

Researchers such as Vandeberg hope to take a skin cell or a white blood cell, or a cell from just about anywhere else in the body and induce it to differentiate into induced pluripotent stem cells that can be used to differentiate into blood vessel precursors that can be used to repair damaged blood vessels.

Three-Dimensional Scaffolds that Support that Regeneration of Tissues by Stem Cells


When cultured in the laboratory, stem cells can form tissues that are commonly found in our own body. However, the size, shape and organization that what stem cells make in culture tends to not resemble what is observed in our bodies.

There are ways to coax stem cells to make tissues that more closely resemble those in our bodies. This includes growing stem cells on “biomimetic” scaffolds that have the same shape and organization as our own tissues. Such scaffolds direct the growth and organization of the stem cells and the tissues that form so that they more closely resemble our own.

Researchers from Singapore at the Nanyang Technological University of Singapore and collaborators, led by Zu-yong Wang, have invented a clever and innovative method that creates a stretched polymer scaffold that supports complex tissue architecture. By stretching this polymer (poly ε-caprolactone for the interested) thin-film, it can actually produce scaffolds that can support the growth of mesenchymal stem cells.

This stretching process generates a nice three-dimensional with micro-grooves that are oriented on the surface of the film. These grooves direct stem cells to grow in a neatly aligned fashion that can develop into tissues as the stem cells grow on and eventually degrade and absorb the scaffold.  Such a finding advances tissue engineering research, the goal of which is to use stem cells to remake new organs to replace damaged or disease ones.

Induced Pluripotent Stem Cells Used to Define Proper Treatment for Heart Patient


Scientists from Columbia University Medical Center in the laboratory of Robert Kass have used a heart patient’s cells to make induced pluripotent stem cells (iPSCs) that were then differentiated into heart muscle cells. These heart muscle cells were used to test drug strategies to keep the patient’s heart going.

This patient, you see, suffers from Long QT syndrome (LQTS), which is caused by an abnormal ion channel in the heart. LQTS affects the patient’s heart rhythm, which result in fast, chaotic heartbeats. These rapid heartbeats may trigger sudden fainting or a seizure. In some cases, the heart may beat erratically for so long that it can cause sudden death.

Long QT syndrome is treatable by means of decreased physical activity, and certain medicines. Other patents will need surgery or an implantable device.

In this case, the four-year-old patient responded poorly to medicines. Therefore, to find the right combination of drugs, The child had a mutation in the SCN5A gene, which encodes the alpha subunit of the voltage-gated sodium channel. However, this child also had a mutation in the KCNH2 gene, which encodes a potassium channel. This child’s LQTS, therefore, was particularly severe and did not respond to the usual drug regimens.

By using an electrophysiological test called “voltage clamping” on the heart muscle cells made from the patient-specific iPSCs, heart doctors were able to find a drug treatment strategy that eventually stabilized the patient’s heart and saved his small life.

Voltage clamping is a technique used to control the voltage across the membrane of a small or area of a nerve or heart muscle cell by means of an electronic feedback circuit. By sucking a small part of the cell membrane into a micropipette that has a tiny wire in it (yes it sounds hard and yes it is hard), the voltage is increased gradually and the circuit required to hold the voltage at each level is measured. This current is the same as the ionic current that flows across the membrane in response to the applied voltage. This ionic current tells the heart specialist all about what ion channels are present and how well they work in the presence or absence of particular drugs.

Voltage Clamp

These results demonstrate the power of iPSCs in culture as a model system to determine patient-specific therapies.

Cholesterol Derivatives Push Neural Stem Cells to Become Cells for Parkinson’s Disease Treatments


When we hear the word cholesterol we often have very negative thoughts of clogged arteries, heart attacks and strokes. However, cholesterol serves several vital roles in our bodies. It regulates the fluidity of the membranes of our cells, serves as a precursor for the synthesis of steroid hormones (such as estrogen, testosterone, cortisol and others), and is an important signaling molecule for several biological processes. Therefore. cholesterol is not all bad. Cholesterol when we get too much of it and our bodies handle the excess cholesterol poorly. Then wandering cells called macrophages have to mop up the excess cholesterol, but it makes them sick, and they get lost underneath the inner layers of blood vessels. That, however, is for another blog post.

In the present study, scientist from Karolinska Institutet in Sweden have identified two molecules, both of which are derivatives of cholesterol, that can help turn brain cells into the kind of cells that die during Parkinson’s disease. This finding might be useful for producing large quantities of neurons in the laboratory for therapeutic purposes.

As I have blogged before Parkinson’s disease results from the death of midbrain neurons that use the neurotransmitter dopamine. Because these midbrain neurons project to, in part, regions of the brain involved in voluntary movement, the death of the dopamine-using neurons in the midbain produces pronounced defects in voluntary movement and resting stability. Several experiment in humans and laboratory animals have definitively shown that cell transplantation experiments can improve the symptoms of patients with Parkinson’s disease. Therefore, cultivating and growing dopamine-using neurons in the laboratory is of cardinal importance in the treatment of this devastating disease.

Workers in the laboratory of Ernest Arenas investigated molecules known to play a role in the differentiation of midbrain neurons. They discovered that a group of receptors collectively known as “liver X receptors” or LXRs are necessary for making ventral midbrain neurons from neural stem cells. However, they were unsure what molecules bound to the LXRs in order to activate them.

Enter cholesterol stage right. By subjecting LXRs to a cocktail of molecules from living organisms and analyzing by means of mass spectrometry, they discovered that two molecules, cholic acid (a bile salt), and 24,25-EC, both of which are derivatives of cholesterol, bind to LXR and activate it.

Cholesterol
Cholesterol
Cholic Acid
Cholic Acid

24,25-Epoxycholesterol24,25-Epoxycholesterol

Cholic acid binds to LXR and stimulates the neural stem cells to form a group of midbrain cells known as the “red nucleus.” The red nucleus receives signals from several different parts of the brain to coordinate the movements of several different parts of the body. The other molecule, 24,25-EC binds to LXR and induces the formation of dopamine-using midbrain neurons – the ones that die off during Parkinson’s disease.

These data could open the possibility that cholesterol derivatives can be used to produce dopamine-using neurons from neural stem cells to treat Parkinson’s disease.

Ernest Arenas, professor of stem cell neurobiology in the department of biochemistry and biophysics, who led this study said: “We are familiar with the idea of cholesterol as a fuel for cells, and we know that it is harmful for humans to consume too much cholesterol. What we have shown now is that cholesterol has several functions, and that it is involved in extremely important decisions for neurons. Derivatives of cholesterol control the production of new neurons in the developing brain. When such a decision has been taken, cholesterol aids in the construction of these new cells, and in their survival. Thus cholesterol is extremely important for the body, and in particular for the development and function of the brain.”

How Stem Cells Stay Ready


Embryonic stem cells and induced pluripotent stem cells have a characteristic known as “pluripotency,” which simply means that they can become any cell type found in the adult human body. When these cells are given the proper cues, they can differentiate into muscle, skin, heart, blood, brain, or kidney cells, just to name a few. How do they do this?

A new study from the laboratory of Ali Shilatifard a scientist at the Stowers Institute for Medical Research in Kansas City, Mo, has shown that the plasticity of pluripotent stem cells partially comes from the stationing of a protein called EII3 at various stretches of DNA in the genome of these cells.

The vast majority of the cells in our bodies have a nucleus that houses the chromosomes, which are made of DNA. The DNA contains stretches known as genes that encode RNAs that are either translated into protein or have some other function. DNA stores genetic information and this genetic information is accessed by a complex set of enzymes called RNA polymerases and make these RNA molecules.

How do cells know when are where to make these RNA molecules? The signals for when to make a gene resides in sequences in or near the gene called “enhancer” sequences. Enhancers bind particular proteins and the assembly of particular proteins on the enhancers of a gene activate the expression of that gene.

EII3 is a member of a family of proteins known as the EII or (get ready) the “eleven-nineteen-lysine-rich-leukemia gene” (told you) family of elongation factors. ELongation factors increase the rate at which genes are expressed, and in pluripotent stem cells, EII3 parks itself at the enhancers of a variety of developmentally regulated genes, even ones that are silent in pluripotent cells.

According to Shilatifard: “We now know that some enhancer misregulation is involved in the pathogenesis of solid and hematological (that means blood-based) malignancies. But a problem in the field has been how to identify inactive or poised enhancer elements. Our discovery that EII3 interacts with enhancers in ES cells gives us a hand-hold to identify and to study them.”

EII3 was initially thought to be a dud because it was expressed at high levels in testes, which is a notoriously uninteresting tissue to work on from a gene expression perspective. All that changed when a postdoctoral research fellow in Shilatifard’s lab named Chengqi Lin searched all the potential places in the genome that EII3 could potentially bind in mouse embryonic stem cells. For this study, Lin collaborated with Alesander Garruss, another postdoctoral fellow in the Shilatifard lab, who is a specialist in bioinformational technologies. Lin and Garruss showed that EII3 occupies more than 5,000 enhancers, and many of these are affiliated with genes that regulate stem cell differentiation into spinal cord tissues, kidney cell types, blood cells and so on.

Lin put it this way: “What was interesting was that EII3 marked enhancers that are active and inactive, as well as enhancers that are known as “poised.” That indicated that EII3’s major function might be to prime activation of genes that are just about to be expressed during development.”

The fact that EII3 could prime silent genes for immediate expression under the right conditions was not a surprise, since researchers knew that enzymatic machine that copies DNA into RNA – RNA polymerase II, also known as Pol II, often pauses at the start of some genes. Pol II acts very like like a race car that has started its engine and is revving the engine in anticipation of the green light. When researchers in Shilatifard’s lab removed EII3 from the stem cells by means of a genetic trick, they discovered that the paused Pol II molecules disappeared these genes. This shows that EII3 oreferentiually marks stem cells enhancers and its presence there is necessary to keep an idling Pol II molecule there ready for action.

When the conditions are right, the EII3-Pol II complex interacts with the “Super Elongation Complex to give Pol II the green light and transcribe the gene. Without EII3, these genes are never expressed, even under the right conditions.

As a layer of icing on this remarkable research cake, Fengli Guo at the Stowers Institute use electron microscopy to prepare highly magnified images of mouse sperm DNA with Pol II and EII3 bound to this DNA. The significance of this come from development. Once fertilization occurs, the zygotes begins to divide, and the daughter cells are gradually committed to various developmental possibilities. The fact that EII3 is necessary for differentiation and that it is carried into the embryo by the sperm explains how the blastomeres of the early embryo are so exquisitely poised to readily differentiate into the various cell types that they eventually become.

“It is very significant that EII3 and other factors that regulate transcription are found in sperm,” said Lin. He continued to note that that it “would be exciting to further investigate whether transcription factors found in sperm could contribute to the decondensation of sperm chromatin or even further gene activation after fertilization by serving as epigenetic markers.”

Shilatifard is cautious about not overinterpreting these results, but he does think that they have fundamental implications for development and also, perhaps, cancer research. “This work has opened up a whole new area of research in my lab,” said Shilatifard, who has in the last decade focused on aberrant gene expression associated with leukemia. “If we find that transcription factors bind to specific regions of chromatin in germ cells, I may focus on germ cells in the next few decades. This would open a huge door enabling us to determine the role of these factors in early development.”

What Does Breast Cancer Have to Do With Skin Stem Cells?


BRCA1 is a gene that plays a huge role in breast cancer. Particular mutations in BRCA1 predispose women increased risks of breast cancer cervical, uterine, pancreatic, and colon cancer and men to increased risks of pancreatic cancer, testicular cancer, and early-onset prostate cancer.

BRCA1 encodes a protein that helps repair damage to chromosomes. When this protein product does not function properly, cells cannot properly repair acquired chromosomal damage, and they die or become transformed into cancer cells.

What does this have to do with stem cells? A study led by Cédric Blanpain from the Université libre de Bruxelles showed that BRCA1 is critical for the maintenance of hair follicle stem cells.

Peggy Sotiropoulou and her colleagues in Blanpain’s laboratory showed that when BRCA1 is deleted, hair follicle cells how very high levels of DNA damage and cell death. This accumulated DNA damaged drives the follicle stem cells to divide furiously until they burn themselves out. This is in contrast to the other stem cell populations in the skin, particularly those in the sebaceous glands and epidermis, which are maintained and seem unaffected by deletion of BRCA1.

Sotiropoulou said of these results: “We were very surprised to see that distinct types of cells residing within the same tissue may exhibit such profoundly different responses to the deletion of the same crucial gene for DNA repair.”

This work provides some of the first clues about how DNA repair mechanisms in different types of adult stem cells are employed at different stages of stem cells activation. Blanpain and his group is determining if other stem cells in the body are also affected by the loss of BRCA1. These results might elucidate why mutations in BRCA1 causes cancer in the breast and ovaries, but not in other tissues.

Neural Stem Cells Slow the Progression of ALS


A research project that includes work done by 11 different institutions has tested the ability of neural stem cells to treat Lou Gehrig’s disease, which is also known as amyotrophic lateral sclerosis or ALS. ALS affects neurons in the central nervous system, particular in those cells in the spinal cord that allow voluntary movement (motor neurons). These neurons die off, and the patient loses the ability to move and, eventually, to breath. There is no presently no cure for this catastrophic and horrific disease.

Stem cell treatments have shown some success in laboratory animals, and this recent study examined the ability of neural stem cells, which have the ability to form neurons or those cells that support neurons, glial cells, to treat mice with a form of ALS that seems to closely resemble the disease presented by human ALS patients.

Neural Stem Cells
Neural Stem Cells

The combined work of these 11 different institutions showed that when these cells were transplanted into the spinal cords of laboratory mice afflicted with a form of ALS, symptoms of the disease decreased and the progression of the disease was greatly altered. When treated mice were compared with untreated mice, their movement ability and breathing were much better. Even more remarkable was the ability of these stem cells to slow the progression of ALS. Twenty-five percent of the treated mice survived for one year or more, which is three-four times longer than the untreated mice.

Even though neural stem cells can form neurons and other types of nervous system-specific cells, the neural stem cells in this experiments did not benefit mice by differentiating into new neurons. Instead, the transplanted stem cells prolonged the life of the troubled tissues by secreting molecules that are beneficial to the health of neurons and other cells in the nervous system. This menagerie of helpful molecules made by neural stem cells also stimulates other native cells in the nervous system to make their own fair share of protective molecules.

The transplanted neural stem cells also decreased the production of toxins by the diseased tissues and also diminished inflammation in the spinal cord.

In the words of the senior author of this study, Evan Y. Snyder, the director of Sanford-Burnham‘s Stem Cell and Regenerative Biology Program: “We discovered that cell replacement plays a surprisingly small role in these impressive clinical benefits. Rather the stem cells change the host environment for the better and protect the endangered verve cells. This realization is important because most diseases are now being recognized as multifaceted in their cause and their symptoms – they don’t involve just one cell type or one malfunctioning process. We are coming to recognize that the multifaceted actions of the stem cell may address a number of these disease processes.”

These studies demonstrate the potential neural stem cells hold for treating ALS and other nervous system disorders. However, Snyder tempered these results with this measured optimism: “While not a cure for human ALS, we believe that the careful transplantation of neural stem cells, particularly into areas that can best sustain life – respiratory control centers, for example – may be ready for clinical trials.”


Stem cells are used to reverse dementia in an 80-year old patient. Remarkable!!

The Stem Cell Blog

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Stem Cells Shown to Reverse Dementia

Mary Holler, age 80, of Marco Island, Florida is smiling again.  Mary was suffering from dementia and felt her ability to function on a daily basis was slipping away.  Now, after undergoing a successful stem cell treatment in early December 2012, Mary is more like her old self again. She no longer suffers the frustration and agitation of being told she had already asked that question several times. Peter Holler , age 82, had become very concerned that his wife of 60 years was slowly losing her memory. She had been on medications for memory loss for several years but the deterioration in her recall accelerated in the last six months.  It was not unusual for him to answer the same question 4 to 5 times over the course of a day.  He felt he was losing his wife right in front of his…

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Induced Pluripotent Stem Cells Do Not Induce Immune Response


In 2007, scientists from Japan and the United States discovered that cells from a person’s own body could be reprogrammed to an embryo-like cell known as induced pluripotent stem cells or iPSCs. These cells seemed to provide an endless source of pluripotent stem cells that are matched to the patient to treat diseases or for model systems for research.

The formation of iPSCs seems to provide a solution to the ethical concerns that surround the derivation of embryonic stem cells. However, worries about the use of iPSCs emerged with the publication of a 2011 study published in Nature that threw doubt on one of the key advantages of iPSCs. In this study, iPS cells could provoke an immune response when injected into the mice from which they had been derived (Zhao, T., Zhang, Z.-N., Rong, Z. & Xu, Y. Nature 474, 212–215 (2011).

However, a more recent study also published in Nature has shown that iPSCs do not induce immune responses in the laboratory animals from which they were derived. Masumi Abe, who works as a geneticist at the National Institute of Radiological Sciences in Chiba, Japan, and co-workers took iPS cells derived from mice and injected them back into the animals. As a control, they injected other mice with embryonic stem (ES) cells. Contrary to the 2011 study, which observed that injected iPS cells induced an immune response but injected ES cells did not, Abe’s team were not able to find any differences between the immune responses elicited by iPSCs or ES cells. Abe and his colleagues also transplanted skin and bone-marrow cells derived from iPS or ES cells into mice and achieved similar success rates between the groups. The immune responses to both sets of tissues were, in the words of Abe, “indistinguishable.”

Konrad Hochedlinger, a stem-cell scientist at Massachusetts General Hospital in Boston, says that the result will probably “calm people down” about iPS cells. “It is definitely reassuring,” he says.

These findings from Abe’s laboratory come on the heels of another positive study on iPS cells that was published late last year. This study showed that the reprogramming process caused fewer mutations than previously thought. Flora Vaccarino, a Yale University neuroscientist (New Haven, Connecticut) and her colleagues used high-resolution DNA analysis to compare the genomes of iPS cells and the adult cells from which they were derived. They discovered that most of the DNA mutations in the iPS cells did not arise as a result of reprogramming but had been present in the parent cells (Abyzov, A. et al. Nature 492, 438–442 (2012).

The co-author of the 2011 study, Yang Xu, a stem-cell scientist at the University of California, San Diego, is skeptical about this present study and says that the new work does not dispel all concerns about the immune response provoked by iPS cells. Xu pointed out that the skin and bone-marrow cells used in the latest study were not grown from iPS cells in culture, as they would be for clinical use. Instead, the researchers mixed iPS cells into early mouse embryos to make “chimeric” embryos. They then used skin and bone-marrow tissues that arose from iPS cells after the embryos grew into adult mice for their transplantation experiments. It is possible, says Xu, that the most immunogenic cells were rejected as the mice developed, which would explain why Abe and his colleagues observed a limited immune response. According to Xu, transplanting tissues from chimeric mice is “flawed.”

Chimeric embryo production is a standard technique for testing whether mouse iPS cells have been fully reprogrammed, says Jakub Tolar, a clinician at the University of Minnesota in Minneapolis. Tolar also noted that differentiating cells in culture outside the body is much harder. Tolar hopes to use iPS cells to treat the childhood skin disease epidermolysis bullosa, and added that iPS-cell therapies will use human cells, which could behave quite differently from mouse cells. “It’s helpful that they’ve done this, but it is absolutely different when you go to something that is cultured,” he says.

Hochedlinger believes that iPS cells show just as much promise for cell transplantation as ES cells, although many issues stand between the lab and the clinic. According to Hochedlinger, the differences between the two kinds of stem cell are minor compared with the differences in how individual cell lines grow and differentiate in culture.

“Based on what we know at this time from mice,” he says, “iPS cells are as good as ES cells, and should be as safe.” See Araki, R. et al. Nature 493, 145 (10 January 2013) doi:10.1038/493145a.

Tissue Engineers Use New Biomaterial to Repair Knee Cartilage


Tissue engineers from Johns Hopkins University School of Medicine’s Translational Tissue Engineering Center (TTEC) have used a biomaterial to stimulate and facilitate the growth of new cartilage in human patients.

An illustration of the cartilage repair surgical procedure. A mini-incision exposes the cartilage defect (top left-hand panel), and any dead tissue is removed from the edges. (B) The adhesive is then applied to the base and walls of the defect, followed by microfracture. (C) Lastly, the hydrogel solution is injected into the defect. (D) Bleeding from the microfracture holes is trapped in and around the hydrogel.Science Translational Medicine/AAAS
An illustration of the cartilage repair surgical procedure. A mini-incision exposes the cartilage defect (top left-hand panel), and any dead tissue is removed from the edges. (B) The adhesive is then applied to the base and walls of the defect, followed by microfracture. (C) Lastly, the hydrogel solution is injected into the defect. (D) Bleeding from the microfracture holes is trapped in and around the hydrogel.
Science Translational Medicine/AAAS

This was a rather small study that only examined 15 patients. All 15 patients had cartilage defects and were scheduled to undergo “microfracture surgery.” Microfracture surgery uses a drill to bore tiny holes in the bone. These small holes allow bone marrow stem cells to leak into the joint space and make new bone and cartilage. In this study, hydrogel scaffolding was troweled into the wound to in order to support and nourish the healing process. The results from this study were published in the Jan. 9 issue of Science Translational Medicine. According to the authors, this study is a proof of concept trial that paves the way for larger trials to test the hydrogel’s safety and effectiveness.

“Our pilot study indicates that the new implant works as well in patients as it does in the lab, so we hope it will become a routine part of care and improve healing,” says Jennifer Elisseeff, the Jules Stein Professor of Ophthalmology and director of the Johns Hopkins University School of Medicine’s TTEC. Cartilage damage results from overuse, injury, disease or faulty genes. Microfracture surgery is a standard of care for cartilage repair, but when holes in cartilage are caused by joint injuries, microfracture surgery often either fails to stimulate new cartilage growth or grows cartilage that is less hardy than the original tissue

To address this problem, tissue engineers, such as Elisseeff, have postulated that the bone marrow mesenchymal stem cells need a nourishing scaffold on which to grow in order to make the right type of cartilage and enough of it. Unfortunately, demonstrating the clinical value of hydrogels has been slow, difficult, and expensive. By experimenting with various materials, Elisseeff and her colleagues have developed a promising hydrogel, and an adhesive that sticks the hydrogel to the bone.

After testing the combination for several years in the lab and in goats, the hydrogel seemed ready for human trials. Elisseeff and her group collaborated with orthopedic surgeons to conduct their first clinical study. 15 patients with holes in the cartilage of their knees received a hydrogel and adhesive implant along in combination with microfracture surgery. In order to compare the efficacy of their hydrogel, another three patients were treated with microfracture surgery alone. After six months, it was clear that the hydrogel implants had caused no major problems. Furthermore, magnetic resonance imaging of these patient’s knees showed that patients with implants had new cartilage filling an average 86% of their defects in their knees, and patients that had received only microfracture surgery had an average of 64% of their tissue replaced. Patients with the implant also reported a greater decrease in knee pain in the six months following surgery, according to the investigators.

As the trial continues, more patients have enrolled. This clinical trial is presently managed by a company called Biomet. These data from this trial is part of an effort to earn European regulatory approval for the device.

Elisseeff and her team have begun developing a next-generation implant in which the hydrogel and adhesive will be combined in a single material. Elisseeff and others are also interested in technologies for joint lubrication that reduce joint pain and inflammation

Lance Armstrong Admits Taking Performance-Enhancing Drugs


Professional cycling icon, Lance Armstrong, gave an interview with cultural icon Oprah Winfrey, during which, he admitted using performance-enhancing drugs. Even though this interview has yet to air, media sources have leaked that Armstrong admitted that he had used PEDs during the two-and-a-half-hour interview.

For years, Armstrong vehemently denied cheating while winning a record seven Tours de France. While there was no official evidence that Armstrong took performance-enhancing drugs (PEDs), the circumstantial case against him was enormous.

I have blogged before on the case against Lance Armstrong here and here. Testimony from former teammates, support staff, and other witnesses, some of whom would have no reason to lie about Armstrong’s PED use, constituted the official case against Armstrong.  Also, evidence of various associations between rogue characters, such as the Italian physician Michele Ferrari and Armstrong, were rather damning.

For those of you who sent me nasty emails about my Armstrong posts, this is my official “I told you so.” There are simply too many ways to beat the drug testers, and a history of clean tests means nothing.  In the case of Armstrong, there were positive tests, but these tests were never officially confirmed or recognized. Therefore, cases must be made by other means. This is called darn good investigating.

Incidentally, my sister was an amateur cyclist who has done more than her fair share of bike races.  Also, my good friend Dr. Michael Baumann, professor of Religion at Hillsdale College has also raced at the national level. Neither my sister nor Dr. Baumann believed that Armstrong raced clean for one reason – Armstrong’s times were better than those athletes who were known to have doped. There was simply no way Armstrong could have beat those times unless he himself also doped.

Now the jig is up and the word is out – Armstrong has confessed. It’s official; he cheated.

Stem Cell Treatments for Retinitis Pigmentosa Inch Toward Clinical Trials


Retinitis Pigmentosa or RP is the most common form of inherited blindness. There are many different genes involved in the onset of RP. Molecular defects in more than 40 different genes can cause “isolated RP” and defects in more than 50 different genes can cause “syndromic RP.” Not only are there a host of different genes involved in RP, two patients with exactly the same molecular lesion can have a type of RP that differs substantially in its presentation.

The retina at the back of the eye is composed of two thick layers known as the inner neural retina and the outer pigmented retina. The neural retina consists of an outer layer of photoreceptors that are connected to an inner layer of bipolar cells. The bipolar cells connect with ganglion cells that have axons that extend to the optic nerve. The photoreceptor cells have their tips embedded in the pigmented retina, and the pigmented retina maintain and nourish the photoreceptors.

Pigmented Retina

If the pigmented retina does not function properly, then the effects are most profoundly displayed in the photoreceptors. Photoreceptors respond to light and the constant exposure to light causes the photoreceptors to take a beating. The byproducts of all that light-induced damage accumulates at the tips of the photoreceptors cells, and these rubbish-filled tips are taken a gulped down by the cells of the pigmented retina. The pigmented retina cells degrade the damaged byproducts and recycle the precursor molecules. Without properly functioning pigmented retina cells, the photoceptors cells accumulate toxic light damage and then eventually die. Photoreceptor cell death is the end product of RP, and it results in blindness.

There is no cure for RP, and the treatments available are very hit-and-miss. For this reason, cell therapies have been examined in a variety of animal models of RP, which, in many cases, closely mimic the human disease to some degree.

Two different experimental treatments, one with induced pluripotent stem cells (iPSCs) and another with gene therapy have produced long-term improvement in visual function in mice with RP. These studies have been conducted at the Columbia University Medical Center (CUMC).

Stephen Tsang, associate professor of pathology, cell biology and ophthalmology who led both studies commented: “While these therapies still need to be refined, the results are highly encouraging. We’ve never seen this type of improvement in retinal function in mouse models of RP. We hope we may finally have something to offer patients with this form of vision loss.”

In one study, CUMC researchers tested the long-term safety and efficacy of iPSC grafts into the pigmented retina to restore visual function in a mouse model of RP. The mice were injected with undifferentiated iPSCs when they were five years old, and the cells differentiated into retinal pigmented epithelial (RPE) cells and integrated into the retinas. None of the mice that received these transplantations developed tumors over their lifetimes.

To test the effects of the implanted cells on the vision of the mice, Tsang’s group used electrophysiological measurements of the retina. In RP mice, as they become blind, the electrophysiology of the retina becomes rather abnormal, but in these mice implanted with the iPSCs, the electrophysiology of their retinas were not only normal, but stayed normal for a long period of time.

According to Tsang: “This is the first evidence of lifelong neuronal recovery in an animal model using stem cell transplants, with vision improvement persisting throughout the lifespan.”

In 2011, the FDA approved clinical trials of embryonic stem cell (ESC) transplants for the treatment of macular degeneration, but this treatment requires the application of drugs that suppress the immune system. Such drugs have rather nasty side effects.

“Our study focused on patient-specific iPS cells, which offer a compelling alternative,” Tsang said. “The iPS cells can provide a potentially unlimited supply of cells for functional rescue and optimization. Also, since they would come from a patient’s own body, immunosuppression would not be necessary to prevent rejection after transplantation.”

Theoretically, iPSC transplants, could also be used to treat age-related macular degeneration, which is the leading cause of vision loss in older adults.

In a second approach to treating RP, CUMC scientists tested a gene therapy protocol in RP mice. A specific type of RP that results from mutations in a PDE6alpha gene was used as a model system for gene therapy protocol. This particular type of RP is rather common in humans. The CUMC scientists injected a virus into one of the eyes of afflicted mice. This virus was engineered to express the PDE6alpha gene when it entered cells. Because this virus is the AAV or adenovirus-associated virus, it only spreads in the presence of adenovirus. Without a helper adenovirus in the retina, the engineered virus particles will infect the cells they initially contact, but they will not produce a productive infection. However, ferry the genes inside them to the cell they initially infect. This the engineered AAV particles are excellent vehicles for getting genes inside cells without causing an infection.

Examination of the mice six months later, the photoreceptors in the AAV-treated eyes were healthy and these eyes were able to see, but the uninjected eyes were unable to see and their photoreceptors were mostly dead.

Again Tsang commented: “These results provide support that RP due to PDE6alpha deficiency in humans is also likely to be treatable by gene therapy.”

CUMC and its teaching-hospital affiliate, New York-Presbyterian Hospital are part of an international consortium that was recently formed to bring this PDE6A gene therapy to patients. Pending FDA approval, clinical trials could begin within a year.

See  Li, Y., Tsai, Y.T., Hsu, C.W., Erol, D., Yang, J., Wu, W.H., Davis, R.J., Egli, D., and Tsang, S.H. Long-term safety and efficacy of human induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa. Mol Med. 2012 Aug 9. doi: 10.2119/molmed. 2012.00242. [Epub ahead of print] (2012).

Wert KJ, Davis RJ, Sancho-Pelluz J, Nishina PM, Tsang S.H. Gene therapy provides long-term visual function in a pre-clinical model of retinitis pigmentosa. Hum. Mol. Genet. (2012) doi: 10.1093/hmg/dds46

Glucosamine, Chondroitin and Delaying Osteoarthritis


I have a confession to make. I have been taking 1200 mgs of glucosamine sulfate for the past 5-6 years for my knee cartilage. I do not presently have osteoarthritis, but I am trying to stave it off by taking this supplement.

Does this supplement work? That’s hard to say for certain because the studies disagree. There are theoretical reasons to suspect that glucosamine would help with cartilage deposition. Cartilage is very rich in a group of sticky, sugary compounds called “glycosaminoglycans,” which have the unfortunate acronym of GAGs. GAGs consist of repeating two-sugar motifs, and the building block for the vast majority of these two-sugar motifs is glucosamine. Therefore, glucosamine is a main building block of a prominent component of cartilage.

What about chrondroitin? Chondroitin is a GAG that usually comes attached to a protein. This complex of GAG + backbone protein is called a “proteoglycan.” The chondroitin you get in the store is a repeating polymer of a two-sugar motif, and this complex molecule is either degraded in your digestive system by bacteria, or by our own gastrointestinal tract.  The degradation and absorption of chondroitin probably varies considerably from person to person.  If chondroitin is absorbed then the building blocks of chondroitin can potentially help build cartilage, since chondroitin-containing proteoglycans are important structural components of cartilage.  There is also the possibility that chondroitin precursors prevent the breakdown of cartilage.

Chondroitin_sulfate-over

In 2006, a good-sized study called the GAIT study was published in the New England Journal of Medicine (Clegg, D.O. et al. (2006). Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. New Eng. J. Med. 354(8):795-808). In this study, 1583 patients with symptomatic knee osteoarthritis were randomly assigned to different treatment subgroups. These groups were:

a) chondroitin sulphate alone (400 mg 3x a day)
b) glucosamine hydrochloride alone (500 mg 3x a day)
c) combined glucosamine hydrochloride/chondroitin sulphate (same doses but combined)
d) celecoxib (Celebrex®) (200 mg per day)
e) placebo (inactive dummy tablet)

Daily dosages for glucosamine and chondroitin were 1500 mgs and 1200 mgs, respectively. The efficacious dosage for these supplements have yet to be determined. Therefore, these dosages are a best guess. Celecoxib was included as a positive control for the GAIT study, since celecoxib is FDA approved for the management of osteoarthritis pain. Therefore, investigators therefore expected participants in this group to experience some pain relief, which would serve to validate the results of the GAIT study.

The GAIT study found that when patients were divided into two groups based on pain levels, 1,229 had mild pain and 354 had moderate to severe pain. With regard to the effectiveness of these supplements, neither glucosamine nor chondroitin sulphate either on their own or in combination were effective in reducing pain. However, when only those patients with moderate to severe pain was analyzed the combination of glucosamine and chondroitin sulphate was effective for pain relief. Unfortunately, no cartilage thickness studies were performed to determine if the supplements augment cartilage thickness. The GAIT study was publicly funded, and therefore, accusations of conflict of interest could not be used to discredit this study.

in 2005, results from the GUIDE study were presented at the 2005 Annual Meeting of the American College of Rheumatology. This study was funded by glucosamine manufacturers and examined of pain and mobility in 318 osteoarthritis sufferers between the ages of 45 and 75 at 13 European hospitals. Participants in this study were divided into three groups:

a) glucosamine sulphate in soluble powder form 1500mg daily
b) acetaminophen (e.g. Tylenol® and paracetamol) 3000mg daily
c) placebo

In addition, subjects in all three groups were allowed to take ibuprofen as needed as a ‘rescue’ for pain relief.

The GUIDE study found that glucosamine sulphate and acetaminophen were more effective in reducing pain than placebo. Patients who took glucosamine sulphate experienced greater pain relief than patients on acetaminophen.

The GUIDE and GAIT studies were positive for glucosamine and chondroitin, but there are negative studies too. In October 2004, Jolanda Cibere and others published a study in the journal Arthritis Care and Research in which they gave glucosamine or a placebo to arthritis suffers and then discontinued them. 42% of the patients receiving the placebo experienced a disease flare-up and 45% of the glucosamine-receiving patients experienced a flare-up. Also, the time to disease flare was not significantly different in the glucosamine compared with placebo group. Thus Cibere and others concluded that “this study provides no evidence of symptomatic benefit from continued use of glucosamine sulfate.”

The bottom line on all this is the glucosamine and chondroitin perform inconsistently in controlled studies. When poor-quality studies are excluded, glucosamine seems to delay arthritis. The highly respected Cochrane Library published a summary of human clinical trials with glucosamine and when the poor-quality trials were excluded, Towheed and his colleagues concluded that glucosamine provided relief of the symptoms of arthritis and also, based on X-rays, helped delay the onset of osteoarthritis.

However, the European Food Safety Authority reviewed over 60 articles on glucosamine and came to a completely different conclusion. In 2012, the EFSA concluded that “The Panel concludes that a cause and effect relationship has not been established between the consumption of glucosamine and maintenance of normal joint cartilage in individuals without osteoarthritis.”

In 2009, in the Journal, Arthroscopy, Vangsness, Spiker, and Erickson came to a somewhat blasé conclusion, “glucosamine sulfate, glucosamine hydrochloride, and chondroitin sulfate have individually shown inconsistent efficacy in decreasing OA pain and improving joint function.”

The long and the short of it is that these supplements might work. Furthermore, my best guess at this point is that they probably work better for some people than for others. So should you take glucosamine or even chondroitin? All our information at this point says that it is safe to do so. No serious or even moderate side effects have been observed by taking these supplements. Secondly, they might work for some people. How do know if you are one of them? By taking the supplement.

I realize that this post is probably very unsatisfying to many of you, but some are very enthusiastic about glucosamine and chondroitin, and I think that this enthusiasm needs to be tempered by a hard dose of reality.  There is much we simply do not know at this time about the efficacy of these supplements, and more work needs to be done before we can say anything definitive about them.   A recent study shows that large doses of chondroitin (1200 mgs) are effective at reducing symptoms in patients with osteoarthritis of the knee, but given the vagaries of chondroitin absorption (see above), it is unlikely that we can make any hard and fast conclusions about it.

One more note about these supplements.  Several studies have shown that the quality of over-the-counter glucosamine vary considerably.  Be careful what you buy and from whom you buy your supplements.  Consumer Reports has shown that some supplements are even spiked with prescription drugs!  So caveat emptor and do not believe the marketer’s own statements about their supplements.

Using Stem Cells to Make Two Different Building Blocks of Blood Vessels


A research team from Johns Hopkins University has discovered methods that use stem cells to make two different types of tissues that help construct blood vessels.

Even though many experiments have used a variety of stem cells to make blood vessels in living laboratory animals and human patients, making blood vessels in the laboratory from scratch has been a colossal headache. Because patients with vascular diseases need new blood vessels, being able to grow blood vessels in the laboratory for clinical use is an important step in tissue engineering.

Sharon Gerecht, an assistant professor of chemical and biomolecular engineering, led this research team. Through this work, they hope to provide material that can be used to treat patients with diabetes, heart disease, and patients with other types of vascular illnesses.

In an interview, Gerecht said: “That’s our goal: to give doctors a new tool to treat patients who have problems in the pipelines that carry blood through their bodies. Finding our how to steer these stem cells into becoming critical building blocks to make these blood vessel networks is an important step.”

Gerecht and her team focused on smooth muscle cells (SMCs). SMCs are found in the walls of blood vessels and by contracting or relaxing, they regulate the diameter of blood vessels, the rates of blood flow and blood pressure. They are two main types of SMCx: synthetic and contractile. Synthetic SMCs migrate through surrounding tissue and continue to divide. They primarily support newly-formed blood vessels. Contractile smooth muscle cells remain in place and stabilize the growth of new blood vessels. Contractile SMCs also control blood pressure.

To make SMCs in the laboratory, Gerecht and her colleagues used embryonic stem cells and induced pluripotent stem cells. in earlier work, Gerecht’s team differentiated pluripotent stem cells into SMC-like cells that were close to SMCs, but not completely like them. However, by modifying their protocol, Gerecht’s team were able to differentiate pluripotent stem cells into synthetic SMCs. This modified protocol included high concentrations of growth factors and serum, but they also modified their protocol further and were able to induce pluripotent stem cells to differentiate into contractile SMCs.

“When we added more of the growth factor and serum, the stem cells turned into synthetic smooth muscle cells,” Gerecht said. “When we provided a much small er amount of these materials, they became contractile smooth muscle cells.”

This ability to make one type or another type of SMC in the laboratory could be critical in developing new blood vessel networks, since SMCs are such a vital part of blood vessels. Gerecht sad as much when she noted that when you are “building a pipeline to carry blood, you need the contractile cells to provide structure and stability.” Gerecht continued” “But in working with very small blood vessels, the migrating synthetic smooth muscle cells can be more useful.”

This work also carries additional bonuses, since cancer cells induce the formation of small blood vessels to nourish the growing tumor. The current work could also help researchers understand how blood vessels are formed and stabilized in tumors, which could be useful in the treatment of cancer.

Gerecht concluded: “We still have a lot more research to do before we can build complete new blood vessels networks in the lab, but our progress in controlling the fate of these stem cells appears to be a big step in the right direction.”

See M. Wanjare, Cardiovascular Research 2012; DOI: 10.1093/cvr/cvs315.