Implantation of Irradiated Embryonic Stem Cells into the Heart Improves Heart Function After a Heart Attack

Adult stem cell transplantation has been used to treat heart attack patients in several different clinical trials. While the results have not been consistent, adult stem cells, it is clear that adult stem cells, primarily from bone marrow, and in some cases fat, help improve heart function. However, a major criticism of the use of adult stem cells is that they do not differentiate into heart muscle cells, but only improve the heart through “paracrine mechanisms,” which means that they secrete molecules that help heal the heart. This criticism is only represent part of the picture, since bone marrow stem cells transdifferentiate into heart muscle and blood vessel cells, albeit at a rather low rate, and fuse with endogenous cells to form hybrid cells that show improved function (Strauer BE, Steinhoff G. J Am Coll Cardiol. 2011 Sep 6;58(11):1095-104. doi: 10.1016/j.jacc.2011.06.016). In addition, adult stem cells activate endogenous cardiac stem cells to divide and replace lost heart muscle cells and make new blood vessels (Loffredo FS, et al., Cell Stem Cell. 2011 Apr 8;8(4):389-98. doi: 10.1016/j.stem.2011.02.002).

Embryonic stem cells, on the other hand, are thought to differentiate into heart muscle cells that integrate into the heart and directly replace the dead heart muscle cells, Animal studies do show such improvements (Caspi O, et al., J Am Coll Cardiol. 2007 50(19):1884-93). However, there is a caveat to all this: Most of the animal experiments with heart muscles derived from embryonic stem cells have only analyzed heart function for up to four weeks after transplantation. Experiments that examined heart function for longer than four weeks have not been able to show that these improvements are sustained after four weeks (van Laake LW, et al., Stem Cell Res. 2007 Oct;1(1):9-24. doi: 10.1016/j.scr.2007.06.001). Therefore, could it be possible that embryonic stem cell-derived cells also help the heart mainly through paracrine mechanisms?

A new paper from Piero Anversa’s and Richard Burt’s laboratories has shown that implantation of embryonic stem that were hit with radiation so that they cannot divide significantly improves heart function after a heart attack.

Experiments were conducted with mice and rhesus monkeys, and mouse and human embryonic stem cells (ESCs) were used. The ESCs were treated with 20 to 100 Grays of radiation, which completely abolished their ability to divide (a gray is the absorption of one joule of energy, in the form of ionizing radiation, per kilogram of matter).

The irradiated ESCs or iESCs were implanted into mice and Rhesus monkeys that had suffered a heart attack. Control animals were implanted with conditioned culture media from the ESC culture dishes.

In the mice and the Rhesus monkeys, the control animals showed little improvement and their hearts continued to deteriorate after the heart attack. However, the animals that had been implanted with the iESCs showed significant improvement of their heart function.

The authors in the discussion suggest that the iESCs might have suppressed the inflammatory response that occurs in the heart after a heart attack, but tissue sections of the hearts after the experiment showed that the iESC-implanted hearts had just as many immune cells infiltrating the tissue as the hearts of the control animals. Mesenchymal stem cells, however, do a very fine job of suppressing inflammation in the heart after a heart attack (see the recent paper by van den Akker et al., Biochimica et Biophysica Acta 1830 (2013): 2449-58). Therefore, the mechanisms by which ESCs improve heart function might be more paracrine-based than anything else. If this is the case, then why are embryonic stem cells being pursued for clinical purposes? Adult stem cells heal by means of paracrine mechanisms and can also sidestep the problem of immune rejection. Also, adult stem cells treatments do not require the dismemberment of young human beings at the embryo stage of their existence. Therefore, even though the present ESC lines are certainly appropriate for clinical and biological research, deriving more of them for clinical treatments is inappropriate, and even murderous.

Happy Easter to All My Readers

From Mark 16:1-8:

1 When the Sabbath was over, Mary Magdalene, Mary the mother of James, and Salome bought spices so that they might go to anoint Jesus’ body. 2 Very early on the first day of the week, just after sunrise, they were on their way to the tomb 3 and they asked each other, “Who will roll the stone away from the entrance of the tomb?”

4 But when they looked up, they saw that the stone, which was very large, had been rolled away. 5 As they entered the tomb, they saw a young man dressed in a white robe sitting on the right side, and they were alarmed.

6 “Don’t be alarmed,” he said. “You are looking for Jesus the Nazarene, who was crucified. He has risen! He is not here. See the place where they laid him. 7 But go, tell his disciples and Peter, ‘He is going ahead of you into Galilee. There you will see him, just as he told you.’”

8 Trembling and bewildered, the women went out and fled from the tomb. They said nothing to anyone, because they were afraid

Stem Cells From Gum Tissue Help Replace Missing Teeth

Researchers from King’s College London, UK have developed a new method that replaced missing teeth with bioengineered material made from a patient’s own gum cells.

If a patient loses a tooth, the dentist or oral surgeon will typically replace it with an implant. The vast majority of dental implants used today are root-form endosseous implants. Such implants have a similar look to an actual tooth root and are placed within the bone of the jaw. The bone of the jaw fuses the surface of the implant with the surrounding bone (a process known as osseointegration). Because dental implants lack the periodontal ligament they will feel slightly different from natural teeth during chewing. Also, the friction from chewing and from other jaw movements can cause loss of bone around the implant.


Research by members of Paul Sharpe‘s laboratory at King’s College London has brought us closer to the reality of bioengineered teeth to replace toss teeth. Bioengineered tooth research has focussed primarily on producing immature teeth that can grow into adult teeth. Typically, such tooth buds are grown in culture and then transplanted into the gums. The gum actually provides and adequate environment for embryonic tooth buds to develop and form adult teeth. Therefore, the prospect of forming bioteeth certainly seems viable. The only question is identifying the cells and materials that can combine to properly form a normal adult tooth.


Sharpe noted, “What is required is the identification of adult sources of human epithelial and mesenchymal cells that can be obtained in sufficient numbers to make biotooth formation a viable alternative to dental implants.”

Sharpe and his colleagues surmised that gum tissue might provide the right cells for this project. Therefore, they isolated adult human gum tissue samples from patients at the Dental Institute at King’s College and grew it in culture in the laboratory. Next, Sharpe’s group combined this gum tissue with mouse embryonic tooth mesenchyme cells, which are stem cells that can induce tooth formation.  This gum-tooth combination created teeth with surrounding gum tissue that could be transplanted into the mouths of mice. The teeth had dentine, enamel and viable roots.

The epithelial cells from human gum were able to respond to tooth-inducing signals from the embryonic tooth mesenchymal cells in a manner that allowed them to contribute to the tooth crown and the roots, and formed all the available cell types necessary for normal tooth formation. Thus, it appears that gum biopsies can provide a realistic source for human biotooth production.

The next step in this research is the formidable challenge of finding a mesenchymal stem cell population that can induce tooth formation. Presently, only embryonic mesenchymal cells can do this, according to Sharpe, but it is possible that adult mesenchymal stem cells can be manipulated to become tooth-inducing cells.

Polycomb Proteins Pave the Way for Proper Stem Cell Differentiation

Embryonic stem cells have the ability to differentiate into one of the more than 200 cell types. Differentiation requires a strictly regulated program of gene expression that turns certain genes on at specific times and shuts other genes off. Loss of this regulatory circuit prevents stem cells from properly differentiating into adult cell types, and an inability to differentiate has also been linked to the onset of cancer.

Researchers at the BRIC, University of Copenhagen have identified a crucial role of the molecule Fbx110 in embryonic stem cell differentiation. Kristian Helin from the BRIC said, “Our new results show that this molecule is required for he function of one of the most important molecular switches that constantly regulated the activity of our genes. If Fbx110 is not present in embryonic stem cells, the cells cannot differentiate properly and this can lead to developmental defects.”

What is the function of Fbx110? Fbx110 recruits members of the “Polycomb” gene family to DNA. Polycomb proteins, in particular PRC1 & 2, are known to modulate the structure of chromatin, even though they do not bind DNA. Fbx110,, however binds DNA, but it also binds PRC1 . Therefore, Fbx110 seems to serve as an adapter that recruits Polycomb proteins to DNA.

Polycomb proteins bound to nucleosomes
Polycomb proteins bound to nucleosomes

Postdoctoral fellow Xudong Wu, who led the experimental part of this investigation, said, “Our results show that Fbx110 is essential for recruiting PRC1 to genes that are to be silenced in embryonic stem cells. Fbx110 binds directly to DNA and to PRC1, and this way it serves to bring PRC1 to specific genes. When PRC1 is bound to DNA it can modify the DNA associated proteins, which lead to silencing of the gene to which it binds.”

Timing of gene activity is crucial during development and must be maintained throughout the lifespan of any cell. Particular genes are active at a certain times and inactive at other times, and PRC1 seems to be part of the reason for this coordination of gene activity. PRC1 is dynamically recruited to and dissociates from genes according to the needs of the organism.

When cancer arises, this tight regulation of gene activity is often lost and the cells are locked in an inchoate state. This loss of terminal differentiation causes increases cell proliferation and the accumulation of other mutations that allow the cancer cells to undergo continuous self-renewal through endless cell divisions. Such an ability is denied to mature cells because of their tightly controlled programs of gene expression.

Wu added, “Given the emerging relationship between cancer and stem cells, our findings may implicate that an aberrant activity of Fbx110 can disturb PRC function and promote a lack of differentiation in our cells. This makes it worth studying whether blocking the function of Fbx110 could be a strategy for tumor therapy.”

In collaboration with a biotech company called EpiTherapeutics, the BRIC researchers want to develop Fbx110 inhibitors as potential novel therapies for cancer.

The Role of Astrocytes in Lou Gehring’s Disease

A study from Columbia University and Harvard University has uncovered a complex interplay between neurons and support cells known as astrocytes that contributes to the pathology of ALS. Such an intricate interplay complicates regenerative therapies for this disease.

In the spinal cord, a group of neurons called motor neurons extend their axons to skeletal muscles and provide the neural signals for the muscles to contract, which allows movement. Motor neurons also have associated support cells known as glial cells, and a specific group of glial cells known as astrocytes associate with motor neurons in the spinal cord.

Astrocytes are star-shaped cells that surround neurons in the brain and spinal cord, and they outnumber neurons 50:1. Astrocytes are very active in the central nervous system, and serve to maintain, support, and repair the nervous tissue that they serve, and are responsible, in large part, for the plasticity of the nervous system.

astrocytes1 (1)

Motor neurons die off during the course of ALS, which leads to paralysis and death within two to fives years of diagnosis. ALS also affects neurons in the brain and it completely robs the individual of the ability to initiate movement or even breathe. There is, at present, no cure and no life-prolonging treatment for ALS.

Data from the ALS Association group suggests that astrocytes in ALS patients go from supporting neurons to strangling them. According to Lucie Bruijn, the chief scientist at the ALS Association in Washington D.C.,, these results seem to “strengthen the case that astrocytes are central to the ALS disease process.” She continued: “Furthermore, the results are based on an exciting new disease model system, one that will allow us to test important hypotheses and search for new therapeutic targets.”

In a cell culture system of ALS, in which neurons derived from embryonic cells were co-cultured with normal and ALS astrocytes, Bruijn’s team found that gene expression patterns in those neurons associated with ALS astrocytes were abnormal. In this experiment, neurons derived from embryonic stem cells with co-cultured with normal and ALS affected astrocytes. In a time course experiment in which gene expression profiles were analyzed from the neurons after specific amounts of time, the gene expression patterns from the normal astrocytes co-cultured with neurons were compared with those of the ALS-affected astrocytes co-cultured with neurons. From these experiments, it became clear that the ALS-affected astrocytes did not communicate properly with the nearby neurons.

Even though neurons communicated with each other by means of the release of neurotransmitters, astrocytes and other glial cells also communicate with each other by means of the release of various molecules. This astrocyte-neuron communication maintains healthy neuron function. However, in the case of ALS, the neuron-astrocyte communication is “profoundly disrupted” and is disruption is not neuron dependent, since in this experiment, the neurons were normal. Without proper communication with their astrocytes, motor neurons the spinal cords of ALS patients are not able to function properly.

According to Bruijn, “This study points out several potential points for treatment intervention.” The protection of motor neurons is the goal, since the astrocytes seem to be doing little to protect and support the neurons and also might be hurting them.

An added bonus to this study is that when spinal cords from mice with a disease that shows some similarities to ALS have their gene expression profiles compared to these gene expression profiles observed in the cultured neurons, the results are remarkably similar. This shows that culture system does recapitulate what goes on in the spinal cord.

The next step is to show that the molecular abnormalities discovered in this system mimics those that occur in human disease. This publication utilized mouse cells, and the human disease, while similar, is not exactly the same.

A Protein Responsible for Cancer Stem Formation Provides a Drug Target

Eighty-five percent of all tumors are carcinomas, which are tumors that form in layers of cells that line surfaces.  Such cell layers are known as an epithelium. When carcinomas form, they undergo an “epithelial-mesenchymal” transformation” or EMT.  EMT means that cells go from being closely aligned and tightly bound to each other in a an organized layer to cells that have little to do with each other and grow in unorganized clumps.  Is there a molecule that unites the carcinomas and if so is this molecule a potential drug target for cancer treatments?

Mammary Carcinoma
Mammary Carcinoma

Researchers at the University of Texas MD Anderson Cancer Center have identified a protein that seems to play a pivotal role in EMT.  This protein, FOXC2, may lay at the nexus of why some carcinomas resist chemotherapy and grow uncontrollably and spread.  FOXC2 could, conceivably represent a novel drug target for chemotherapy.

Sendurai Mani, assistant professor of Translational Molecular Pathology and co-director of the Metastasis Research Center at MD Anderson, said, “We found that FOXC2 lies at the crossroads of the cellular properties of cancer stem cells and cells that have undergone EMT, a process of cellular change associated with generating cancer stem cells.”

Cancer stem cells are fewer in number than other tumor cells, yet research has tied them to cancer progression and resistance to treatment.  Abnormal activation of EMT can actually create cancer stem cells, according to Mani.

Mani continued, “There are multiple molecular pathways that activate EMT.  We found many of these pathways also activate FOXC2 expression to launch this transition, making FOXC2 a potentially efficient check point to block EMT from occurring. ”  Mani’s research group used experiments with cultured cells and mice to discover these concepts, but the next step will require assessing the levels of FOXC2 expression in human tumors samples.

In the meantime, these new data from Mani’s research team may have profound implication for the treatment of particular types of carcinomas that have proven to be remarkably stubborn.  Breast cancers, for example, are typically carcinomas of the mammary gland ductal system.  A specific group of breasts cancers are very notoriously resistant to treatment, and FOXC2 seems to be at the center of such breast cancers.

The anti-cancer drug sunitinib, which is marketed under the trade name Sutent, has been approved by the US Food and Drug Administration (US FDA) for three different types of cancers.  In this study, sunitinib proved effective against these particularly stubborn types of breast cancer; the so-called “triple-negative, claudin-low” breast cancers.


Mani explained why such breast cancers are so resistant to treatment:  “FOXC2 is a transcription factor, a protein that binds to DNA in the promoter region of genes to activate them.  For a variety of reasons, transcription factors are hard to target with drugs.”

However, sunitinib seems to target these triple-negative breast cancers.  When mice with triple-negative breast cancer were treated with sunitinib, the treated mice had smaller primary tumors, longer survival, and fewer incidences of metastasis.  The cancer cells also showed a marked decreased in their ability to form “mammospheres,” or balls of cancer stem cells (this is an earmark of cancer stem cells).  Thus sunitinib seem to attack cancer stem cells.

As it turns out, FOXC2 activates the expression of the platelet-derived growth factor receptor-beta (PDGFRc-beta).  Activation of PDGFRc-beta drives cell proliferation in FOXC2-positive cells, and sunitinib inhibits PDGFRc-beta and inhibits cells that have active FOXC2 and undergoing EMT.

Triple-negative breast cancer cells lack receptors that are used by the most common anti-cancer drugs.  These deficiencies are responsible for the resistance of these cancers to treatment.  Such cancer cells also tend to under go EMT because they lack the protein claudin, which binds epithelial cells together.  Without claudin, these cancer cells become extremely aggressive.

Since cells undergoing EMT are heavily expressing FOXC2, Mani and his colleagues used a small RNA molecule that makes a short hairpin and inhibits FOXC2 synthesis.  Unfortunately, blocking FOXC2 had no effect on cell growth, but it did alter the physical appearance of the cells and reduced their expression of genes associated with EMT and increased the expression of E-cadherin, a protein necessary for epithelial cell organization.  Breast cancer cells also became less invasive when FOXC2 was inhibited, and they down-regulated CD44 and CD24, which are markers of cancer stem cells..  Additionally, triple-negative breast cancer cells that had FOXC2 inhibited had a reduced ability to make mammospheres.  Thus, FOXC2 expression is elevated in cancer stem cells, and inhibition of FOXC2 decreased the ability of the cancer stem cells to behave as cancer stem cells.


Mani’s group also approached these experiments from another approach by overexpressing FOXC2 in malignant mammary epithelial cells.  This forced FOXC2 expression drove cells to undergo EMT and become much more aggressive and metastatic (the cancer spread to the liver, hind leg, lungs, and brain).  Breast cancer cells without forced FOXC2 overexpression showed no tendency to metastasize.

Finally, Mani’s group examined metastatic mammary tumors that were highly aggressive when implanted into nude mice (mice that cannot reject transplants).  Two of the tumors were claudin-negative and both of these tumors showed elevated FOXC2 expression.  When FOXC2 expression was blocked by Mani’s hairpin RNA, the claudin-negative tumors became less aggressive and grew more as mesenchymal cells.  The cells that underwent EMT also showed high levels of PDGF-RC-beta expression.

Mani said of these data: “We thought PDGF-B might be a drugable target in these FOXC2-expressing cells.”  Mani’s group also showed that suppressing FOXC2 reduced the expression of PDGFRC-Beta.  Thus, this small molecule might be an effective therapeutic strategy for treating these hard-to-treat breast cancers.

MD Anderson has filed a patent application connected to this study.

See Hollier B.G., Tinnirello A.A., Werden S.J., Evans K.W., Taube J.H., Sarkar T.R., Sphyris N., Shariati M., Kumar S.V., Battula V.L., Herschkowitz J.I., Guerra R., Chang J.T., Miura N., Rosen J.M., and Mani S.A.,. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Research. e-Pub 2/2013.

Mesenchymal Stem Cells Rarely Engraft But Work in a “Hit and Run” Manner

Even though this paper was published in 2012, it is a very important study that deserves a wide reading and lots of discussion.

The paper is “Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation” from Kathleen Le Blanc’ s laboratory, which was published in Stem Cells 2012;30:1575–1578.

In this paper, Le Blanc and her colleagues examined autopsies from patients who had received mesenchymal stem cell transplants. Since many scientists consider mesenchymal stromal cells (MSCs) a novel treatment for a variety of medical conditions, it is crucial that the fate of MSCs after infusion is better understood. Also, the long-term safety profile of MSCs is also quite important. DO they cause malignant transformation and ectopic tissue formation? Autopsies are an excellent way to address this questions.

The Le Blanc laboratory examined autopsy material from 18 patients who had received MSC transplants from people other than themselves. They analyzed 108 tissue samples from 15 patients by means of polymerase chain reaction (PCR) to search for the DNA of MSCs from donors in the tissue. If such foreign DNA was present in the tissues of the stem cell recipients, this would indicate that the MSCs had engrafted into the tissues of the patient. Unfortunately, MSC donor DNA was detected in only one or several tissues including lungs, lymph nodes, and intestine in eight patients at very low levels (from 1/100 to <1/1,000). Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, which simply means that the more time had elapsed since the time of the MSc transplant, the less likely it was that MSC DNA was found in the patient. For example, MSC DNA was detected in nine of 13 patients whose MSC infusions had been given within 50 days before sampling, in only two of eight of those infusions that had been given earlier.

On a more positive note, there were no signs of ectopic tissue formation or malignant tumors of MSC-donor origin upon macroscopic or histological examination of the tissues of the autopsied individuals.

What does all this mean? MSCs appear to mediate their healing capacities through the molecules that they secrete. This is called a “paracrine” mechanism. and MSCs seem to engraft into host tissues only very rarely. Instead MSCs come to a damaged tissue and stimulate the endogenous healing mechanisms already present. After doing this job, MSCs do not typically stick around. Thus, MSCs seem to work in, what Le Blanc calls a “hit and run” mechanism.

Because MSCs do not seem to engraft over a long period of time, the potential adverse reactions to these cells seems to be largely limited. Thus these cells are quite safe, but their effects are almost certainly indirect to some extent.

Rejuvenating the Blood of Older People With New Stem Cells

Like it or not, the blood of young people and older people is different. Can the blood of an older person be rejuvenated and made young again?

In an article published recently by the scientific journal Blood, a research group at Lund University in Sweden details a series of experiments in which they rejuvenated the blood of mice by reversing, or re-programming, the blood cell-making stem cells.

Stem cell populations throughout the body form and replace cells in the body and help repair organs. Stem cells have the capability to divide an unlimited number of times, and when they divide, one cell remains a stem cell and the other matures into another cell type needed by the body.

Martin Wahlestedt, a doctoral student in stem cell biology at the Faculty of Medicine at Lund University, and principal author of the article explained, “Our ageing process is a consequence of changes in our stem cells over time.” Wahlestedt continued, “Some of the changes are irreversible, for example damage to the stem cells’ DNA, and some could be gradual changes, known as epigenetic changes, that are not necessarily irreversible, even if they are maintained through multiple cell divisions. When the stem cells are re-programmed, as we have done, the epigenetic changes are cancelled.”

Shinya Yamanaka was awarded the Nobel Prize in Medicine last year for this very discovery.

Blood composition changes as we age. For example, blood from a young person contains a certain mix of B- and T-lymphocytes and myeloid cells, but in older people, according to Wahlestedt, “In older people, the number of B- and T-lymphocytes falls, while the number of myeloid cells increases.” Therefore, when an elderly person is affected by leukemia, the cancer usually originates in the myeloid cells, since the elderly have more myeloid cells. Being able to refurbish the blood, as Martin and his colleagues have done in their mouse studies, therefore, presents interesting possibilities for future treatment.

“There is a lot of focus on how stem cells could be used in different treatments, but all that they are routinely used for in clinical work today is bone marrow transplants for diseases where the blood and immune systems have to be regenerated”, said Martin Wahlestedt, continuing:  “A critical factor that gives an indication of whether the procedure is going to work or not is the age of the bone marrow donor. By reversing the development of the stem cells in the bone marrow, it may be possible to avoid negative age-related changes.”

Even if the composition of the blood in old and young mice is remarkably like that in young and elderly people, Martin Wahlestedt stressed that at this stage; the technology is only at the basic research stage and is far from a functioning treatment. The research group is pleased with the results, because they indicate that it may not primarily be damage to DNA that causes blood to age, but rather the reversible epigenetic changes.

Neural Cells Made from Monkey Skin Cells Integrate into Monkey Brains and Form Neurons

Stem cell scientists from the University of Wisconsin at Madison have transplanted neural cells that were made from a monkey’s skin cells into the brain of that same monkey. The transplanted cells formed variety of new brain cells that were entirely normal after six months.

This experiment is a proof-of-principle investigation that shows that personalized medicine in which regenerative treatments are designed for specific individuals is possible. These neural cells were derived from the monkey’s skin cells and were, therefore, no foreign. Therefore, there is no risk of them being rejected by the host immune system.

Su-Chun Zhang, professor of neuroscience at the University of Wisconsin-Madison, said: “When you look at the brain, you cannot tell that it is a graft. Structurally the host brain looks like a normal brain; the graft can only be seen under the fluorescent microscope.”

Marina Emborg, associate professor of medical physics at UW-Madison and one of the lead co-authors of the study, said: “This is the first time I saw, in a nonhuman primate, that the transplanted cells were so well-integrated, with such a minimal reaction. And after six months, to see no scar, that was the best part.”

The skin-derived neural cells were implanted into the monkey brain by means of a state-of-the-art surgical procedure whereby the surgeon was guided by a live MRI. The three rhesus monkeys used in the study at the Wisconsin National Primate Research Center had brain lesions that caused Parkinson’s disease. Up to one million Americans suffer from Parkinson’s disease, and some 60,000 new patients are diagnosed with it each year. Parkinson’s disease results from the death of midbrain neurons that manufacture the neurotransmitter dopamine.

The cells that were transplanted into the brain were derived from induced pluripotent stem cells (iPSCs), which, like embryonic stem cells, can develop into virtually any cell in the adult human body.

Once the iPSC lines were established, Zhang and his colleagues differentiated them into neural progenitor cells (NPCs), which have the ability to form a wide variety of brain-specific cells. Zhang was the first scientist to ever successfully differentiate iPSCs into NPCs, and therefore, this paper utilized his unique expertise.

According to Zhang, “We differentiate the stem cells only into neural cells. It would not work to transplant a cell population contaminated by non-neural cells. By taking cells from the animal and returning them in a new form to the same animal, this is a first step toward personalized medicine. Now we want to more ahead and see if this leads to a real treatment for this awful disease.”

Another positive sign was the absence of any signs of cancer, which is a troubling but potential outcome of stem cell transplants. Zhang jubilantly but guardedly announced that the appearance of the cells is “normal, and we also used antibodies that mark cells that are dividing rapidly, as cancer cells are, and we do not see that. And when you look at what the cells have become, the become neurons with long axons, as we’d expect. The also build oligodendrocytes that are helping build insulating sheaths for neurons, as they should. That means they have matured correctly, and are not cancerous.”

Zhang and his colleagues at the Waisman Center on the UW-Madison campus designed this experiment as a proof of principle investigation, but because they did not transplant enough dopamine-making cells into the brain, the animal’s behavior did not improve. Thus, although this transplant technique is certainly very promising, it is some ways from the clinic.

As noted by Emborg: “Unfortunately, this technique cannot be used to help patients until a number of questions are answered: Can this technique improve the symptoms? Is it safe? Six months is not long enough.” Emborg continued, “And what are the side effects? You may improve some symptoms, but if that leads to something else, then you have not solved the problem.”

Regardless of these shortcomings, this study still represents a genuine breakthrough. “By taking cells from the animal and returning them in a new form to the same animal, this is a first step toward personalized medicine,” said Emborg.

Human Amniotic Fluid Stem Cells Embedded in Beads for Heart Atacks

Human amniotic fluid stem cells (hAFSCs) have been isolated from the “water” that surrounds the baby when it is born. Amniotic fluid is the material is lost when a pregnant woman’s “water breaks.” If the amniotic fluid is retrieved before it ruptures, a specific stem cell population can be isolated from it, and these stem cells grow very well in culture, and can differentiate into a multiple of adult cell types.


When it comes to the heart, hAFSCs have a bit of a mixed record. One publication from Anthony Atala’s laboratory showed that implantation of hAFSCs into the heart of a laboratory animal after a heart attacked was followed by the formation of bony nodules in the heart tissue (see Chiavegato et al., J Mol Cell Cardiol. 42 (2007) 746-759). However, a follow-up publication, showed that the conditions used in the previous experiments caused the formation of bony nodules in the heart regardless of whether or not hAFSCs were implanted into the heart (Delo DM et al., Cardiovasc Pathol 2011 20(2):e69-78).  Other papers showed that implanted cAFSCs could protect the heart from further deterioration (Bollini S et al., Stem Cells Dev. 2011 20(11):1985-94).  However, a perennial problem is the poor retention of the cells in the heart after injection.  Therefore, one group tried implanting hAFSCs into cellular goo (extracellular matrix). This caused the hAFSCs to stay put in the heart and differentiate into heart muscle cells and blood vessels (Lee WY et al., Biomaterials. 2011 32(24):5558-6).

On the heals of this success comes a paper from Taiwanese researchers who have embedded hAFSCs into polylactic-co-glycolic acid (PLGA) beads and implanted these into the heart of a laboratory animal after a heart attack.  These beads are made of material that is completely biogradable, but the hAFSCs survive and grow well in them.  Also, once they are implanted into the heart, the beads are large enough to prevent them from being displaced.  Once the beads disintegrate inside the heart tissue, the cells are already so deeply implanted into the heart tissue, that they do not become washed out by circulating blood and other fluids.  

Poly lactic-co-glycolic acid
Poly lactic-co-glycolic acid

The implanted hAFSCs differentiated into heart muscle cells and blood vessels.  The blood vessels density in these hearts of the hAFSC implanted animals twice that of the control animals in the area of the infarct and almost three times that of the control outside the area of the infarct.  The scar shrunk in the hAFSC-implanted hearts by ~30%, and the structure of the hAFSC-implanted hearts was much more robust and thick relative to the controls.  Finally, the contraction of the heart muscle was (4 weeks after treatment) twice as strong in the hAFSC-treated hearts compared to the control.  Ejection faction was not measured, and that is a deficiency in this paper, but all the cardiac parameters that were measured were vastly improved in the hAFSC-treated hearts relative to the untreated controls.

This paper shows that the porous PLGA beads are not toxic, deliver cells to the chosen target, and quickly disintegrate without affecting the target tissue, in this case the heart. Clearly hAFSCs have a part to play in the future of regenerative medicine.

Turning Muscle Stem Cells into Brown Fat

Michael Rudnicki’s laboratory at the Ottawa Hospital Research Institute has managed to convert stem cells from skeletal muscle into brown fat. Because brown fat burns calories, studies have shown that trimmer people tend to have more brown fat, Therefore, Rudnicki’s findings are being viewed as a potential treatment for obesity.

According to Rudnicki, “This discovery significantly advances our ability to harness this good fat in the battle against bad fat and all the associated health risks that come with being overweight and obese. Rudnicki is a senior scientist and director for the Regenerative Medicine Program and Sprott Center for Stem Cell Research at the Ottawa Hospital Research Institute.

Obesity is the fifth leading risk death, globally speaking, and an estimated 2.8 million people dying every year from the effects of being overweight or obese, according to the World Health Organization. The Public Health Agency of Canada estimates that 25% of Canadian adults are obese.

in 2007, Rudnicki and his research team demonstrated the existence of a stem cell population in skeletal muscle. In this new publication, Rudnicki and others show that these adult muscle stem cells not only have the ability to produce muscle fibers, but can also make brown fat.

An even more important aspect of this paper (Yin, et al., Cell Metabolism 17(2) 2013: 210), is that it shows how adult muscle stem cells become brown fat. The main switch is a regulatory molecule called microRNA-133 or miR-133. When miR-133 is present, the muscle stem cells produce muscle fibers, but when the intracellular concentration of miR-133 is reduced, the muscle stem cells form brown fat.

Graphic Abstract

Rudnicki’s research staff developed a molecule that could reduce the concentration of miR-133 in cells. This molecule an antisense oligonucleotide or ASO that is complementary to miR-133. When injected into mice, the ASO caused the mice to produce more brown fat and prevented obesity. Additionally, when injected into the hind leg muscle, the metabolism of the mouse increased, and this effect lasted for four months after the ASO injection.

Even though antisense oligonucleotides are being used in clinical trials, such trials with miR-133 ASOs are still years away.

Rudnicki noted that “we are very excited by this breakthrough.” He continued: “While we acknowledge that it’s a first step there are still many questions to be answered, such as: Will it help adults who are already obese to lose weight? How should it be administered? How long do the effects last? Are there any adverse effects we have not yet observed?”

Surely these questions will be addressed in good time, and Rudnicki’s lab is probably working on them as you read this entry.

Taste Stem Cells Identified

Researchers at the Monell Center in Philadelphia, PA have successfully identified the location and markers for taste stem cells on the tongue. These findings will almost certainly allow scientists to grow and manipulate taste cells for clinical and research purposes.

Neurobiologist Robert Margolskee with the Monell Center who was also one of the authors of this study said: “Cancer patients who have taste loss following radiation to the head and neck and elderly individuals with diminished taste function are just two populations who could benefit from the ability to activate adult taste stem cells.”

Taste cells are located in rosette-like clusters known as taste buds in bumpy structures called papillae on the upper surface of the tongue. Two types of taste cells contain chemical receptors that initiate the perception of sweet, bitter, unami salty, and sour taste qualities. A third type of taste cell appears to serve as a support cell for these taste cells.


A truly remarkable characteristic of these sensory cells is that they regularly regenerate, and all three taste cells undergo frequent turnover. The average lifespan of these cells is 10-16 days, which means that constant regeneration must occur in order for these cells to replace the cells that constantly die.

For decades, scientists who study taste have tried to identify the stem cell population that form these different taste cells. Scientists were also completely uncertain as to the location of these taste cell progenitors. Where they in the taste buds, near the taste buds, or someplace entirely different?

Monell scientists drew upon the strong association between oral taste cells and endocrine cells in the intestine. They reasoned that the cell-surface markers for stem cells in the intestine might also serve as markers for stem cells in the tongue. By using antibodies to a surface protein called Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5), the Monrell team observed two strong expression patterns for this marker in the tongue. One signal was underneath taste papillae at the back of the tongue and the second signal was an even weaker signal underneath taste buds in those papillae.

The Monell group hypothesized that the two levels of expression could indicate two different populations of cells that expressed Lgr5 at different levels. The stronger-expressing cells are probably the actual stem cells and those that more weakly express Lgr5 are those progeny of these stem cells that are beginning to differentiate. Therefore, the expression of the stem cell marker in these cells is fading.

Additional work showed that Lgr5-expressing cells were capable of differentiating into any of the three different types of taste cells.

Peihua Jiang. who is also a neurobiologist at the Monell Center, said: “THis is just the tip of the iceberg. Identification of these cells opens up a whole new area for studying taste cell renewal, and contributes to stem cell biology in general.”

In the future, the Monell group plans to program the Lgr5-expressing cells to differentiate into the different taste cell types, and explore how to grow these cells in culture. This will create a renewable source of taste receptor cells for research and perhaps even clinical use.

See Karen Yee, et al., “Lgr5-EGFP Marks Taste Bud Stem/Progenitor Cells in Posterior Tongue.” Stem Cells 2013 DOI: 10.1002/stem.1338.

Stem Cells from Human Adipose Tissue Used to Chase Migrating Cancer Cells

From The Stem Cell Blog – very nice article about fat-based mesenchymal stem cells chasing metastatic cancers. Enjoy.

The Stem Cell Blog

Stemness of primary AMSC lines demonstrated with differentiation along three mesenchymal lineages, Adipocyte (a, d [48], g), Osteocyte (b [48], e, h), and Chondrocyte (c [48], f, i), documented via lineage specific staining with Oil Red O, Alizarin Red, and Collagen II, respectively. (Credit: Pendleton et al. Mesenchymal Stem Cells Derived from Adipose Tissue vs Bone Marrow: In Vitro Comparison of Their Tropism towards Gliomas. PLoS ONE, 2013; 8 (3): e58198 DOI: 10.1371/journal.pone.0058198)
Using Fat to Fight Brain Cancer: Stem Cells from Human Adipose Tissue Used to Chase Migrating Cancer Cells

Mar. 12, 2013 — In laboratory studies, Johns Hopkins researchers say they have found that stem cells from a patient’s own fat may have the potential to deliver new treatments directly into the brain after the surgical removal of a glioblastoma, the most common and aggressive form of brain tumor.

The investigators say so-called mesenchymal stem cells (MSCs) have…

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Transplantable Hematopoietic Stem Cells Made From Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are made from adult cells by genetic manipulation. In short, four different genes, all of which encode DNA-binding proteins that direct gene expression, are introduced into adult cells. The four proteins direct a gene expression program that dedifferentiates a small proportion of the cells to become stem cells that greatly resemble embryonic stem cells.

These iPSCs have the capacity to differentiate into any cell type in the adult body, but there are particular cell types that have proven difficult for iPSCs to make. One of these is the blood cell-making stem cell that normally resides in bone marrow. This stem cells, the hematopoietic stem cell or HSC. Several different types of blood cells have been made from iPSCs, but, again, making HSCs from iPSCs has proven elusive.

A paper from the laboratories of Leslie Silberstein and Daniel Tenen at the Harvard Stem Cell Institute and Harvard Medical School has used a new approach to make HSCs from iPSCs. In this paper, Giovanni Amabile and colleagues injected undifferentiated HSCs into mice whose immune systems were compromised to prevent them from rejecting the implanted cells. The iPSCs formed tumors known as teratomas that contained a wide variety of cells types that included HSCs. Isolation of these HSCs from the teratomas produced pure cultures of HSCs that could be used to reconstitute the immune system of mice.

Isolation of HSCs from teratomas is actually rather easy, since very high-affinity antibodies can bind to the surfaces of HSCs and facilitate their isolation. Once isolated, Amabile and others used them to reconstitute the immune system of imunodeficient mice. This demonstrates that HSCs isolated in this manner are transplantable.

Embryonic stem cells can be converted to HSCs by co-culturing them with OP9 cells, a special mouse bone marrow-derived cell line. If iPSCs were injected into mice with OP9 cells, the number of HSCs they made in culture greatly increased.

OP9 cells
OP9 cells

The cells produced by the HSCs were evaluated for functionality, and the white blood cells made all the right molecules, ate bacteria like they should and also moved like white blood cells. Antibody making cells all made antibodies and T cells responded just as they should and made all the right molecules in response to stimulation. Thus, these HSCs were normal HSCs and produced blood cells that were completely normal from a functional perspective.

This technique could provide a way to make HSCs for human antibody production, drug screening, and, possibly, transplanation. Unfortunately, if these cells have been passed through an animal, there is no way they can be used for human treatments, since they might have picked up animal viruses and animal sugars on their surfaces. If these procedure could be refined to eliminate passing the iPSCs through an animal , then this technique could certainly be used to make transplantable HSCs for the treatment of human diseases of the blood.

See Amabile et al., Blood 121(8):1255-1264.

Platelet-Rich Plasma Enhances the Clinical Outcomes of Microfracture Surgery in Older Patients

Osteoarthritis occurs when the cartilage that covers the opposing bones at a joint erodes away and the bare opposing bones smash into each other causing the bone to crack, fragment and chip. The result is extensive inflammation of the joint and further destruction of the bone, which prompts a knee replacement.

Because knee replacement surgeries are so painful and because they only last about two decades at the most, replacing the lost cartilage is a better option. One surgical treatment for osteoarthritis is microfracture surgery. Microfracture surgery involves the drilling of small holes in the tips of the bones of the joint to serve as conduits for stem cells in the bone to come to the surface and make cartilage.

Unfortunately, there are some problems with microfacture surgery, the most prominent of which is that it works better in younger patients than in older patients. Patients older than 40 years old show a precipitous drop in success after microfracture surgery. Thus, finding some way to increase the activity of cartilage production by endogenous stem cells would be a welcome finding for orthopedic surgeons.

Platelet-rich plasma (PRP) has been used to augment the cartilage-making activities of mesenchymal stem cells from bone marrow. Therefore, some surgeons from South Korea decided to try adding PRP to the knees of patients who had just had microfracture surgery. They examined 49 patients with early arthritis. All of these patients were subjected to arthroscopic microfracture surgery for a cartilage lesion that was less than four cubic centimeters in size. These patients were all between the ages of forty to fifty years old, which means that they were outside the age range for successful microfracture surgery.

These 49 patients were randomly divided into two groups. The first group was a control group of 25 patients that only had arthroscopic microfracture surgery. The second group consisted of 24 patients and they had arthroscopic microfracture surgery and injections of PRP into the knee. 10 patients from each group had follow-up arthroscopies four to six months after the procedure to determine the extent of cartilage restoration. Further evaluations were also done 2 years after the procedure.

The results? There were significant improvements in clinical results between preoperative evaluation and postoperative at 2 years post surgery in both groups (p = 0.017). However in the group that received PRP injections plus microfracture surgery the results were significantly better than those of the control group. These patients had better range of motion and less pain (p = 0.012). In the 2nd look arthroscopies, the cartilage of the patients that received PRP and microfracture surgery was harder and showed increased elasticity than the cartilage of patients that received only microfracture surgery.

The conclusion of these authors: “The PRP injection with arthroscopic microfracture would be improved the results in early osteoarthritic knee with cartilage lesion in 40-50 years old, and the indication of this technique could be extended to 50 years.” (Lee GW et al., “Is platelet-rich plasma able to enhance the results of arthroscopic microfracture in early osteoarthritis and cartilage lesion over 40 years of age? European Journal of Orthopedic Surgery. 2012 Jul 5., epub ahead of publication)  If PRP could improve the outcomes of microfracture surgery, then maybe such a technique could extend the groups of patients who are successfully served by this procedure.

While this is an exciting result, we must temper our excitement with the realization that this is a small study and MRIs were not used to measure cartilage thickness. Therefore, while this study is useful and frankly, ingenious, it has its limitations.

Genome of HeLa Cells Sequenced: It’s a Genetic Mess

HeLa cells are cultured cancer cells that have been used extensively in research. Historically, HeLa cells were used in the development of the polio vaccine and other types of research that led to two Nobel prizes. Over 60,000 publications have used HeLa cells in their research.

HeLa Cells

These cells received their name from the unfortunate young woman Henrietta Lacks, who died of cervical cancer and whose cancer cells were cultured to eventually produce the HeLa cell line, without her approval and without any compensation. The derivation of this cell line is the subject of a very fine book entitled “The Immortal Life of Henrietta Lacks” by Rebecca Skloot.

A recent paper has examined the genome of HeLa cells, and there were certainly some surprises. This paper was published in G3: Genes, Genomes, and Genetics, and this research project was led by Lars Steinmetz of the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany (Landry, J., et al. 2013. The genomic and transcriptomic landscape of a HeLa cell line. G3, doi: 10.1534/g3.113.005777). In this paper, the whole genome sequencing of HeLa cells showed that these cells have a haphazard combination of gene duplications and chromosomal rearrangements. Several chromosomes in the HeLa genome have been shattered and then randomly glued back together, and several genes exist in five or more copies apiece. All this has produced aberrant gene expression pathways that differ dramatically from normal human tissues. These findings could have a profound impact on how HeLa cells are used in the laboratory, according to the authors.

Even though HeLa cells grow well in the laboratory and are very hardy, scientists have known for some time that HeLa cells are not normal. However, according to Steinmetz, “nobody had sequenced the genome to figure out, at nucleotide resolution, where the rearrangements are in this genome. . . I was really struck by how abnormal these cells are.”

Given how heavily HeLa cells are used, the HeLa genome had never been sequenced. However, with the vast decrease in sequencing costs, Steinmetz and his EMBL team sequenced both DNA and RNA—1.1 billion DNA reads, each 101 base pairs in length, and 450 million RNA sequences—from the HeLa Kyoto cell line.

Their analysis of the HeLa genome showed that a dramatic phenomenon called chromosome shattering had occurred to a large degree in HeLa cells. Many chromosomes appear to have broken apart and then reassembled with countless chromosomal regions inverted or in the wrong order. Chromosome shattering is a recently described phenomenon that is associated with 2-3% of all cancers. In the case of HeLa cells, the chromosome shattering was probably original to Henrietta Lacks’ cervical tumor.

Other HeLa cell characteristics probably came about as the cells adapted over decades to life in the laboratory culture dishes. Steinmetz’s team used RNA analysis to show that HeLa gene expression differs dramatically from gene expression in normal human tissues. Cell cycle and DNA repair pathways are upregulated, which is expected for rapidly dividing cells. However, the genes associated with the immune system and environmental sensing are downregulated, which is expected for cells adapted to an isolated, nutrient-rich lab setting.

“We’re using these cells as our workhorse to study human biology,” said Steinmetz, “and if we have these genomic rearrangements, that’s clearly going to have some impact on the interpretation of gene function that we’re carrying out.”

For experiments in which genomic abnormalities don’t matter and scientists just need a lot of biological material quickly, HeLa cells are still suitable. But for genetic studies, the researcher must decide if HeLa cells are an appropriate model for addressing the research problem at hand. If that is the case, scientists can now use the HeLa genome rather than the Human Genome Project reference genome as a basis to better interpret experiments.

Mesenchymal Stem Cell Article

I wrote this review article for the Mesenchymal Stem Cell site.  Unfortunately, this site has now become defunct.  Therefore, I have moved it here for your enjoyment:

“Critical Distinctions between Mesenchymal Stem Cells from Bone Marrow and Alternative Sources”

Michael Buratovich Ph.D (Author)
Supplied Courtesy of BioInformant Worldwide, LLC

Mesenchymal stem cells (MSCs) are adult, multipotent stem cells that have been isolated from circulating blood (Kuznetsov et al 2001), umbilical cord blood (Beibacket al 2004; Lee et al 2004b), placenta (Iguraet al 2004), heart (Warejckaet al 1996), amniotic fluid (Tsai et al2004), adipose tissue (Katzet al 2005), synovium (Fickert et al 2003), skeletal muscle (Younget al 1995), pancreas (Hu et al 2003), deciduous teeth (Estrelaet al 2011), and bone marrow (Charbord 2010). Bone marrow-derived MSCs (BMSCs) are the most heavily-studied of all MSCs, and, therefore, tend to be the standard against which MSCs from other sources are evaluated. BMSCs can differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts, hepatocytes, neural cells, etc., and can give rise to cartilage (Kadiyala et al 1997), bone (Bruder et al 1997; 1998), tendon (Young et al 1998), muscle (Galmiche et al 1993; Ferrari et al 1998), and many other tissues. Do MSCs from tissues other than bone marrow have similar differentiation potentials, and if not how does the potency of these MSCs from alternative sources compare with those from bone marrow? Fortunately stem-cell scientists have examined this question in some detail, but a central question remains: Do MSCs from diverse bodily locations represent distinct or the same cell types?

If MSCs throughout the body are similar cell types then we would expect them to have similar embryological origins. However, this is not the case, since MSCs develop from several different embryonic tissues. The first wave of MSCs arises from Sox-1-expressing neuroepithelial cells during embryonic development. However, later MSCs come from multiple sources (Takashima et al 2007), including neural crest cells (Nagoshi et al 2008; Morikawaet al 2009). Therefore, MSCs from various tissues almost certainly have distinct embryological origins. Additionally, MSCs are located in different sites in the body, and are influenced by specific microenvironments. Thus MSCs from different tissue sources might represent distinct cell types, and could potentially display distinct differentiation profiles and express particular genes. Despite these differences in developmental origin and environmental influences, MSCs from various sources have very similar morphologies and share a common array of surface markers (Mitchell et al 2003; Lee et al 2004a; Wang et al 2004; Tsai et al 2007). However, several studies have established that MSC populations are rather heterogeneous (Dominici et al 2009), and, therefore, surface markers expressed on some cells of an MSC population are not always expressed in all the cells of that population (Mafi et al 2011). Also, the growth kinetics of cultured MSCs differs remarkably with respect to their source (Kang et al 2004b; Yoshimura et al 2007; Troyer and Weiss 2008).

Despite the shared array of cell surface markers, presently there are no cellular markers or cell surface proteins that are unique to MSCs. In order to provide a more unified approach to MSC biology, the International Society of Cryotherapy has proposed three criteria for the identification of MSCs. Under these criteria, MSCs must: (1) be plastic-adherent when maintained in standard culture conditions; (2) express the following cell surface molecules CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules, and; (3) be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro (Dominici et al 2006). Despite these definitions, flow cytometric analyses of MSCs from several different populations have shown some significant differences in cell surface markers (Boeuf and Richter 2010). For example, even though the absence of CD34 is generally considered a criterion for the definition of MSCs, various investigators have reported low expression of CD34 in ADSCs (ADSCs; De Ugarte et al 2003a; Rebelatto et al 2008; Roche et al 2009) and BMSCs (Zvaifler et al 2000; Gronthos et al 2003; Yu et al 2010). Likewise, many investigators have shown that MSCs from multiple sources do not express CD45 (Zvaifler et al 2000; Zuk et al 2002; Igura et al 2004; Dominici et al 2006; Wongchuensoontorn et al 2009), but BMSCs are CD45 positive (Yu et al 2010).

Other cell marker differences include CD271,which shows high levels of expression in BMSCs and ADSCs (Jones et al 2002; Quirici et al 2010), but is not expressed in synovial membrane MSCs (SMSCs; De Bari et al 2001; Van Landuyt et al 2010). Another molecule that is highly expressed in the vast majority of MSC population is STRO-1 (Gronthos et al 1991; Simmons and Torok-Storb 1991; Gronthos et al 1994; Gronthos et al 1999; Stewart et al 1999; Walsh et al 2000; Zuk et al 2002; Miura et al 2003; Kadar et al 2009), but other studies have shown that ADSCs are STRO-1 negative (Gronthos et al 2001). Signal transduction receptors also show varied expression in distinct MSC populations. For example, platelet-derived growth factor receptor (CD140a/PDGFRα) is involved in proliferation and migration of osteoblasts and MSCs. This receptor is much more highly expressed in SMSCs than BMSCs (Nimura et al 2008). Finally the vascular cell adhesion molecule CD106/VCAM1, which is involved in hematopoietic stem cell homing (Simmons et al 1992), is more highly expressed in BMSCs than ADSCs (De Ugarte et al 2003a; Kern et al 2006; Rider et al 2008; Roche et al 2009). This cell surface difference almost certainly is related to the specific microenvironment in which BMSCs are found and their specific roles in maintaining hematopoietic stem cell growth.

Comparative gene array analyses of MSCs from different sources have revealed some differences in gene expression between these distinct MSC populations, but overall the gene expression profiles between these cells are relatively similar (Winter et al 2003; Lee et al 2004a; Djouad et al 2005; Wagner et al 2005; Aranda et al 2009; Jansen et al 2010). Proteomic comparisons of distinct MSC populations using two-dimensional gel electrophoresis analysis came to very similar conclusions (Roche et al 2009). MSCs from intra-articular tissues (synovial membrane and anterior cruciate ligament) and chondrocytes show gene expression profiles that were more similar to each other than to MSCs from extra-articular locations (Segawa et al 2009). These data suggest that MSCs from varied sources probably represent similar, but distinct cell types that express a core of common genes, but also clusters of distinct genes. These gene expression differences convey different differentiation potentials upon specific MSC populations and varied requirements for these particular MSC populations to differentiate into specific cell types (Gimble et al 2008; Rastegar et al 2010).

MSC Differentiation
With respect to the differentiation potential of MSC populations, the general rule of thumb is the closer the MSC source tissue is to the target tissue, the more effectively that particular MSC population differentiates into the target tissue. A few examples should suffice. Yoshimura and colleagues found that rat SMSCs derived from the synovial tissue of the knee, which is closest to the target tissue of chondral cartilage, formed cartilage better than BMSCs, ADSCs, or MSCs from periosteum or muscle (Yoshimura et al 2007). Likewise, gene expression profiles of human BMSCs or umbilical cord-derived MSCs (UCSCs from Wharton’s jelly) definitively showed that BMSCs express a variety of osteogenic genes (RUNX2, DLX5 and NPR3) not observed in UCSCs. Under osteogenic induction, BMSCs produced far more bone than UCSCs. However, UCSCs express angiogenesis genesand fewer genes involved in the immune response than BMSCs, suggesting that UCSCs are superior for allogeneic transplantation. When cocultured with allogeneic macrophages,UCSCs prevented the macrophages from producing immunomodulatory cytokines tumor necrosis factor and Interleukin-6 (Hsieh et al 2010). Finally, Niemeyer and coworkers showed that BMSCs and ADSCs formed bone with similar efficiencies in vivo (Niemeyer et al 2007), but in animals studies, BMSCs produced better repair of tibial osteochondral defects in sheep when compared to ADSCs (Niemeyeret al 2010).

MSC Chondrogenesis
Initiation of cartilage development during animal development begins with the condensation of mesenchymal precursor cells (Woods, Wang and Beier 2007). These cell-cell contacts are mediated by N-cadherin, whose expression is highly upregulated in human MSCs after being subjected to chondrogenic induction (Tuli et al 2003). N-cadherin is required for chondrogenesis of chick limb mesenchymal cells in vitro and in vivo (Oberlender and Tuan 1994). Prior to MSC condensation prechondrocytic MSCs secrete extracellular matrix rich in hyaluronic acid, collagen type I and IIa. Initiation of MSC condensation also correlates with the expression of neural cadherin (N-cadherin) and neural cell adhesion molecule (N-CAM). The secreted signaling molecule transforming growth factor-β (TGF-β) is one of the earliest signals in chondrogenic condensation. TGF-β activates production of the extracellular matrix protein fibronectin, which up-regulates N-CAM, and also stimulates the synthesis of Sox transcription factors (Sox-5, -6 and -9), which are essential for cartilage formation. Other extracellular matrix molecules made by chondrogenic MSCs include tenascins, thrombospondins, and cartilage oligomeric protein (COMP). These extracellular matrix molecules interact with cell adhesion molecules to activate intracellular signaling pathways that initiate the transition from chondroprogenitor cells to fully committed chondrocytes. Proliferating chondroprogenitor cells synthesize hyaluronan, collagen II, IX and XI, and the cartilage-specific proteoglycan core protein (or chondroitin sulfate proteoglycan 1) known as aggrecan. Aggrecan (encoded by the ACANgene) is a member of the aggrecan/versican proteoglycan family, and is the most predominant proteoglycan in the extracellular matrix of articular cartilage. Aggrecan helps cartilage withstand compression. N-cadherin and N-CAM expression fade and disappear in differentiating chondrocytes (Golding, Tsuchimochi and Ijiri 2006).

When grown under chondrogenic conditions, MSCs in monolayer culture respond by condensing into high-density three-dimensional cell aggregates (Winter et al 2003). In order to realistically recapitulate chondrogenesis in culture, researchers deposit centrifuged MSC pellets that contain ~200,000 – 500,000 cells in a two-dimensional culture. This culture system, which is one of the most widely used in chondrogenesis research, is called a pellet, aggregate or spheroid culture. To induce chondrogenesis, pellets are cultured in a basal medium (typically low- or high-glucose Dulbecco’s Modified Eagles Medium, otherwise known as DMEM, or fetal calf serum) that contains dexamethasone, ascorbate, proline, insulin, transferrin and selenous acid (Johnstone et al 1998; MacKay et al 1998; Puetzer, Petitte and Loboa 2010). Classically, the growth factor used to induce chondrogenesis in this type of medium is 10 ng/ml of transforming growth factor-β (TGF-β). TGF-β1, 2, and 3 are the only well-established full inducers of chondrogenesis that, when added as single factors, induce proteoglycan and collagen type II deposition (MacKay et al 1998; Barry et al 2001). Other chondrogenic inducers have been described; bone morphogen protein-2 (BMP-2) for BMSCs (Schmitt et al 2003) and BMP-6 for ADSCs (Estes, Wu and Guilak 2006). However, other studies have failed to confirm the chondrogenic efficacy of these two growth factors (Winter et al 2003; Indrawattana et al 2004; Xu et al 2006; Hennig et al 2007; Weiss et al 2010), and there is even a chance that these two growth factors might only work in a donor-specific fashion.BMP-2, -4, and -6, and insulin-like growth factor-1 (IGF-1) seem to promote chondrogenesis in MSCs when given in combination with TGF-β (Schmitt et al 2003; Im, Shin and Lee 2005; Sekiya et al 2005; Liu et al 2007).

Presently, a significant controversy exists over whether ADSCs or BMSCs are better sources for orthopedic tissue repair (Frisbee et al 2009). Both BMSCs and ADSCs have been successfully differentiated into chondrocytes in vitro (John stone et al 1998; Erickson et al 2002) and used for cartilage repair in vivo (Wakitani et al 1994; Im et al 2001; Centeno et al 2011). However, harvesting adipose tissue is much less painful than bone marrow aspirations, which makes ADSCs much more preferable for orthopedic therapies.

With respect to MSC chondrogenesis (cartilage induction), several studies have reported relatively robust chondrogenesis by ADSCs in two-dimensional (Zuk et al 2002; Erickson et al 2002; Gimble and Guilak 2003) and three-dimensional culture systems (Awad et al 2004; Estes, Wu, and Guilak 2006). However, several head-to-head comparisons of BMSCs and ADSCs have produced contradictory results, with some studies reporting equivalent chondrogenic capacities (Zuk et al 2001; De Ugarte et al 2003b; Rebelatto et al 2007), but many others concluding that human and equine BMSCs show superior chondrogenic ability (Winter et al 2003; Im, Shin and Lee 2005; Sakaguchi et al 2005; Vidal et al 2008). Because the same MSC populations from different donors show different differentiation potentials (Bieback et al 2004; Chang et al 2006a Kern et al 2006), head-to-head comparisons of donor-matched MSC populations are essential in order to compare the chondrogenic potential of MSCs that share the same genetic background. Such donor-matched studies have consistently shown that BMSCs show superior chondrogenic potential over ADSCs (Huang et al 2005; Afizah et al 2007). Additionally, gene array studies indicate that during chondrogenic induction, BMSCs show gene expression profiles that more closely resemble native cartilage than ADSCs (Winter et al 2003). If grown in three-dimensional culture, which is thought to be an essential aspect of chondrogenic differentiation (Johnstone et al 1998; Yoo et al 1998; Erickson et al 2002), once again BMSCs outperform ADSCs if seeded in a hyaluronic acid scaffold (Jakobsen et al 2010) or encapsulated in alginate (Mehlhorn et al 2006). BMSCs also show superior chondrogenesis to UCSCs in a three-dimensional culture in which cells were seeded in a polygycolic acid (PGA) matrix (Wang et al 2009).

These data do not necessarily mean that BMSCs are the best cartilage-making MSCs in the body. First of all, head-to-head comparisons treated both MSC populations with the same chondrogenic induction protocol, which implicitly assumes that culture conditions optimized for BMSCs are also be optimal for ADSCs. This assumption, however, ignores the intrinsic differences between these two MSC populations. Kim and Im have shown that ADSCs display a chondrogenic potential equal to that of BMSCs if ADSCs are treated with higher concentrations of growth factors (Kim and Im 2009). Additionally, Diekman and colleagues have shown that chondrogenesis of BMSCs and ADSCs is highly dependent on the presence and concentration of particular growth factors, the presence or absence of serum, and the composition of the scaffold in which the cells are embedded for the chondrogenic induction. ADSCs made significantly more aggrecan in response to BMP-6 than to TGF-β, but the opposite was true for BMSCs. Likewise, ADSCs produced more type II collagen in the presence of serum whereas BMSCs produced more type II collagen without serum. Finally when seeded in alginate beads, the quantity of glycosaminoglycan (GAG) made by BMSCs were significantly higher in the dual-growth factor cocktail of TGF-β and BMP-6 as compared to TGF-β alone. However, when these same cells were grown in a cartilage-derived matrix, those grown in the TGF-β-alone cocktail had higher viability and produced higher amounts of GAG when compared to those grown in dual cocktail (TGF-β + BMP-6). Thus the growth scaffold greatly influences the response of MSCs to particular growth factors, but these data also underscore that BMSCs and ADSCs are probably distinct cell types (Diekman et al 2010).

Secondly, keeping with the original rule that the closer the source tissue is to the desired target tissue, the more effectively MSCs from those tissue sources differentiate into the target tissue, Sakaguchi and colleagues showed that MSCS from bone marrow, synovium, and periosteum made more cartilage than ADSCs or skeletal muscle-derived MSCs, but SMSCs clearly made the most cartilage (Sakaguchi et al 2005). Interestingly, this result was replicated in rat MSCs (Yoshimura et al 2007). Equine BMSCs, however, do show superior chondrogenesis to UCSCs and MSCs from amniotic fluid (Lovati et al 2011), and human fetal and adult BMSCs exceed the chondrogenic potentials of fetal lung-, and placenta-derived MSCs (Bernardo et al 2007).

The varied responses of MSCs from various sources to different growth factors also have been well documented. For example, TGF-β alone is sufficient for chondrogenesis of BMSCs (Afizah et al 2007), but not ADSCs (Awad et al 2003: Estes, Wu and Guilak 2005). Additionally, the combination of TGF-β and dexamethasone stimulates chondrogenesis in BMSCs, but in ADSCs, TGFβ is required for chondrogenesis but dexamethasone tends to suppress chondrogenesis (Awad et al 2003). The reduced chondrogenic induction of ADSCs by TGF-β is probably due to reduced expression of the TGF-β receptor in these cells. However, BMP-6 treatment induces expression of the TGF-β receptor ALK-5 in ADSCs and combined application of TGF-β and BMP-6 restores chondrogenesis in this MSC population (Hennig et al 2007). A published protocol to successfully differentiate ADSCs into chondrocytes makes use of the combination of TGF-β and BMP-6 (Estes et al 2010).

Differential responses to BMP-6 are also observed in different types of MSCs. As previously mentioned, BMP-6 strongly induces chondrogenesis in ADSCs, but not in BMSCs. BMP-6 in combination with TGF-β inhibits hypertrophy in ADSCs (Estes, Wu and Guilak 2003), but in BMSCs, BMP-6 promotes hypertrophy and endochondral ossification (Sekiya, Colter and Prockop 2001; Sekiya et al 2002; Indrawattana et al 2004).

These varied responses to growth factors by distinct MSC populations might also be a reflection of the assorted levels of “stemness” found among the cells of each MSC population. As previously noted, MSC populations tend to be highly heterotropic, and clonal analyses of ADSCs have shown that these cell populations are a mixture of cells that can form bone, cartilage and fat (tripotent), those that can only form two of these tissues (bipotent), and others that can only form only cell type (monopotent). The ratios of these tripotent, bipotent to monopotent clones seems to vary from study to study. Guilak and colleagues found that 21% of ADSCs clones were tripotent and approximately 30% were bipotent (Guilak et al 2006), but Zuk and others found that only 1.4% of all ADSC clones were tripotent (Zuk et al 2002). The disparities between these studies seem to be due to the media conditions used, the age of the adipose tissue donors, and the overall design of the experiment. However, these studies certainly show that distinct MSC populations consist of cells at varying levels of “stemness,” with some being more committed to a particular cell type and others being less developmentally committed to a particular cell fate. The heterogeneity of these populations almost certainly influences the response of these cell populations to particular growth factors.

MSC Osteogenesis
Runt-related transcription factor-2 (Runx-2) is considered a master regulator of early osteogenic differentiation (Fujita et al 2004). In combination with TGF-β, Runx-2 up-regulates the expression of interleukin-11 (IL-11), which reduces adipogenesis (fat formation) and promotes chondrocytic and osteocytic differentiation (Enomoto et al 2004). Runx-2 also promotes the expression of osterix, another important osteogenic inducer. Osterix suppresses chondrogenesis at low concentrations and promotes osteogenesis at high concentrations (Tominaga et al 2009).

Continuous exposure of BMSCs or ADSCs to ligands for the glucocorticoid receptor (e.g., dexamethasone)and/or the vitamin D receptor (e.g., 1,25 dihydroxyvitamin D3), plus ascorbic acid and β-glycerophosphate induces them to produce mineralized extracellular matrix within three weeks (Gimble et al 2008).Exposure of MSCs to BMPs and Wnt signaling proteins also results in successful differentiation into osteoblasts (Peng et al 2003; Shea et al 2003; Kang et al 2004a; Luo et al 2004; Peng et al 2004; Si et al 2006; Luu et al 2007; Deng et al 2008; Tang et al 2009). Additionally, magnetic field stimulation and can also stimulate osteogenic differentiation of MSCs (Singh, YashRoy and Hoque 2006).

Several studies have found that ADSCs and BMSCs from humans and other animals show equal osteogenic potential (Zuk et al 2001; Zuk et al 2002; De Ugarte et al 2003b; Winter et al 2003; Cowan et al 2004; Lee et al 2004a; Romanov et al 2005; Wagner et al 2005; Kern et al 2006). However, other studies argue that BMSCs display superior osteogenic potential to ADSCs (Im, Shin and Lee 2005; Sakaguchi et al 2005; Musina et al 2006; Lui et al 2007; Yoshimura et al 2007). Yet another study insists that ADSCs have superior osteogenic potential than BMSCs (Izadpanah et al 2006).

In head-to-head comparisons with other types of MSCs, the osteogenic potential of BMSCs was approximately the same as SMSCs, and only slightly better than periosteum-derived MSCs (Sakaguchi et al 2005). However, in another study SMSCs from healthy donors expressed significantly lower levels of osteogenic markers after induction of osteogenesis (Djouad et al 2005). Another comparison between human umbilical cord perivascular cells (HUCPVCs) and BMSCs found that HUCPVCs had higher osteogenic potential than BMSCs (Baksh, Yao and Tuan 2007). However, other studies compared the gene expression profiles and osteogenic potential of UCSCs and BMSCs not only showed a pronounced expression of osteogenic genes in BMSCs, but also established their superior osteogenic potential in in vitro differentiation assays (Hsieh et al 2010; Majore et al 2011). It is unclear if these two experiments analyzed the same umbilical cord cell populations. MSCs isolated from human umbilical cord blood also showed a distinctly greater osteogenic potential in comparison to BMSCs (Chang et al 2006a). Also human UCSCs show superior osteogenic potential in comparison to chorionic plate-derived MSCs (Kim et al 2011).

MSC Adipogenesis
Adipocytes are specialized cells that store triacylglycerols (fats). MSC differentiation into adipocytes requires the activity of a transcription factor called peroxisome proliferator activator receptor-gamma (PPAR-γ). PPAR-γ regulates the function of many adipocyte specific genes (Rosen 2000), and interacts with members of the CCAAT/enhancer binding protein (C/EBP) family to regulate adipogenesis (Farmer 2005). Osteogenic transcription factor Runx2 inhibits adipogenesis by directly interacting with PPAR-γ (Akune et al 2004).

Adipogenic induction of cultured MSCs requires the use of compounds that increase intracellular levels of the signaling molecule 3’,5’-cyclic adenosine monophosphate (cAMP) such as phosphodiesterase inhibitors (e.g., isobutylmethylxanthine or theophylline), and ligands for the glucocorticoid receptor (e.g., dexamethasone), and PPAR-γ, (i.e., rosiglitazone, which is marketed as the anti-diabetic insulin sensitizer AvandiaTM). Additionally, most adipogenic cocktails also include insulin, and some protocols also include indomethacine (Mosna, Sensebe and Krampera 2010). MSCs exposed to these agents form intracellular droplets composed of neutral lipid and express key adipogenic markers (e.g., adiponectin, fatty acid binding protein, aP2) within three-to-nine days (Gimble et al 2008; Muruganandan, Roman and Sinal 2009).

Head-to-head comparisons of MSCs from varied tissue sources have shown that ADSCs have an adipogenic potential that is superior (Sakaguchi et al 2005; Izadpanah et al 2006; Musina et al 2006; Liu et al 2007; Yoshimura et al 2007; Rider et al 2008) or equal to that of BMSCs (Zuk et al 2001; 2002; De Ugarte et al 2003b; Winter et al 2003; Lee et al 2004a; Romanov et al 2005;Wagner et al 2005; Kern et al 2006). SMSCs also showed an adipogenic potential that was equal to that of ADSCs and superior to that of periosteum-derived MSCs (Sakaguchi et al 2005; Yoshimura et al 2007). Some studies suggest that UCSCs show poor adipogenic ability in comparison to BMSCs and ADSCs (Rebelatto et al 2008; Hsieh et al 2010), but another study found that HUCPVCs had superior adipogenic potential when compared to BMSCs (Bask, Tao and Tuan 2007). Chorionic-plate-derived MSCs showed superior adipogenic potential to UCSCs (Kim et al 2011), but umbilical cord and umbilical cord blood seem to contain more than one MSC population, all of which display different adipogenic potentials (Chang et al 2006b; Kestendjieva et al 2008; Cheong et al 2010; Lu et al 2010; Majore et al 2011).

MSC Muscle Differentiation
Myogenesis (muscle formation) is regulated by a family of transcription factors known as the myogenic regulatory factors (MRFs). During embryonic development, two basic helix-loop-helix (bHLH) transcription factors, MyoD and Myf5, establish the skeletal muscle lineage and drive myocyte differentiation (Rudnicki et al 1993). Later events in myogenesis that consist of myocyte fusion into myotubes and the synthesis of muscle-specific contractile proteins is associated with the expression of another bHLH transcription factor, myogenin (Hasty et al 1993; Nabeshima et al 1993). Muscle injury activates a muscle stem cell population called satellite cells that recapitulate the MRF expression program (Smith et al 1994; Yablonka-Reuveni and Rivera 1994; Cornelison and Wold 1997; Cooper et al 1999).

Many different types of MSCs can form skeletal, smooth and cardiac muscle. Maintaining MSCs in 10%-20% serum causes them to express smooth muscle markers like α-smooth muscle actin (Abedin, Tintut and Demer 2004; Gimble et al 2008). When transplanted in vitro, MSCs make smooth muscle rather easily (Galmiche et al 1993; Wakitani, Saito and Caplan 1995; Prockop et al 1997; Ferrari et al 1998; Pittenger et al 1999; Caplan and Bruder 2001; Jiang et al 2002).

Exposing MSCs to low serum concentrations or horse serum leads to the expression of skeletal muscle markers such as myogenin and the formation of multi-nuclear myotubes. However, MSCs do not differentiate into mature, skeletal muscles as readily as they do smooth muscles, and the culture conditions under which the cells are grown seem to be extremely important. Co-culturing BMSCs (Lee, Kosinski and Kemp 2005; Beier et al 2011) or ADSCs (Di Rocco et al 2006) with skeletal muscles can induce myotube formation and the expression of myogenic genes by MSCs. The efficiency of skeletal muscle formation with this procedure is almost doubled by exposing MSCs to the chromatin remodeling reagent trichostatin A (Collins-Hooper et al; 2011). Incubation of MSCs with conditioned medium prepared from chemically damaged, but not undamaged, muscle cells also induces MSC myotube formation and expression of MyoD (Santa Maria, Rojas and Minguell; 2004). Treatment of MSCs with particular molecules such as Galectin-1 (Chan et al 2006), TWEAK (Gigenrath et al 2006) and 5-azacytidine (Kocaefe et al 2010; Natasuke et al 2010) can also induce myogenesis, as can hypoxic preconditioning (Leroux et al 2010).

Dezawa and colleagues have published a protocol for differentiating BMSCs into skeletal muscle. They treated mouse BMSCs for three days with a mixture of bFGF, forskolin, which is known to increase intracellular concentrations of cAMP, platelet-derived growth factor and neuregulin. After the three-day culture period, they transfected the cells with a plasmid that encoded the intracellular domain of the Notch receptor, and selected only those cells that had successfully taken up the plasmid. To augment the ability of the remaining cells to form myotubes, they exposed the cells to either 2% horse serum or ITS (insulin-transferrin-selenite) in serum-free medium. Both of these media promoted myogenic differentiation of MSCs to myoblasts that formed myotubes, and were able to integrate into existing muscle and repair muscle in mdx mice (Dezawa et al 2005). mdx Mice harbor a loss-of-function mutation in the gene that encodes the dystrophin protein, which, in humans, is defective in individuals who are afflicted with Duchenne Muscular Dystrophy (Muntoni, Torelli and Ferlini 2003). Therefore, even though it shows a relatively mild phenotype, the mdx mouse is a model system for muscular dystrophy (Sicinski et al 1998).

Treatment of MSCs with a drug called 5-azacytidine directs them to transdifferentiate into cells that resemble cardiomyocytes (heart muscle cells). In cells, 5-azacytidine is incorporated into DNA where it inhibits DNA methylation, and DNA hypomethylation leads to activation of particular genes (Christman 2002). Treatment of BMSCs (Fukuda 2001; Shim et al 2004; Xu et al 2004; Antonitsis et al 2007; 2008), ADSCs (Rangappa et al 2003b; Lee et al 2009) or UCSCs (Cheng et al 2003) with 5-azacytidine drives them to form cells that have a fibroblast-like morphology, synchronously beat, and express many cardiac-specific genes like troponin T, atrial natriuretic protein (ANP), GATA-4, Nkx2.5, TEF-1, and MEF-2C (Fukuda 2001; 2002; Yang et al 2012). Some work has even shown that these differentiated MSCs respond to adrenergic and muscarinic stimulation (Fukuda 2002), and can integrate into the heart of a laboratory animal and form functional connections with native cardiomyocytes (Hattan et al 2005).

MSCs can also be converted into cardiomyocytes by being co-cultured with living (Rangappa et al 2003a; Yoon et al 2005b; Arminan et al 2009; Peran et al 2010) or apoptotic cardiomyocytes (He et al 2010). Also treatment with particular growth factors, such as BMP-2, Fibroblast growth factor -2 (FGF-2) and IGF-1 (Yoon et al 2005a; Bartunek et al 2007; Hahn et al 2008), can push MSCs to become cardiomyocytes, as can transfection with particular genes like Wnt-11 (He et al 2011), GATA-4 (Li et al 2011), or a combination of GATA-4 and Nkx2.5 (Gao, Tan and Wang 2011). Some controversy exists over cardiomyocyte-induced MSCs, since some studies suggest that differentiated MSCs retain their stromal phenotypes and are, at best, only immature cardiomyocytes (Gallo et al 2007; Rose et al 2008).

Because MSC populations tend to form smooth muscle rather readily, there have been few head-to-head comparisons of the efficiency of smooth muscle formation in distinct MSC populations.

Comparisons of the ability of various MSC populations to differentiate into skeletal muscles include in vitro differentiation of MSCs from bone marrow, spleen, thymus, and liver. This study showed that BMSCs, liver- and thymus-derived MSCs all made skeletal muscle in culture, but splenic-derived MSCs did not (Gornostaeva, Rzhaninova and Gol’dstein 2006). Comparisons of the in vivo ability of BMSCs, ADSCs, and SMSCs to form skeletal muscle when implanted showed that ADSCs had the greatest ability to integrate into existing muscles (de la Garza-Rodea et al 2011).

Interestingly, a small fraction of BMSCs can form myotubes and integrate into existing muscle when injected into laboratory animals, whether that muscle is damaged or not (Ferrari et al 1998), a characteristic also shared by SMSCs (De Bari et al 2003). However, when UCSCs were injected into the tail vein of mdx mice, the cells were able to integrate into the muscle but unable to differentiate in vivo into mature, skeletal muscles (Vieira et al 2010; Zucconi et al 2011). Different MSCs show varying efficiencies of cardiomyocyte differentiation. UCSCs, for example, show particularly low transdifferentiation rates (Martin-Rendon et al 2008). ADSCs, however, transdifferentiate into cardiomyocytes with the highest efficiency (Zhu et al 2008; Tobita, Orbay and Mizuno 2011; Paul et al 2011;Yong et al 2012). In fact, when grown in a semisolid methycellulose medium enriched with growth factors, ADSCs spontaneously form beating ventricular- and atrial-like cardiomyocytes (Planat-Benard et al 2004). This makes ADSCs an attractive source of material for cardiac regenerative therapies.

MSCs and Tooth Formation
Tooth formation results from a complex set of interactions between the overlying stomadial epithelium and underlying mesenchymal cells. Dental mesenchymal cells develop from neural crest cells derived from midbrain and hindbrain cranial neural crest cells. In mice, these two cell populations are in place by day 8.5 (E8.5) and by day 10.5 (E10.5) tooth-forming sites and tooth types are determined. At E11.5, a localized thickening of the dental epithelium that results from cell shape changes forms the “dental placode.” Between E12.5-E13.5, the dental placode proliferates and invaginates to form the epithelial bud around which mesenchymal cells condense (Peters and Bailing 1999). At E14.5, the cap stage, the epithelial component of the developing tooth folds and forms a transient cluster of non-dividing cells called the “enamel knot.” The enamel knot is a signaling center that produces many powerful growth factors, including Sonic hedgehog (Shh), BMP-2, BMP-4, BMP-7, FGF-4 and FGF-9 (Thesleff and Mikkola 2002). The cap stage is followed by the bell stage, and at this time the epithelially-derived ameloblasts and the mesenchymally-derived odontoblasts differentiate. The ameloblasts form enamel and the odontoblasts produce the dentine. MSCs also generate the alveolar bone that forms the sockets for the teeth. Human tooth development occurs in a very similar fashion (Zhang et al 2005).

In adult animals, dentinal repair results from odontoblasts that differentiate from a precursor cell population that resides in dental pulp tissue. These dental pulp stem cells (DPSCs) have been isolated from adult human teeth (Gronthos et al 2002). In culture, DPSCs show robust growth and a high proliferation rate and, even after extensive subculturing, have the ability to form a dentin/mineralized complex with a mineralized matrix when grafted into the dorsal surface of immunocompromised mice (Gronthos, et al 2002; Batouli et al 2003). In a rabbit model of tooth regeneration, DPSCs are able to support the formation of functional teeth (Hung et al 2011), and in mouse and dog models, DPSCs regenerated alveolar tooth socket bone in the jaw (Yamada et al 2010; 2011; Ito et al 2011).

Four other dental-associated, MSC-like stem cell populations have been isolated and characterized. The first of these, stem cells from human exfoliated deciduous teeth (SHED), like DPSCs, have many similarities to MSCs. However, SHEDs differ from DPSCs in that they have a higher proliferation rate and can differentiate into odontoblasts, which form a dentin-pulp-like structure without the mineralized matrix, but not ameloblasts (Miura et al 2003). Transplantation experiments have established that SHEDs can make vascularized bone and endothelial cells, and when implanted into the jaws of laboratory animals SHEDs can effectively regenerate jaw bone (Cordeiro et al 2008; Nakamura et al 2009; Yamada et al 2010; 2011; Ito et al 2011). The second cell population, periodontal ligament stem cells (PDLSCs), expresses a subset of neural crest cell and MSC markers (Seo et al 2004; Nagatomo et al 2006; Gay et al 2007; Fujita et al 2007; Coura et al 2008; Huang et al 2009), and shows some ability to repair periodontium (Seo 2004; Grimm et al 2011). The third population, stem cells from apical papillae (SCAP) readily makes dentin-pulp-like complexes and expresses several neuronal markers (Sonoyama et al 2006; 2008). The fourth stem population, dental follicle precursor cells (DFPCs), form fibrous and rigid tissue when transplanted into laboratory animals but not dentin, cementum or bone (Morsczecket al 2005; 2008).

In a head-to-head comparison of the ability of DPSCs and ADSCs to replace teeth in a rabbit model, the teeth produced by ADSCs were very similar to those generated by DPSCs. Both sets of replacement teeth were living teeth with nerves and vascular systems, but the ADSCs grew at faster rate and were more resistant to senescence (Hung et al 2011). BMSCs, like DPSCs, are also able to form calcified deposits in vitro (Gronthos et al 2000). Likewise, gene microarray analyses of these two stem cell populations show similar levels of expression for more than 4000 genes, with only a few differences (Shi, Robey and Gronthos 2001). Head-to-head comparisons of BMSCs, DPSCs, and SHEDs have shown that these stem cells have an equivalent the ability to regenerate alveolar tooth socket bone in the jaws of laboratory animals (Yamada et al 2010; 2011; Ito et al 2011). Comparison of BMSCs and SHED gene expression profiles by means of DNA microarray and real-time reverse transcriptase polymerase chain reaction has shown that 2753 genes in SHEDs show a more than two-fold difference in expression level in comparison to BMSCs. The genes that show the greatest differences in expression in SHEDs are those involved in BMP signaling, and the protein kinase A (PKA), c-Jun-N-terminal kinase (JNK), and apoptosis signaling-regulating kinase-1 (ASK-1) signaling cascades. Therefore SHEDs have specific characteristics that differ from BMSCs, and the osteogenic and odontogenic differentiation of SHEDs and BMSCs are probably regulated by different mechanisms (Hara et al 2009).

BMSCs can probably serve as a source for dental regenerative treatments, but the faster growth rates and easier isolation of ADSCs probably makes them a superior choice.

MSC Neural Differentiation
To date, neural differentiation of MSCs remains controversial, since many stem cell biologists think that the neuron-like cells formed by MSCs after neural induction do not represent true neurons. However, protocols have been published for converting MSCs into specific types of neurons. One method (Tropel et al 2006) cultures MSCs at low density (3,000 cells / cm2) on poly-lysine-coated plates for seven days in low-glucose DMEM, 10% fetal calf serum, glutamine (2mM), and bFGF (25ng/mL). A second protocol incubates MSCs with bFGF (5ng / mL) for 24 hours, followed by complete medium substitution with DMEM, N2 supplement, butylated-hydroxyanisole, KCl, valproic acid, and forskolin (Krampera et al 2007; Anghileri et al 2008). When subjected to either protocol, MSCs show dramatic morphological changes after 24-48 hours. They begin to sprout long branches and axon-like structures. Molecularly, neurally induced MSCs up-regulate synthesis of the neuron-specific intermediate filament nestin, which is typically only made by dividing neurons and disappears from terminally differentiated neurons (Michalczyk and Ziman 2005). Neurally induced MSCs also initiate expression of several neuronal and glial markers that include light neurofilament (NF-L), β-tubulin III (β3-tub), peripheral myelin protein-22 (PMP-22), glial fibrillary acidic protein (GFAP), and NeuN or neuronal nuclear antigen (Krampera et al 2007). They also express functional neuronal receptors and pharmacologically sensitive voltage-gated calcium channels (Wislet-Gendebien et al 2005; Tropel et al 2006). Unfortunately, MSC neuronal induction is reversible, and as soon as neural induction ceases MSCs revert back to their ground state. Interestingly, co-culturing neutrally induced MSCs with Schwann cells locks the neutrally induced MSCs in their neuronal state (Krampera et al 2007).

Despite reports that MSCs can be differentiated into functional neurons, several studies have failed to recapitulate these results (Scuteri et al 2010). Time-lapse photography of rat BMSCs that had undergone neural induction showed that instead of extending neurites, the cells merely shrunk and retracted their cell extensions so that only two extensions remained. This was interpreted to be a response to toxic or stressful conditions, and treatment of MSCs with chemicals and conditions known to stress cells (extremes of pH, high-molarity NaCl or detergents) produced similar “pseudoneuronal” morphology and increased MSC staining for neuronal markers. Strangely, pretreatment of MSCs with cycloheximide (an antibiotic that inhibits translation) failed to abrogate this response, suggesting that no new gene expression is required for cells to assume this pseudoneuronal morphology. These findings suggest that neural induction of MSCs in culture is largely an artifact (Lu, Blesch and Tuszynski 2004). Other studies have implanted MSCs into the brains of laboratory animals in the hope that a neural environment can induce neuronal differentiation in MSCs, but the implanted cells showed a spherical morphology with few extensions and connections with other cells (Zhao et al 2002).

Despite these negative results, genetic engineering of MSCs with the intracellular domain of Notch (Dezawa et al 2004; Xu et al 2010), neurogenin-1 (Kim et al 2008), neurotrophin-3 after retinoic acid pretreatment (Zhang et al 2006), siNRSF (Yang et al 2008) and brain-derived neurotrophic factor (Lim et al 2011), have all successfully transdifferentiated MSCs into functional neurons. Furthermore, MSC treatment with various combinations of growth factors (Long et al 2005; Bae et al 2011; Trzaska and Rameshwar 2011), signaling molecules (Kondo et al 2011) and small molecules (Wang et al 2011) have also transdifferentiated MSCs into neurons, and in some cases into dopaminergic neurons. Finally, sequential analysis of gene expression (SAGE) and microRNA expression profiles of MSCs before and after neural induction have shown high level expression of several neural specific genes that are not expressed in MSCs before neural induction. Also cell the expression of reprogramming factors like Oct4, Klf4, and c-Myc are modulated during differentiation (Crobu et al 2011).

With respect to MSC neuronal differentiation, BMSCs have definitely received the most attention. However, other types of MSCs have the capacity to form neuron-like cells (Chen, He and Zhang 2009; Chang et al 2010; Jiang et al 2010; Lim et al 2010). To date there have been few head-to-head comparisons of the efficiency of neural induction between distinct MSC populations, and this is probably a function of the variability of MSC neural induction. One study found that neural induction of UCSCs and BMSCs produces dopaminergic neurons with roughly equal efficiencies (Datta et al 2011).

Also, there are few comparisons with dentally-derived MSCs, but these cells descend from neural crest cells. Consequently, they demonstrate more neural properties than other types of MSCs (Karaoz et al 2011). Such MSCs begin with more neural characteristics, and, therefore, neural differentiation of dental-derived MSCs probably requires fewer molecular steps (Nourbakhsh et al 2011).

Are BMSCs significantly different or relatively similar to MSCs from other tissue sources? The extensive research on BMSCs has provided a wealth of data that we can use for comparison with other MSCs. Work on MCSs from other tissues strongly suggests that genuine similarities exist between BMSCs and other types of MSCs. All these MSCs, with a few exceptions, display roughly the same set of cell surface proteins (De Ugarte et al 2003a; Musina, Bekchanova and Sukhikh 2005). For the most part, clonal differences in specific MSC populations notwithstanding (Zuk et al 2002; Guilak et al 2006), can differentiate into osteocytes, chondrocytes, or adipocytes (Pittenger et al 1999; Pontos et al 2006), and BMSCs and ADSCs utilize common pathways to differentiate into these distinct cell types (Liu et al 2007). They also express a common core of genes and proteins that distinguish them from other cell types.

Despite these similarities, there are also some stark differences between various MSCs from assorted tissues. First of all, the efficiencies with which these different MSC populations differentiate into osteocytes, chondrocytes, and adipocytes widely differ. Secondly, even though BMSCs and ADSCs use a set of common genes for early differentiation into all three lineages, they recruit different sets of genes for later differentiation and maturation into fully differentiated cells (Liu et al 2007; Kim and Im 2010). Thirdly, varied MSC populations differ with regards to their stemness. UCSCs share more genes in common with embryonic stem cells than BMSCs, and are, therefore, more primitive. They also express more angiogenesis and growth related genes. On the other hand, the gene expression profiles of BMSCs are much more significantly altered under different culture conditions, and express more osteogenesis genes (Hsieh et al 2010). Fourth, even though MSC populations commonly express a core set of genes(Winter et al 2003; Lee et al 2004a; Djouad et al 2005; Wagner et al 2005; Aranda et al 2009; Jansen et al 2010), gene expression profiles of distinct MSC populations differs substantially. For example, UCSCs and umbilical cord blood-derived MSCs(UBSCs) show remarkable differences in gene expression. Gene expression profiles from UBSCs revealed that genes involved in anatomical structure and multicellular organism development, osteogenesis and the immune system were expressed at high levels. However in UCSCs, genes related to cell adhesion, neurogenesis, morphogenesis, secretion and angiogenesis were more highly expressed (Secco et al 2009). Fifth, even though distinct MSC populations express very similar sets of proteins (Roche et al 2009), there are significant differences (Maurer 2011). Finally, the differentiation requirements for each MSC population differ, and these differences are a result of the signature gene expression profiles of each MSC population.

Thus, MSCs represent a familial cell type, but each distinctive MSC population represents a particular subfamily of this cell type family. While some subfamilies are clearly more closely related to some than others, these MSC subfamilies constitute the constituents that compose the MSC cell type.

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Michael Buratovich received his Ph.D. in Cell and Developmental Biology from UC Irvine in the laboratory of Peter Bryant where he worked on tumor suppressor genes in Drosophila melanogaster. He worked as a postdoctoral research fellow at Sussex University with Robert Whittle on the role of Wnt proteins in patterning the peripheral nervous system, and at University of Pennsylvania with Betsy Wilder on Wnt signaling during development. Since 1999, he has been a member of the faculty of Spring Arbor University (Spring Arbor, MI) in the Biochemistry department. He also served as a visiting scientist at Boston University in the laboratory of Joseph Ozer where he worked on the role of basal transcription factors in stem cell differentiation, and has collaborated with Amr Amin at the University of Al-Ain on cancer research. He is zealous about communicating science to the public and passionately blogs at

Researchers from the University of Minnesota Use Genetically Corrected Stem Cells To Repair Muscles

University of Minnesota researchers from the Lillehei Heart Institute have combined genetic engineering techniques to repair mutations in abnormal muscle cells with cellular reprogramming to generate stem cells that can repair and regenerate muscle regeneration in a mouse model for Duchenne Muscular Dystrophy (DMD). This research is a proof-of-principle experiment that determines the feasibility of combining induced pluripotent stem cell technology and genetic engineering techniques that correct mutations to treat muscular dystrophy. Experimental strategies such as these could represent a major step forward in autologous cell-based therapies for DMD. Furthermore, it might pave the way for clinical trials to test this approach in reprogrammed human pluripotent cells from muscular dystrophy patients.

University of Minnesota researchers combined three groundbreaking technologies to achieve effective muscular dystrophy therapy in a mouse model of DMD. First, researchers reprogrammed skin cells into induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into any of the mature cell types within an adult organism. In this case, the University of Minnesota researchers generated pluripotent cells from the skin of mice that carry mutations in two genes; the dystrophin and utrophin genes. Mice with mutations in both the dystrophin and utrophin genes develop a severe case of muscular dystrophy that resembles the type of disease observed in human DMD patients. This provided a model system platform that successfully mimicked what would theoretically occur in humans.

The second technology employed is a genetic correction tool developed at the University of Minnesota. In this case, they used a transposon, which is a segment of DNA that can jump from one location to another within the genome. The specific transposon used is the “Sleeping Beauty Transposon.” The use of this transposon allowed them to transport genes into cells in a convenient manner. The Lillehei Heart Institute researchers used the Sleeping Beauty transposon to deliver a gene called “micro-utrophin” into the iPSCs made from the DMD mice.

Sleeping Beauty Transposon

Human micro-utrophin can support muscle fiber strength and prevent muscle fiber injury throughout the body. However, there is one essential difference micro-utrophin and dystrophin: dystrophin is absent in muscular dystrophy patients, but if it is introduced into the bodies of DMD patients, their immune system will initiate a devastating immune response against it. However, in those same patients, utrophin is active and functional, which makes it essentially “invisible” to the immune system. This invisibility allows the micro-utrophin to replace dystrophin build and repair muscle fibers within the body.


The third technology utilized is a method to produce skeletal muscle stem cells from pluripotent cells. This procedure was developed in the laboratory of Rita Perlingeiro, who is also the principal investigator of this latest study.

Rita Perlingeiro Ph.D.
Rita Perlingeiro Ph.D.

Perlingeiro’s technology gives pluripotent cells a short pulse of a muscle stem cell protein called Pax3, which nudges the pluripotent cells to become skeletal muscle stem cells, which can then be exponentially expanded in culture. These Pax3-induced muscle stem cells were then transplanted back into the same strain of DMD mice from which the pluripotent stem cells were originally derived.

Pax3 and 7

When combined, these platforms created muscle-generating stem cells that would not be rejected by the body’s immune system. According to Perlingeiro, the transplanted cells performed very well in the dystrophic mice, and they generated functional muscle and responded to muscle fiber injury.

“We were pleased to find the newly formed myofibers expressed the markers of the correction, including utrophin,” said Perlingeiro, a Lillehei endowed scholar within the Lillehei Heart Institute and an associate professor in the University of Minnesota Medical School. “However, a very important question following transplantation is if these corrected cells would self-renew, and produce new muscle stem cells in addition to the new muscle fibers.”

By injuring the transplanted muscle and watching it repair itself, the researchers demonstrated that the transplanted muscle stem cells endowed the recipient mice with fully functional muscle cells. This latest project provides the proof-of-principle for the feasibility of combining induced pluripotent stem cell technology and genetic correction to treat muscular dystrophy.

“Utilizing corrected induced pluripotent stem cells to target this specific genetic disease proved effective in restoring function,” said Antonio Filareto, Ph.D., a postdoctoral fellow in Perlingeiro’s laboratory and the lead author on the study. “These are very exciting times for research on muscular dystrophy therapies.”

These studies pave the way for testing this approach in a clinical trial that would use reprogrammed human pluripotent cells from muscular dystrophy patients.

According to Perlingeiro, “Developing methods to genetically repair muscular dystrophy in human cells, and demonstrating efficacy of muscle derived from these cells are critical near-term milestones, both for the field and for our laboratory. Testing in animal models is essential to developing effective technologies, but we remained focused on bringing these technologies into use in human cells and setting the stage for trials in human patients.”

This research was published in Nature Communications.

A Gene that Prevents Induced Pluripotent Stem Cell Formation Linked to Cancer Severity

A Mount Sinai research team has published some remarkable observations in the journal Nature Communications. Emily Bernstein, PhD, and her team at Mount Sinai have discovered a particular protein that prevents normal cells from being reprogrammed into induced pluripotent stem cells (iPSCs). Since iPSCs resemble embryonic stem cells, these data might provide significant insights into how cells lose their plasticity during normal development, which has wide-reaching implications for how cells change during both normal and disease development.

Previously, Bernstein and others showed that during the formation of particular tumors known as melanomas in mice and human patients, the loss of a specific histone variant called macroH2A (a protein that helps package DNA) correlated rather strongly to the growth and metastasis of the tumor. In this current study, Bernstein and her team determined if macroH2A acted as a barrier to cellular reprogramming during the derivation of iPSCs (see Costanzi C, Pehrson JR (1998). “Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals”. Nature 393 (6685): 599–601).

In collaboration with researchers at the University of Pennsylvania, Bernstein evaluated mice that had been genetically engineered to lack macroH2A. When skin cells were used from macroH2A(-) mice were used to make iPSCs and compared with skin cells from macroH2A(+) mice, the cells from macroH2A(-) mice that lacked macroH2A were much more plastic and were much more easily reprogrammed into iPSCs compared to the wild-type or macroH2A(+) mice. This indicates that macroH2A may block cellular reprogramming by silencing genes required for plasticity.

Bernstein, who is an Assistant Professor of Oncological Sciences and Dermatology at the Graduate School of Biomedical Sciences at Mount Sinai, and corresponding author of the study, said: “This is the first evidence of the involvement of a histone variant protein as an epigenetic barrier to induced pluripotency (iPS) reprogramming.” She continued: “These findings help us to understand the progression of different cancers and how macroH2A might be acting as a barrier to tumor development.”

In their next group of experiments, Bernstein and her team plan to create cancer cells in a culture dish by inducing mutations in genes that are commonly abnormal in particular types of cancer cells and then couple those mutations to the removal of macroH2A to examine whether the cells are capable of forming tumors.

Induced Pluripotent Stem Cells Make Stem Cells to Treat MS

Many nerves inside and outside the central nervous system are insulated by a sheath rich in a protein called “myelin.” This myelin-enriched sheath greatly increases the speed at which nerve impulses travel through these nerves. You have probably experienced such fast nerve impulse conduction. Remember the last time you had your hands in water that was overly hot. First there was a very sharp pain that caused you to withdraw your hand as fast as you could, but it was followed by a dull ache that became more and more painful until it abated. This is an example of the fast-moving pain impulses that help protect our limbs from further damage and the slower moving pain impulses that convey the dull ache associated with soft tissue damage.

In some cases the myelin sheath is damaged, or, in some cases, people are born with damaged myelin sheaths. Either way, such a condition is catastrophic, and multiple sclerosis is an example of a disease that results from progressive damage to and loss of the myelin sheath. Spinal cord injuries also strip the myelin sheath from many neurons, thus decreasing the effectiveness with which nerve impulses are conducted. The loss of the myelin sheath can also, in some cases, causes the death of the nerve.

Can the myelin sheath be replaced? Almost certainly. Cells make the myelin sheath and this is a cue for regenerative medicine. Many different types of stem cells can differentiate into myelin sheath-making cells. Embryonic stem cells, for example, can be differentiated into myelin sheath-making cells. This was the basis for Geron Corporation’s clinical trial with embryonic stem cell (ESC)-derived cells that could make myelin sheaths. Myelin sheath-making cells in the central nervous system are known as “oligodendrocytes,” and “oligodendrocyte progenitor cells,” which are mercifully abbreviated OPCs, give rise to oligodendrocytes. Differentiation of ESCs into OPCs led to the Geron clinical trial. However, Geron prematurely terminated this trial, and it is unclear if these embryonic stem cell-derived OPCs can restore sensation and nerve function to spinal cord injury patients.


Other cells, however, can form OPCs, and one of these is induced pluripotent stem cells (iPSCs). Since these cells are derived from the patient’s own cells, they should be recognized by the immune system as part of the patient’s own tissue and not a foreign group of cells.

Su Wang and colleagues from Steven Goldman’s lab at the Center for Translational Neuromedicine at the University of Rochester in Rochester, NY, have made patient-specific iPSCs from which they made patient-specific OPCs. Wang and his colleagues devised a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into OPCs.

In this publication, Wang and others made three hiPSC lines, from which they made human OPCs. They used a very convenient methods to isolate the OPCs – fluorescence-activated cell sorting. hiPSC OPCs differentiated very efficiently into oligodendrocytes and other cell types found in the nervous system.

Next, Wang and others used their iPSC-derived OPCs to recoat nerves of mutant mice that lack myelin sheaths. Mice that have the “shiverer” mutation lack meylin sheaths, and they shake and shiver as a result of it. When implanted with Wang and companies’ iPSC-derived OPCs, these cells recoated with nerves very efficiently. When they compared the efficiency of the iPSC-derived OPCs with that of fetal OPCs, the iPSC-derived OPCs were clearly superior. The recoating of the nerves definitely increased the survival of the siverer mice. No tumors were observed in any of the mice implanted with iPSC-derived OPCs. implanted mice.

Goldman said of this study, “This study strongly supports the utility of hiPSCs as a feasible and effective source of cells to treat myelin disorders.” Goldman continued: “The new population of OPCs and oiligodendrocytes was dense, abundant, and complete. In fact, the re-myelination process appeared more rapid and efficient than with other cell sources.” This is significant because Goldman’s team also made OPCs from ESCs and their iPSC-derived OPCs outperformed the ESC-derived OPCs as well.

Goldman is part of a collaborative research consortium with scientists from Rochester, Syracuse, and Buffalo that wants to conduct a clinical trial that uses OPCs to treat patients with multiple sclerosis. This research group is called the Upstate MS Consortium and the early stages of this study are scheduled to begin in 2015, and it will focus on cells derived from various tissue sources. Goldman anticipates that his HiPSCs-derived OPCs will be included in this project.