Synthetic version of Oct4 robustly supports induced pluripotent stem cell formation


A recent paper by members of the Danish Stem Cell Centre in Copenhagen, the MRC Centre for Regenerative Medicine in Edinburgh, and Memorial Sloan Kettering Cancer Center in New York has used a synthetic version of the Oct4 protein to dissect the precise role of this protein in stem cells, and to more effectively generate induced pluripotent stem cells.
Oct4 is a member of the “class V POU” transcription factors. POU stands for Pit-Oct-Unc, which are the founding members of this group of transcription factors. Transcription factors are proteins that bind to specific sequences of DNA and turn on gene expression. The POU family of transcription factors was originally defined on the basis of a common region of ~150–160 amino acids that was identified in the transcription factors Pit-1, Oct-1, and Oct-2, which were known from mammals, and the nematode factor Unc-86. This common POU protein domain is the DNA binding region that consists of two subdomains joined by a common linker.

Embryonic development in mammals is controlled by regulatory genes, many of which regulate the expression of other genes. These regulators activate or repress patterns of gene expression that mediate the changes characteristic of development. Oct4, like other members of the POU family of transcription factors, activates the expression of their target genes by binding an eight-base sequence motif that usually has some similarity to this sequence: AGTCAAAT. During embryonic development, Oct4 is expressed initially in all the cells of the embryo, but eventually becomes restricted to the Inner Cell Mass (ICM) and Oct4 expression fades in the outer cells (known collectively as the trophectoderm). At maturity, Oct4 expression is confined exclusively to the developing germ cells. Disruption of Oct4 in mice produces embryos without a pluripotent ICM. This suggests that Oct4 is required for maintaining pluripotency.

Given the importance of Oct4 in early development, it is no surprise that it plays an important role in embryonic stem cell maintenance. Oct4 also plays an essential role reprogramming adult cells from their mature state to the embryonic state. In the absence of Oct4, embryonic stem cells differentiate. Oct4 plays a powerful role in regulating stem cell genes. However, while large quantities of Oct4 are needed, too much of it can hamstring the properties of stem cells.

Given these data, does Oct4 maintain pluripotency by activating the expression of particular genes or by repressing those genes necessary for differentiation? These scientists, whose work is published in the journal Cell Reports, made fusions of Oct4 with proteins that are known to activate gene expression or fusions with proteins known to repress gene expression. Then they accessed the ability of these fused versions of Oct4 to support pluripotency in embryonic stem cells or induce pluripotency in adult cells.

The synthetic version of Oct4 fused to known activator of gene expression were much more efficient in turning on genes that instruct cells how to be stem cells.  Cells also did not require as much of this synthetic Oct4; stem cells required less of the synthetic Oct4 to remain stem cells and adult cells required less to become reprogrammed as stem cells.  Those synthetic versions of Oct4 that were fused to known transcriptional repressors caused cells to differentiate, and such synthetic versions of Oct4 could not replace endogenous Oct4 in stem cells.

Further tests with the activating synthetic Oct4 showed that it could support stem cells under conditions that are usually not conducive to their growth.  This provides a way to generate stem cells in the laboratory when growth conditions are less than optimal.  Because the activator version of synthetic Oct4 could replace endogenous Oct4 and not the repressor version of synthetic Oct4, Oct4 must work primarily as an activator of gene expression rather than a repressor of gene expression.

Professor Joshua Brickman, who is affiliated with The Danish Stem Cell Center (DanStem), University of Copenhagen and Medical Research Council Centre for Regenerative Medicine at the University of Edinburgh. said “Our discovery is an important step towards generating and maintaining stem cells much more effectively.  Embryonic stem cells are characterized, among other things, by their ability to perpetuate themselves indefinitely and differentiate into all the cell types in the body – a trait called pluripotency. But to be able to use them medically, we need to be able to maintain them in a pure state, until they’re needed. When we want to turn a stem cell into a specific cell, such as insulin producing beta cell, or a nerve cell in the brain, we’d like this process to occur accurately and efficiently. This will not be possible if we don’t understand how to maintain stem cells as stem cells. As well as maintaining embryonic stem cells in their pure state more effectively, the artificially created Oct4 was also more effective at reprogramming adult cells into so-called induced Pluripotent Stem cells (iPSCs), which have many of the same traits and characteristics as embryonic stem cells but can derived from the patients to both help study degenerative disease and eventually treat them..”

Exploitation of such technology could improve the efficiency of protocols to generate iPSCs in the laboratory and the clinic.  Such cells could be used to produce individualized cells for developing individualized therapies for degenerative diseases such as type 1 diabetes and neuro-degenerative diseases.

Wesley Smith and the New Stem Cells in Ovaries: The Means for Mass Human Cloning


Wesley Smith, who runs a bioethics blog called “Secondhand Smoke” has blogged about the discovery of stem cells in the human ovary that can make cells that look like human eggs. He fears, and I think rightly so, that this could lead to the “Brave New World project of human self design, genetic engineering, transhumanistic tinkering, human enhancement, and using reproductive technologies to shatter the remaining vestiges of norms surrounding families.”

Human cloning can lead to nothing good. Right now it has stalled because of an acute shortage of human eggs (oocytes). The risk of the egg procurement procedure, and the work of people like my friend Jennifer Lahl and her film Eggsploitation, has dried up the supply of eggs. Thus cloning work has greatly slowed. TIlley’s work, however, could change that. Oh, sure, it will be sold as providing eggs for the infertile, which is a laudatory thing by the way. However, the use of huge numbers of eggs for human cloning purposes is a bridge too far.  A cloned  embryo is still a human embryo and is therefore still a human person.  Cloning for manipulation and enhancement research is a grotesque abuse of human rights.

You can read Smith’s article here.

Human Ovaries Harbor Egg-Making Stem Cell Population


We have read it before, countless times, that women are born with a particular number of eggs and after they die during ovulation or are ovulated, the women is out of eggs and goes through menopause. She does not have the ability to make any more eggs.

Well, another dogma falls by the wayside. As it turns out, egg-making stem cells exist in the woman’s ovaries. An article published in Nature Medicine by Jonathan Tilly and his colleagues who work in a laboratory at Massachusetts General Hospital in Boston, confirms earlier work by Tilly in 2004 that found ovarian stem cells exist in mouse ovaries (Joshua Johnson, et al., Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145-150 (11 March 2004) | doi:10.1038/nature02316), and 2009 publication by a laboratory in Shanghai, China, that confirmed Tilly’s controversial publication (Kang Zou, et al., Production of offspring from a germline stem cell line derived from neonatal ovaries. Nature Cell Biology 11 (2009): 631 – 636).

Despite the rigor of earlier experiments by Tilly’s group and Ji Wu’s, many ovarian experts remained quite skeptical that such a stem cell population existed in humans. This present publications, however, seems to seal the deal. According the Tilly, “This is unequivocal proof that not only was the mouse biology correct, but what we proposed eight years ago was also correct — that there was a human population of stem cells in young adult tissue.” says Tilly.

By listening to their critics, Tilly and is group developed techniques to address the concerns to those skeptical of their findings. They a protocol by which they could isolate and identify mouse ovarian stem cells. This new methods use “fluorescence-activated cell sorting” or FACS. FACS requires the attachment of a fluorescent tag onto the surface of those cells you wish to isolate. Tilly and his group used antibodies linked to a fluorescent dye that could bind tightly to a surface protein called “Ddx4.” Ddx4 is found on the surfaces of ovarian stem cells, but is quickly lost once the stem cells differentiate into egg cells. Treating ovarian cells with the fluorescently-labeled antibodies essentially “painted” them a glowing color. Then the cells were given to a cell sorter than placed cells into one container or another, based on whether or not their surfaces glowed. The cell sorter also distinguishes between whole cells and dead or damaged ones that might fluoresce by accident. In this way, Tilly’s lab invested a protocol for isolating and identifying ovarian stem cells that was highly selective and sensitive.

This new protocol confirmed that Ddx-4-expressing stem cells were present in mouse ovaries. The group did not stop there. They asked if human ovaries had the same stem cell population. They turned to Yasushi Takai, who is a former research fellow in Tilly’s lab, but now works as a reproductive biologist at Saitama Medical University in Japan. Takai provided Tilly with frozen, whole ovaries that had been removed from young women during sexual-reassignment procedures. Tilly says, “It was 9 November when we did the first human FACS sort and I knew immediately that it had worked. I cannot even put into words the excitement — and, to some degree, the relief — I felt.”

From the FACS experiment, Tilly’s group isolated human oogonial stem cells (OSCs). When cultured in the laboratory these OSCs spontaneously produced normal immature oocytes. How did the OSCs do this? To get an inside view of OSC differentiation, Tilly’s team labeled OSCs with green fluorescent protein in order to trace them. Then they injected the green-labeled OSCs into bits of cultured human ovarian tissue, and then transplanted the whole thing under the skin of mice. One to two weeks after transplantation, they found that the OSCs had formed green-glowing cells that greatly resembled ovaries and expressed two ovary-specific genes.

Tilly sounded a cautious note: “There’s no confirmation that we have baby-making eggs yet, but every other indication is that these cells are the real deal — bona fide oocyte precursor cells.” To do this, Tilly must show that the OSC-derived oocytes can be fertilized and form an early embryo. Such work must be done with private funding, since federal funding cannot legally be used for any research that will result in the destruction of a human embryo, regardless of the source of the embryo. Another strategy might be to procure a license from the UK Human Fertilisation and Embryology Authority to do the work with collaborators in the United Kingdom.

Tilly’s experiments have actually converted one scientist who was rather skeptical of his results. Evelyn Telfer, a reproductive biologist at the University of Edinburgh, UK, did not believe Tilly’s initial results. Now, however, she has become a believer. Telfer testified, “I’ve visited [Tilly’s] lab, seen these cells and how they behave. They’re convincing and impressive.” Telfer, has studied in vitro maturation of human eggs, and she wants to work with Tilly to try to grow the OSC-derived eggs to the point at which they are ready for fertilization.

Telfer noted that even though OSCs can form egg-like cells in culture, there is presently no evidence that they can do so in the ovary or that they actually do form new eggs in the ovary. However, the ability to convert OSCs into eggs in vitro might make them usable for in vitro fertilization (IVF), and this achievement would change assisted reproduction forever.

However, Tilly admonishes, “That’s a huge ‘if’.” However, it could means that women who under cancer treatments and experience early menopause could have OSCs removed before treatment and for later fertility use. In fact, according to Tilly, follow-up experiments have shown that OSCs actually exist in the ovaries of women well into their 40s. Even giving women another five years would cover most women affected by IVF.

Brain stem cells in the dentate gyrus make new memories and help keep old ones


When you sit down to study something new and try to commit it to memory, you find yourself retaining some things, but forgetting others. However, learning new material does not tend to prevent you from recalling older material. How do neurons, the cells that are responsible for neural impulse in the nervous system, do this? How do they form new memories without compromising old ones?

To answer this question, neuroscientists at the RIKEN-MIT Center for Neural Circuit Genetics examined neural stem cells to dissection the function of a specific portion of the brain known to be involved in forming new memories. Their results are remarkable, and will be published in the March 30th issue of the journal, Cell. This study connects the cellular basis of memory formation with the birth of new neurons. This discovery could offer new strategies for drug makers to make new classed of drugs to treat memory disorders.

Specific neurons in region of the brain called the “dentate gyrus” play a peculiar role in memory formation. The dentate gyrus is part of a larger structure called the “hippocampus.” The hippocampus is also part of a complex circuit called the limbic system. The limbic system supports a wide variety of functions that include emotion, long-term memory, behavior, and the sense of smell. There are several brain structures in the limbic system, and they have all had function mapped to them. These structures are shown in the figure below and their functions are as follows:

1. Hippocampus – This is required for long-term memories and maintains cognitive maps for navigation.
2. Fornix – transmits neural signals from the hippocampus to the mammillary bodies and septal nuclei.
3. Mammillary body – these structures are essential for the formation of memories.
4. Septal nuclei provide essential interconnections between various parts of the limbic system.
5. Amygdala – signals the cerebral cortex when complex stimuli are received, such as fear, rewards, or sexual mating behaviors.
6. Parahippocampal gyrus – this plays an important role in spatial memory.
7. Cingulate gyrus – regulates bodily functions like the heart rate, blood pressure and the ability of pay attention to particular things.
8. Dentate gyrus – contributes to new memories.

The hippocampus, and in particular the dentate gyrus is the site of stem cell populations in the brain (see Ming GL and Song H, Neuron 2011 70(4):687-702). This stem cell population has been thought to contribute to the production of new memories. However, one remarkable new find is that decreased neurogenesis in the dentate gyrus leads to depression. To read more about this, see this site here.

In the present study from the RIKEN-MIT group, the ability of the dentate gyrus to form new memories depends on whether or not the neural stem cells in the dentate gyrus are old or young. These findings also suggest that imbalances between young and old neurons in the dentate gyrus and possibly other regions of the brain as well could potentially disrupt the formation of memories during post-traumatic stress disorder (PTSD) and aging. This could also explain the link between depression and poor memory formation in patients with depression.

Susumu Tonegawa, 1987 Nobel Laureate and Director of the RIKEN-MIT Center, and lead author of this study said, “In animals, traumatic experiences and aging often lead to decline of the birth of new neurons in the dentate gyrus. In humans, recent studies found dentate gyrus dysfunction and related memory impairments during normal aging.”

In this study, researchers tested two types of memory processed in mice. The first is called “pattern separation,” which the means by which the brain distinguishes differences between different events that are similar, but different. For example, remembering two pepperoni pizzas that have different tastes would be one such example, since the two pizzas might look similar, but they have very different gustatory outcomes. The second, “pattern completion” remembers detailed content with few clues. For example, when you memorize lines from a play, you can start anywhere in the play if someone only gives you the first few words on one sentence. Alternatively, if we stick with our pizza example, remembering who was with you and what they were wearing when you had that great pizza would be an example of using pattern completion.

The formation of new memories on the basis of pattern separation utilizes differences between experiences. Pattern completion, on the other hand, recalls memories by detecting similarities. In patients with brain injury or specific types of trauma, cannot remember people they encounter every day. Others with PTSD cannot forget horrific events. According the Dr. Tonegawa, “Impaired pattern separation due to the loss of young neurons may shift the balance in favor of pattern completion, which may underlie recurrent traumatic memory recall observed in PTSD patients.

It has been largely accepted that pattern completion and pattern separation are the work for separate neural circuits in the brain. Pattern separation has been mapped to the dentate gyrus. The dentate gyrus also is involved in depression, epilepsy and also traumatic brain injury. A second region in the hippocampus called the “CA3 region” was suspected to be involved in pattern completion. However, as is often the case in science, a long-held idea has to be abandoned in the face of new data; the MIT group found that dentate gyrus neurons may perform pattern separation or completion depending on the age of their cells.In the picture below, you can see that the CA3 region is part of the hippocampus (see circled bit in the cross-section).  CA3 stands for “cornu ammonis later 3.”

Pattern separation was assessed in mice that had learned to different, but similar chambers. One of these chambers was safe, but the other was dangerous, since upon entering it the mice received an electric shock in their feet. Since these mice had discriminated between to similar but different chambers, the group tested their pattern completion capabilities. To do this, they gave the mice limited cues to scurry through a maze that they had previously learned. Normal mice were compared with mice that had deficits or either old or new neurons in their dentate gyruses. Interestingly, mice exhibited defects in either pattern completion or separation depending on whether the old or the new neurons were missing.

Study co-author Toshiaki Nakashiba said, “”By studying mice genetically modified to block neuronal communication from old neurons — or by wiping out their adult-born young neurons — we found that old neurons were dispensable for pattern separation, whereas young neurons were required for it. Our data also demonstrated that mice devoid of old neurons were defective in pattern completion, suggesting that the balance between pattern separation and completion may be altered as a result of loss of old neurons.”

This remarkable study will almost certainly be the beginning of an entirely new way of looking at depression. Collaborators in this work included researchers from the laboratories of Michael S. Fanselow at the University of California at Los Angeles; and Chris J. McBain at the National Institute of Child Health and Human Development.

Highly Regarded Cochrane Library Study Shows that Bone Marrow Treatments Help Heart Attack Patients


A whole gaggle of stem cells treatments for heart attack patients have been completed. Some patients are definitely helped, but others are not. Some clinical trials have shown a definitively positive effect from stem cell infusions in combination with standard care. Other trials, however, have failed to show any positive benefits to combining stem cell infusions with standard care. What do these clinical trials as a whole tell us?

This question is the realm of “meta-analyses.” While several clinical trials that have given stem cell treatments on heart attack patients have been subjected to meta-analyses, more stem cell trials have been completed, and further analyses are necessary. Meta-analyses take data from separately published studies that were conducted at different times and places and combine these data into a giant database that is subjected to rigorous statistical analysis. One organization that excels at meta-analyses, and has a solid reputation in the field is the Cochrane Library. The Cochrane Library has just completed a systematic meta-analysis of the data generated in 33 different clinical trials that used adult stem cells to treat the hearts of heart attack patients. The Cochrane Library’s analysis revealed that heart function definitely improves after stem cell treatments. However, these same analyses showed that the data are limited by the predominance of small trials and larger clinical trials are necessary to more rigorously demonstrate if the benefit of stem cell treatments in the heart actually means that the treated patients will benefit from a longer and healthier life.

Heart attacks are caused by blocked coronary arteries that prevent life-giving oxygen from flowing to heart muscle. This lack of oxygen causes the demanding heart muscle cells to die, and this cell death damages the heart and leads to the production of a scar that does not contract or conduct electrical impulses. Clinical trials have used adult stem cells from the patient’s own bone marrow to repair and reduce this damage. Although, unfortunately, this treatment regime is only available in facilities that have close links to medical research facilities.

The Cochrane Library authors (David M Clifford and colleagues), cobbled together data from as many clinical trials that used bone marrow stem cells to treat heart attack patients as they could find. In 2008, Cochrane reviewed 13 clinical trials to address this very question. However, since that time, 20 more clinical trials have been completed, and this year, 33 clinical trials that treated 1,765 patients were analyzed. Since the earlier trials continued patient follow-up, there are new data points from many older clinical trials that were also included. These data provide a more precise indication of the effects of stem cell therapy several years after completion of stem cell treatment.

In the analyzed trials, all 1,765 patients had already undergone angioplasty, which is a conventional treatment for heart attack patients. Angioplasty uses an inflatable balloon that is fed into the coronary artery by means of a fine catheter. This catheter is inserted into a large vein and guided by imaging methods to the blocked coronary arteries. Once in place, the balloon is slowly inflated to push the obstructing material to the sides of the artery. This opens up the blocked artery and allows the flow of blood to the heart muscle. To keep the blood vessel open, sometimes a stent is inserted into the blocked vessel. If angioplasty is combined with bone marrow stem cell treatments, the Cochrane reviews finds that such treatments can produce moderate long-term improvement in heart function that is sustained for up to five years. Unfortunately, there was not enough data to reach firm conclusions about increases in survival rates.

Senior author of this review, Enca Martin-Rendon, from the Stem Cell Research laboratory at the John Radcliffe Hospital in Oxford, UK, said, “This new treatment may lead to moderate improvement in heart function over standard treatments. Stem cell therapy may also reduce the number of patients who later die or suffer from heart failure, but currently there is a lack of statistically significant evidence based on the small number of patients treated so far.”

Will such treatments become part of the treatment for a heart attack? At this point it is difficult to say with any certainty. It is simply too early to establish guidelines for standard practice, since several labs have used differing transplantation and cell isolation and storage methods. According to the Cochrane Review, further work is required to properly standardize the procedure. For instance, there is little agreement on the dosage of cells for the heart, even though several studies have shown a dose-specific effect. Secondly, a standardized protocol for when after the heart attack treatment should be given, and what methods most accurately measure heart function must be constructed before such a procedure is universally offered to patients. Martin-Rendon noted, “The studies were hard to compare because they used so many different methods. Larger trials with standardized treatment procedures would help us to know whether this treatment is really effective.

A larger trial is already in the works, since the task force of the European Society of Cardiology for Stem Cells and Cardiac Repair received a recent, sizable grant from the European Union Seventh Framework Programme for Research and Innovation (EU FP7-BAMI) to initiate such a large trials. The Principal Investigator for this trial (called BAMI) who is also a co-author of this review, Anthony Mathur, said, ”The BAMI trial will be the largest stem cell therapy trial in patients who have suffered heart attacks and will test whether this treatment prolongs the life of these patients.”

Mouse Heart-Specific Stem Cells Potentially Offer Hope For Heart Attack Patients


Biomedical researchers from the University of California, San Francisco (UCSF) have published a stem cell experiment in mice that might provide another way to fix damaged heart muscle in heart attack patients. If these results pan out, they could potentially could increase heart function, minimize scar size, promote the growth of new blood vessels around the heart, and doo all this while avoiding the risk to tissue rejection.

Sounds too good to be true? It was published in PLoS ONE. You can read about it here.

To summarize the experiments, researchers isolated a new heart-specific stem cell from the heart tissue of middle-aged mice. When cultured in a laboratory dish, the cells had the ability to differentiate into heart muscle cells that beat in the culture dish. However, these same cells could also become blood vessels, or smooth muscle, which surrounds blood vessels and regulates the diameter of the vessels. All of these tissues are essential for the heart to work and properly function.

After showing that these cells could be grown in the laboratory in converted into heart-specific cell types, this research group examined the ability of these cells to do the same thing inside a living organism. After expanding the cells in culture, they transplanted them into the hearts of sibling mice that had the same genetic lineage as the mice from which the heart stem cells had been isolated in the first place. This prevented the possibility of the immune system of the recipient mice from attacking and rejecting the implanted cells. The implanted stem cells made blood vessels and also formed smooth muscle. The increased blood flow improved heart function.

Even more exciting, these heart specific stem cells are found in all four chambers of the heart. The “cardiosphere-derived cells” (CDCs) that have been used in other clinical trials are only found in the upper chambers of the heart (atria), and express slightly different cell surface proteins. When grown in culture, these cells grow into spheres of cells that are known as “cardiospheres.”  These new heart-specific stem cells are more widely located in the heart, which means that it is possible to isolate them from patients’ hearts by doing ventricular or atrial biopsies. Biopsies of the right ventricle are among the safest procedures for procuring heart cells from live patients. This procedure is relatively easy to perform and does not adversely affect the patient.

The paper’s first author, Jianqin Ye, PhD, MD, senior scientist at UCSF’s Translational Cardiac Stem Cell Program, said, “These findings are very exciting . . . we showed that we can isolate these cells from the heart of middle-aged animals, even after a heart attack. . . we determined that we can return these cells to the animals to induce repair.”

Senior author Yerem Yeghiazarians, MD, director of UCSF’s Translational Cardiac Stem Cell Program and an associate professor at the UCSF Division of Cardiology, agreed with Ye’s assessment: “The finding extends the current knowledge in the field of native cardiac progenitor cell therapy. Most of the previous research has focused on a different subset of cardiac progenitor cells. These novel cardiac precursor cells appear to have great therapeutic potential.”

Yeghiazarians hopes that those patients who have suffered severe heart failure after a heart attack or have enlargement of the heart (cardiomyopathy) could still be treated with their own heart-specific stem cells to improve their overall health and heart function. Because these cells would come from the patients, there would be no concern of cell rejection after therapy.

These heart-specific stem cells are also known as Sca-1+ stem cells. Sca-1 is a small cell surface protein that is involved in cell signaling. These heart-specific Sca-1+ cells also express a transcription factor called Islet (Isl-1). These cells are known to play an important role in heart development. Most of the previous research on heart stem cells has examined different subset of cells known as “c-kit” cells. Sca-1+ cells, like the c-kit cells, are located within a larger clump of cells called cardiospheres.

To isolate the Sca-1+ cells, Yeghiazarians’ group devised ways to separate the Sca-1-expressing cells that were also expressing high levels of Isl-1. Sca-1 is rather easy to use for isolation, since it is a cell surface protein, but Isa-1 is a nuclear protein and is less useful for isolation purposes.

Yeghiazarians proposed that the co-expression of these two molecules that are also made during heart development suggests a strategy for heart therapy: “Heart disease, including heart attack and heart failure, is the number one killer in advanced countries. It would be a huge advance if we could decrease repeat hospitalizations, improve the quality of life and increase survival.” By giving the heart cells that are extremely similar to those cells that help construct it during development; those same cells could reconstruct the heart when it starts to fail.

More studies are on the board for the future, and these animal studies might lead to future clinical trials.

New Clinicals Trials Use Umbilical Cord Blood Stem Cells to Treat Neurological Conditions and Hearing Loss


Umbilical cord and umbilical cord blood contains a wealth of stem cells for multiple uses. Cord blood contains a blood cell-making stem cell that can be used to constitute bone marrow (see Gluckman E., Blood Rev. 2011;25(6):255-9). It also contains two mesenchymal stem cell populations: Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSCs) and Human Umbilical Cord Perivascular Cells (HUCPVCs). Both of these cell populations have remarkable potential or regenerative medicine (Carvalho MM., et al. Curr Stem Cell Res Ther. 2011;6(3):221-8). Other umbilical cord stem cells include unrestricted somatic stem cells (USSCs; Arien-Zakay H, Lazarovici P, Nagler A., Best Pract Res Clin Haematol. 2010;23(2):291-303), embryonic-like stem cells, blood vessel-based endothelial stem cells, and a stem cell that comes from those cells that cover tissues (epithelial stem cells; see Harris DT., Stem Cell Rev. 2008;4(4):269-74, & Harris DT., Br J Haematol. 2009;147(2):177-84).

The usefulness of cord blood has been recognized by the medical community for some time, and there are now umbilical cord blood registries that bank cord blood for medical use and for research. One of these registries, the Cord Blood Registry (CBR) works with various research laboratories to help discover ways to use a child’s own cord blood stem cells to treat conditions like pediatric brain injury or even acquired hearing loss. Because different laboratories use different protocols or equipment to process umbilical cord blood, the experimental results derived from experiments or clinical trials that use cord blood might vary widely. Therefore, to ensure consistency in the storage and processing of cord blood stems, three separate clinical trials have used cord blood that provided by the CBR in their FDA-authorized protocols. These research institutions include the University of Texas Health Science Center at Houston (UTHealth) in partnership with Children’s Memorial Hermann Hospital, and Georgia Health Sciences University, which is the home of the Medical College of Georgia (MCG). CBR is the only family stem cell bank that pairs researchers conducting clinical trials with prospective patients for their studies.

Heather Brown, MS, CGC, Vice President of Scientific & Medical Affairs at Cord Blood Registry, put it this way: “Partnering with a series of specialists who want to research the use of a child’s own newborn blood stem cells on a variety of disease states allows CBR to help advance medical research for regenerative therapies by connecting the child whose family banked with CBR to appropriate researchers. The pediatric specialists from UTHealth, Children’s Memorial Hermann Hospital, and Georgia Health Sciences University are at the forefront of stem cell research as they evaluate cord blood stem cells’ ability to help facilitate the healing process after damage to nerves and tissue.”

One of the clinical trials examined the ability of cord blood stem cells to treat hearing loss. Hearing loss can result from problems with the middle ear, which conducts sound to the cochlea (conductive hearing loss) or from problems with the inner ear, in which the cochlea itself is damaged or defective (sensorineural hearing). Sensorineural hearing loss affects approximately 6 per 1,000 children by 18 years of age, with 9% of the cases resulting from various external causes (e.g., viral infection and head injury). Samer Fakhri, M.D., surgeon at Memorial Hermann-Texas Medical Center and associate professor and program director in the Department of Otorhinolaryngology – Head & Neck Surgery at UTHealth, heads the research team investigating the use of cord blood to treat sensorineural hearing loss. His collaborator is James Baumgartner, M.D.

The Fakhri-Baumgartner study is a Phase I safety study that uses cord blood-based stem cells to treat children who suffer from acquired hearing loss. The inspiration for this trial comes from animal studies that used cord blood to repair damaged organs in the inner ear. The paper (Revoltella RP., et al., Cell Transplant. 2008;17(6):665-78), used mice that had been made deaf from treatment with aminoglycoside antibiotics, which cause irreversible deafness at particular dosages, and intensely loud noises, which also cause deafness. Intravenous administration of hematopoietic stem cells from umbilical cord blood stimulated some structural recovery in the inner ear that was due to umbilical cord stem cells that had survived and become part of the inner ear tissues.

Parents of children 6 weeks to 2 years old that had experiences hearing soon after birth are eligible for this year-long study. Baumgartner explains, “The window of opportunity to foster normal language development is limited. This is the first study of its kind with the potential to actually restore hearing in children and allow for more normal speech and language development.”

Another clinical trial is examining the ability of cord blood to treat brain trauma. Children who experience brain injury heal better than adults who experienced the same injury. Having said that brain trauma is one of the leading causes of childhood death. Charles S. Cox, M.D., distinguished professor of pediatric surgery and pediatrics at UTHealth, initiated a clinical study that will enroll 10 children ages 18 months to 17 years old, all of whom have umbilical cord blood banked with CBR, and have suffered some type of traumatic brain injury. These children will enroll in the study within 6-18 months of suffering brain injury. This trial grows from a growing corpus of studies that have demonstrated the efficacy of umbilical blood stem cells to treat neurological conditions. Read more about the trial here.

According the Charles Cox, “The reason we have become interested in cord blood cells is because of the possibility of autologous therapy, meaning using your own cells. And the preclinical models have demonstrated some really fascinating neurological preservation effects to really support these Phase 1 trials. There’s anecdotal experience in other types of neurological injuries that reassures us in terms of the safety of the approach and there are some anecdotal hints at it being beneficial in certain types of brain injury.”

James Carroll professor and chief of pediatric neurology at the GHSU in Augusta, Georgia, launched the first FDA-regulated clinical trial to test the ability of cord blood stem cell infusions to improve the condition of children with cerebral palsy. This clinical trial will include 40 children whose parents have banked their umbilical cord blood at CBR and meet all the criteria for inclusion in the trial.

Dr. Carroll explains: “Using a child’s own stem cells as a possible treatment is the safest form of stem cell transplantation because it carries virtually no threat of immune system rejection. Our focus on cerebral palsy breaks new ground in advancing therapies to change the course of these kinds of brain injury–a condition for which there is currently no cure.”

Brain injuries or lack of oxygen either before birth, during birth, or during the first years of life can damage specific motor pathways in the brain and lead to an inability properly move, learn, hear, see, or think normally. According to the Centers for Disease Control, 2-3 / 1,000 children are affected by cerebral palsy.

These clinical trials are part of an innovative push that partners clinical researchers with patients. They also represent a move from preclinical studies with cord blood stem cells in animals, to human clinical trials with genuine human patients. Heather Brown put it this way: “The benefits of cord blood stem cells being very young, easy to obtain, unspecialized cells which have had limited exposure to environmental toxins or infectious diseases and easy to store for long terms without any loss of function, make them an attractive source for cellular therapy researchers today. We are encouraged to see interest from such diverse researchers from neurosurgeons to endocrinologists and cardiac specialists.”

Long Version of Mesenchymal Stem Cell Review Article


This is a long version of the review article published at the Mesenchymal Stem Cell web site.  Enjoy.

Comparisons of Mesenchymal Stem Cells from Bone Marrow and Other Sources

Introduction

Mesenchymal stem cells (MSCs) are adult, multipotent stem cells that have been isolated from circulating blood (Kuznetsov et al 2001), umbilical cord blood (Beiback et al 2004; Lee et al 2004b), placenta (Igura et al 2004), heart (Warejcka et al 1996), amniotic fluid (Tsai et al 2004), adipose tissue (Katz et al 2005), synovium (Fickert et al 2003), skeletal muscle (Young et al 1995), pancreas (Hu et al 2003), deciduous teeth (Estrela et 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; Morikawa et 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 genes and 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 (Niemeyer et 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 up-regulated 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 ACAN gene) 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 (Johnstone 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 “stem-ness,” 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, Sensebé 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 María, 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; Armiñán et al 2009; Perán 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-Bénard 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 hedgehod (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 (Morsczeck et 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-, hydroxzyanisole, 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 (Karaöz 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).

Conclusion

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.

References

Abedin M, Tintut Y, Demer LL. Mesenchymal Stem Cells and the Artery Wall. Circulation Research 2004;95:671-676.

Afizah H, Yang Z, Hui JHP, Ouyang HW, Lee EH. A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors. Tissue Eng 2007;13(4):659-666.

Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung UI, Kubota N, Terauchi Y, Harada Y, Azuma Y, Nakamura K, Kadowaki T, Kawaguchi H. PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest 2004;113(6):846-855.

Anghileri E, Marconi S, Pignatelli A, Cifelli P, Galié M, Sbarbati A, Krampera M, Belluzzi O, Bonetti B. Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stem Cells Dev 2008;17(5):909-916.

Antonitsis P, Ioannidou-Papagiannaki E, Kaidoglou A, Papakonstantinou C. In vitro cardiomyogenic differentiation of adult human bone marrow mesenchymal stem cells. The role of 5-azacytidine. Interact Cardiovasc Thorac Surg 2007;6(5):593-597.

Antonitsis P, Ioannidou-Papagiannaki E, Kaidoglou A, Charokopos N, Kalogeridis A, Kouzi-Koliakou K, Kyriakopoulou I, Klonizakis I, Papakonstantinou C. Cardiomyogenic potential of human adult bone marrow mesenchymal stem cells in vitro. Thorac Cardiovasc Surg 2008;56(2):77-82.

Aranda P, Agirre X, Ballestar E, Andreu EJ, Román-Gómez J, Prieto I, Martín-Subero JI, Cigudosa JC, Siebert R, Esteller M, Prosper F. Epigenetic signatures associated with different levels of differentiation potential in human stem cells. PLoS One 2009;4(11):e7809.

Armiñán A, Gandía C, Bartual M, García-Verdugo JM, Lledó E, Mirabet V, Llop M, Barea J, Montero JA, Sepúlveda P. Cardiac differentiation is driven by NKX2.5 and GATA4 nuclear translocation in tissue-specific mesenchymal stem cells. Stem Cells Dev 2009;18(6):907-918.

Awad HA, Halvorsen YD, Gimble JM, Guilak F. Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells. Tissue Eng 2003;9(6):1301-1312.

Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials 2004;25(16):3211-3222.

Bae KS, Park JB, Kim HS, Kim DS, Park DJ, Kang SJ. Neuron-like differentiation of bone marrow-derived mesenchymal stem cells. Yonsei Med 2011;52(3):401-412.

Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 2007;25:1384-1392.

Barry F, Boynton RE, Liu B, Murphy JM. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 2001;268:189-200.

Bartolini P, Vainzof M, Zatz M. Preclinical studies with umbilical cord mesenchymal stromal cells in different animal models for muscular dystrophy. J Biomed Biotechnol 2011; DOI:10.1155/2011/715251.

Bartunek J, Croissant JD, Wijns W, Gofflot S, de Lavareille A, Vanderheyden M, Kaluzhny Y, Mazouz N, Willemsen P, Penicka M, Mathieu M, Homsy C, De Bruyne B, McEntee K, Lee IW, Heyndrickx GR. Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium. Am J Physiol Heart Circ Physiol 2007;292(2):H1095-H1104.

Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos S, Robey PG, Shi S. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res 2003;82(12):976-981.

Beier JP, Bitto FF, Lange C, Klumpp D, Arkudas A, Bleiziffer O, Boos AM, Horch RE, Kneser U. Myogenic differentiation of mesenchymal stem cells co-cultured with primary myoblasts. Cell Biol Int 2011;35(4):397-406.

Bieback K, Kern S, Kluter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004;22:625–634.

Boeuf S, Richter W. Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors. Stem Cell Res Ther 2010;1(4):31.

Caplan AI, Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med 2001;7:259–264.

Centeno CJ, Schultz JR, Cheever M, Freeman M, Faulkner S, Robinson B, Hanson R. Safety and complications reporting update on the re-implantation of culture-expanded mesenchymal stem cells using autologous platelet lysate technique. Curr Stem Cell Res Ther 2011;6(4):368-378.

Chang YJ, Shih D, Tseng CP, Hsieh TB, Lee DC, Hwang SM. Disparate Mesenchyme-Lineage Tendencies in Mesenchymal Stem Cells from Human Bone Marrow and Umbilical Cord Blood. Stem Cells 2006a;24:679-685.

Chang YJ, Tseng CP, Hsu LF, Hsieh TB, Hwang SM. Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood. Cell Biol Int 2006b;30(6):495-499.

Chang YJ, Hwang SM, Tseng CP, Cheng FC, Huang SH, Hsu LF, Hsu LW, Tsai MS. Isolation of mesenchymal stem cells with neurogenic potential from the mesoderm of the amniotic membrane. Cells Tissues Organs 2010;192(2):93-105.

Charbord P. Bone marrow mesenchymal stem cells: historical overview and concepts. Hum Gene Ther 2010;21(9):1045-1056.

Chen L, He DM, Zhang Y. The differentiation of human placenta-derived mesenchymal stem cells into dopaminergic cells in vitro. Cell Mol Biol Lett 2009;14(3):528-536.

Cheng F, Zou P, Yang H, Yu Z, Zhong Z. Induced differentiation of human cord blood mesenchymal stem/progenitor cells into cardiomyocyte-like cells in vitro. J Huazhong Univ Sci Technolog Med Sci 2003;23(2):154-157.

Cheong HH, Masilamani J, Phan TT, Chan SY. Cord lining progenitor cells: potential in vitro adipogenesis model. Int J Obes (Lond) 2010;34(11):1625-1633.

Christman JK. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002;21(35): 5483-5495.

Collins-Hooper H, Luke G, Cranfield M, Otto WR, Ray S, Patel K. Efficient myogenic reprogramming of adult white fat stem cells and bone marrow stem cells by freshly isolated skeletal muscle fibers. Transl Res 2011;158(6):334-343.

Cooper RN, Tajbakhsh S, Mouly V, Cossu G, Buckingham M, Butler-Browne GS. In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J Cell Sci 1999;112 (Pt 17):2895-2901.

Cordeiro MM, Dong Z, Kaneko T, Zhang Z, Miyazawa M, Shi S, Smith AJ, Nör JE. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. J Endod 2008;34(8):962-969.

Cornelison DD, Wold BJ. Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 1997;191(2):270-283.

Coura GS, Garcez RC, de Aguiar CB, Alvarez-Silva M, Magini RS, Trentin AG. Human periodontal ligament: a niche of neural crest stem cells. J Periodontal Res 2008;43(5):531-536.

Cowan CM, Shi YY, Aalami OO, Chou YF, Mari C, Thomas R, Quarto N, Contag CH, Wu B, Longaker MT. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nat Biotechnol 2004;22(5):560-567.

Crobu F, Latini V, Marongiu MF, Sogos V, Scintu F, Porcu S, Casu C, Badiali M, Sanna A, Manchinu MF, Ristaldi MS. Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile. Mol Biol Rep 2011; DOI: 10.1007/s11033-011-1180-1189.

Datta I, Mishra S, Mohanty L, Pulikkot S, Joshi PG. Neuronal plasticity of human Wharton’s jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells. Cytotherapy 2011;13(8):918-932.

De Bari C, Dell’Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001;44(8):1928-1942.

De Bari C, Dell’Accio F, Vandenabeele F, Vermeesch JR, Raymackers JM, Luyten FP. Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane. J Cell Biol 2003;160(6):909-918.

de la Garza-Rodea AS, van der Velde-van Dijke L, Boersma H, Gonçalves MA, van Bekkum DW, de Vries AA, Knaän-Shanzer S. Myogenic properties of human mesenchymal stem cells derived from three different sources. Cell Transplant 2011;DOI: http://dx.doi.org/10.3727/096368911X580554.

De Ugarte DA, Alfonso Z, Zuk PA, Elbarbary A, Zhu M, Ashjian P, Benhaim P, Hedrick MH, Fraser JK. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol Lett 2003a;89(2-3):267-270.

De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M, Dragoo JL, Ashjian P, Thomas B, Benhaim P, Chen I, Fraser J, Hedrick MH. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003b;174(3):101-109.

Deng ZL, Sharff KA, Tang N, Song WX, Luo J, Luo X, Chen J, Bennett E, Reid R, Manning D, Xue A, Montag AG, Luu HH, Haydon RC, He TC. Regulation of osteogenic differentiation during skeletal development. Front Biosci 2008;13:2001-2021.

Dezawa M, Kanno H, Hoshino M, Cho H, Matsumoto N, Itokazu Y, Tajima N, Yamada H, Sawada H, Ishikawa H, Mimura T, Kitada M, Suzuki Y, Ide C. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J Clin Invest 2004;113(12):1701-1710.

Dezawa M, Ishikawa H, Itokazu Y, Yoshihara T, Hoshino M, Takeda S, Ide C, Nabeshima Y. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309(5732):314-317.

Di Rocco G, Iachininoto MG, Tritarelli A, Straino S, Zacheo A, Germani A, Crea F, Capogrossi MC. Myogenic potential of adipose-tissue-derived cells. J Cell Sci 2006;119(Pt 14):2945-2952.

Diekman BO, Rowland CR, Lennon DP, Caplan AI, Guilak F. Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix. Tissue Eng 2010;16(2):523-533.

Djouad F, Bony C, Häupl T, Uzé G, Lahlou N, Louis-Plence P, Apparailly F, Canovas F, Rème T, Sany J, Jorgensen C, Noël D. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther 2005;7(6):R1304-R1315.

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8(4):315-317.

Enomoto H, Furuichi T, Zanma A, Yamana K, Yoshida C, Sumitani S, Yamamoto H, Enomoto-Iwamoto M, Iwamoto M, Komori T. Runx2 deficiency in chondrocytes causes adipogenic changes in vitro. J Cell Sci 2004;117(Pt 3):417-425.

Erickson GR, Gimble JM, Franklin DM, Rice HE, Awad H, Guilak F. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 2002;290:763-769.

Estes BT, Wu AW, Guilak F. Potent Induction of Chondrocytic Differentiation of Human Adipose-Derived Adult Stem Cells by Bone Morphogenetic Protein 6. Arthritis Rheum 2006;54(4):1222-1232.

Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of adipose derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 2010;5(7):1294–1311.

Estrela C, Alencar AH, Kitten GT, Vencio EF, Gava E. Mesenchymal stem cells in the dental tissues: perspectives for tissue regeneration. Braz Dent J 2011;22(2):91-98.

Farmer SR. Regulation of PPARgamma activity during adipogenesis. Int J Obes (Lond) 2005;29(Suppl 1):S13-S16.

Ferrari G, Cusella-De Angelis G, Coletta M., Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle Regeneration by Bone Marrow-Derived Myogenic Progenitors. Science 1998;279:1528–1530.

Fickert S, Fiedler J, Brenner RE. Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting. Osteoarthritis Cartilage 2003;11:790–800.

Frisbie DD, Kisiday JD, Kawcak CE, Werpy NM, McIlwraith CW. Evaluation of adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis. J Orthop Res 2009;27(12):1675-1680.

Fujita T, Azuma Y, Fukuyama R, Hattori Y, Yoshida C, Koida M, Ogita K, Komori T. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J Cell Biol 2004;166(1):85-95.

Fujita T, Iwata T, Shiba H, Igarashi A, Hirata R, Takeda K, Mizuno N, Tsuji K, Kawaguchi H, Kato Y, Kurihara H. Identification of marker genes distinguishing human periodontal ligament cells from human mesenchymal stem cells and human gingival fibroblasts. J Periodontal Res 2007;42(3):283-286.

Fukuda K. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artif Organs 2001;25(3):187-193.

Fukuda K. Molecular characterized of regenerated cardiomyocytes derived from adult mesenchymal stem cells. Congenit Anom (Kyoto) 2002;42(1):1-9.

Gallo MP, Ramella R, Alloatti G, Penna C, Pagliaro P, Marcantoni A, Bonafé F, Losano G, Levi R. Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes. J Cell Biochem 2007;100(1):86-99.

Galmiche MC, Koteliansky VE, Briere J, Herve P, Charbord P. Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood 1993;82:66–76.

Gao XR, Tan YZ, Wang HJ. Overexpression of Csx/Nkx2.5 and GATA-4 enhances the efficacy of mesenchymal stem cell transplantation after myocardial infarction. Circ J 2011;75(11):2683-2691.

Gay IC, Chen S, MacDougall M. Isolation and characterization of multipotent human periodontal ligament stem cells. Orthod Craniofac Res 2007;10(3):149-160.

Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy. 2003;5(5):362-369.

Gimble JM, Guilak F, Nuttall ME, Sathishkumar S, Vidal M, Bunnell BA. In vitro differentiation potential of mesenchymal stem cells. Trans Med Hemother 2008;35:228-238.

Girgenrath M, Weng S, Kostek CA, Browning B, Wang M, Brown SA, Winkles JA, Michaelson JS, Allaire N, Schneider P, Scott ML, Hsu YM, Yagita H, Flavell RA, Miller JB, Burkly LC, Zheng TS. TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration. EMBO J 2006;25(24):5826-5839.

Golding MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem 2006;97:33-44.

Gornostaeva SN, Rzhaninova AA, Gol’dstein DV. Myogenesis in hemopoietic tissue mesenchymal stem cell culture. Bull Exp Biol Med 2006;141(4):493-499.

Grimm WD, Dannan A, Becher S, Gassmann G, Arnold W, Varga G, Dittmar T. The ability of human periodontium-derived stem cells to regenerate periodontal tissues: a preliminary in vivo investigation. Int J Periodontics Restorative Dent 2011;31(6):e94-e101.

Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84(12):4164-4173.

Gronthos S, Zannettino AC, Graves SE, Ohta S, Hay SJ, Simmons PJ. Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res 1999;14(1):47-56.

Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97(25):13625-13630.

Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW, Gimble JM. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol 2001;189(1):54-63.

Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, Robey PG, Shi S. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81(8):531-535.

Gronthos S, Zannettino AC, Hay SJ, Shi S, Graves SE, Kortesidis A, Simmons PJ. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 2003;116(Pt 9):1827-1835.

Guilak F, Lott KE, Awad HA, Cao Q, Hicok KC, Fermor B, Gimble JM. Clonal Analysis of the differentiation potential of human adipose-derived adult stem cells. J Cell Physiol 2006;206:229-237.

Hahn JY, Cho HJ, Kang HJ, Kim TS, Kim MH, Chung JH, Bae JW, Oh BH, Park YB, Kim HS. Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction. J Am Coll Cardiol 2008;51(9):933-943.

Hara K, Yamada Y, Nakamura S, Umemura E, Ito K, Ueda M. Potential characteristics of stem cells from human exfoliated deciduous teeth compared with bone marrow-derived mesenchymal stem cells for mineralized tissue-forming cell biology. J Endod 2011;37(12):1647-1652.

Hasty P, Bradley A, Morris JH, Edmondson DG, Venuti JM, Olson EN, Klein WH. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 1993;364(6437):501-506.

Hattan N, Kawaguchi H, Ando K, Kuwabara E, Fujita J, Murata M, Suematsu M, Mori H, Fukuda K. Purified cardiomyocytes from bone marrow mesenchymal stem cells produce stable intracardiac grafts in mice. Cardiovasc Res 2005;65(2):334-344.

He XQ, Chen MS, Li SH, Liu SM, Zhong Y, McDonald Kinkaid HY, Lu WY, Weisel RD, Li RK. Co-culture with cardiomyocytes enhanced the myogenic conversion of mesenchymal stromal cells in a dose-dependent manner. Mol Cell Biochem 2010;339(1-2):89-98.

He Z, Li H, Zuo S, Pasha Z, Wang Y, Yang Y, Jiang W, Ashraf M, Xu M. Transduction of Wnt11 promotes mesen chymal stem cell transdifferentiation into cardiac phenotypes. Stem Cells Dev 2011;20(10):1771-1778.

Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F, Richter W. Reduced chondrogenesis potential adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6. J Cell Physiol 2007;211:682-691.

Hsieh JY, Fu YS, Chang SJ, Tsuang YH, Wang HW. Functional module analysis reveals differential osteogenic and stemness potentials in human mesenchymal stem cells from bone marrow and Wharton’s jelly of umbilical cord. Stem Cells Dev 2010;19(12):1895-1910.

Hu Y, Liao L, Wang Q, Ma L, Ma G, Jiang X, Zhao RC. Isolation and identification of mesenchymal stem cells from human fetal pancreas. J Lab Clin Med 2003;141:342–349.

Huang JL, Kazmi N, Durbhakula MM, Hering TM, Yoo JU, Johnstone B. Chondrogenic potential of progenitor cells derived from human bone marrow and adipose tissue: a patient-matched comparison. J Orthop Res 2005;23:1383-1389.

Huang CY, Pelaez D, Dominguez-Bendala J, Garcia-Godoy F, Cheung HS. Plasticity of stem cells derived from adult periodontal ligament. Regen Med 2009;4(6):809-821.

Hung CN, Mar K, Chang HC, Chiang YL, Hu HY, Lai CC, Chu RM, Ma CM. A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration. Biomaterials 2011;32(29):6995-7005.

Igura K, Zhang X, Takahashi K, Mitsuru A, Yamaguchi S, Takashi TA. Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta. Cytotherapy 2004;6:543–553.

Im GI, Kim DY, Shin JH, Hyun CW, Cho WH. Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow. J Bone Joint Surg Br 2001;83:289-294.

Im GI, Shin YW, Lee KB. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthritis Cartilage 2005;13(10):845-853.

Indrawattana N, Chen G, Tadokoro M, Shann LH, Ohgushi H, Tateishi T, Tanaka J, Bunyaratvej A. Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun 2004;320(3):914-919.

Ito K, Yamada Y, Nakamura S, Ueda M. Osteogenic potential of effective bone engineering using dental pulp stem cells, bone marrow stem cells, and periosteal cells for osseointegration of dental implants. Int J Oral Maxillofac Implants 2011;26(5):947-954.

Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble JM, Bunnell BA. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2006;99(5):1285-1297.

Jang S, Cho HH, Cho YB, Park JS, Jeong HS. Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biol 2010;11:25

Jansen BJ, Gilissen C, Roelofs H, Schaap-Oziemlak A, Veltman JA, Raymakers RA, Jansen JH, Kögler G, Figdor CG, Torensma R, Adema GJ. Functional differences between mesenchymal stem cell populations are reflected by their transcriptome. Stem Cells Dev 2010;19(4):481-490.

Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.

Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998; 238:265-272.

Jones EA, Kinsey SE, English A, Jones RA, Straszynski L, Meredith DM, Markham AF, Jack A, Emery P, McGonagle D. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum 2002;46(12):3349-3360.

Kadar K, Kiraly M, Porcsalmy B, Molnar B, Racz GZ, Blazsek J, Kallo K, Szabo EL, Gera I, Gerber G, Varga G. Differentiation potential of stem cells from human dental origin – promise for tissue engineering. J Physiol Pharmacol 2009;60 Suppl 7:167-175.

Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 1997;6:125–134.

Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP, Vanichakarn P, Park JY, Li Y, Haydon RC, He TC. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther 2004a;11(17):1312-1320.

Kang TJ, Yeom JE, Lee HJ, Rho SH, Han H, Chae GT. Growth kinetics of human mesenchymal stem cells from bone marrow and umbilical cord blood. Acta Haematol 2004b;112(4):230-233.

Karaöz E, Demircan PC, Sağlam O, Aksoy A, Kaymaz F, Duruksu G. Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrow-derived mesenchymal stem cells. Histochem Cell Biol 2011;136(4):455-473.

Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells 2005;
23:412–423.

Kern S, Eicher H, Stoeve J, Klüter H, Bieback K. Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord Blood, or Adipose Tissue. Stem Cells 2006;24:1294–1301.

Kestendjieva S, Kyurkchiev D, Tsvetkova G, Mehandjiev T, Dimitrov A, Nikolov A, Kyurkchiev S. Characterization of mesenchymal stem cells isolated from the human umbilical cord. Cell Biol Int 2008;32(7):724-732.

Kim SS, Yoo SW, Park TS, Ahn SC, Jeong HS, Kim JW, Chang DY, Cho KG, Kim SU, Huh Y, Lee JE, Lee SY, Lee YD, Suh-Kim H. Neural induction with neurogenin1 increases the therapeutic effects of mesenchymal stem cells in the ischemic brain. Stem Cells 2008;26(9):2217-2228.

Kim HJ, Im GI. The effects of ERK1/2 inhibitor on the chondrogenesis of bone marrow- and adipose tissue-derived multipotent mesenchymal stromal cells. Tissue Eng Part A 2010;16(3):851-860.

Kim HJ, Im GI. Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: greater doses of growth factor are necessary. J Orthop Res 2009;27: 612-619.

Kim MJ, Shin KS, Jeon JH, Lee DR, Shim SH, Kim JK, Cha DH, Yoon TK, Kim GJ. Human chorionic-plate-derived mesenchymal stem cells and Wharton’s jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells. Cell Tissue Res 2011;346(1):53-64.

Kocaefe C, Balci D, Hayta BB, Can A. Reprogramming of human umbilical cord stromal mesenchymal stem cells for myogenic differentiation and muscle repair. Stem Cell Rev 2010;6(4):512-522.

Kondo T, Matsuoka AJ, Shimomura A, Koehler KR, Chan RJ, Miller JM, Srour EF, Hashino E. Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3. Stem Cells 2011;29(5):836-846.

Krampera M, Marconi S, Pasini A, Galiè M, Rigotti G, Mosna F, Tinelli M, Lovato L, Anghileri E, Andreini A, Pizzolo G, Sbarbati A, Bonetti B. Induction of neural-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus. Bone 2007;40(2):382-390.

Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Bianco P, Robey PG. Circulating skeletal stem cells. J Cell Biol 2001;153:1133–1140.

Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004a;14(4-6):311-324.

Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004b;103:1669-1675.

Lee JH, Kosinski PA, Kemp DM. Contribution of human bone marrow stem cells to individual skeletal myotubes followed by myogenic gene activation. Exp Cell Res 2005;307(1):174-182.

Lee WC, Sepulveda JL, Rubin JP, Marra KG. Cardiomyogenic differentiation potential of human adipose precursor cells. Int J Cardiol 2009;133(3):399-401.

Leroux L, Descamps B, Tojais NF, Séguy B, Oses P, Moreau C, Daret D, Ivanovic Z, Boiron JM, Lamazière JM, Dufourcq P, Couffinhal T, Duplàa C. Hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fiber regeneration after ischemia through a Wnt4-dependent pathway. Mol Ther 2010;18(8):1545-1552.

Li H, Zuo S, Pasha Z, Yu B, He Z, Wang Y, Yang X, Ashraf M, Xu M. GATA-4 promotes myocardial transdifferentiation of mesenchymal stromal cells via up-regulating IGFBP-4. Cytotherapy 2011;13(9):1057-1065.

Lim JH, Boozer L, Mariani CL, Piedrahita JA, Olby NJ. Generation and characterization of neurospheres from canine adipose tissue-derived stromal cells. Cell Reprogram 2010;12(4):417-425.

Lim JY, Park SI, Kim SM, Jun JA, Oh JH, Ryu CH, Jeong CH, Park SH, Park SA, Oh W, Chang JW, Jeun SS. Neural Differentiation of Brain-derived Neurotrophic Factor-expressing Human Umbilical Cord Blood-derived Mesenchymal Stem Cells in Culture via TrkB-mediated ERK and β-catenin Phosphorylation and following Transplantation into the Developing Brain. Cell Transplant 2011;DOI: 10.3727/096368910X557236:1-32.

Liu TM, Martina M, Hutmacher DW, Hui JH, Lee EH, Lim B. Identification of common pathways mediating differentiation of bone marrow- and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells 2007;25(3):750-760.

Long X, Olszewski M, Huang W, Kletzel M. Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells. Stem Cells Dev 2005;14(1):65-69.

Lovati AB, Corradetti B, Consiglio AL, Recordati C, Bonacina E, Bizzaro D, Cremonesi F. Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells. Vet Res Commun 2011;35:103-121.

Lu P, Blesch A, Tuszynski MH. Induction of bone marrow stromal cells to neurons: Differentiation, transdifferentiation or artifact? J Neurosci Res 2004;77(2):174-191.

Lu X, Alshemali S, de Wynter EA, Dickinson AM. Mesenchymal stem cells from CD34(-) human umbilical cord blood. Transfus Med 2010;20(3):178-184.

Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, Li X, Luu HH, Luo J, Montag AG, Haydon RC, He TC. Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem 2004;279(53):55958-55968.

Luu HH, Song WX, Luo X, Manning D, Luo J, Deng ZL, Sharff KA, Montag AG, Haydon RC, He TC. Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res 2007;25(5):665-677.

Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 1998;4:415-428.

Mafi P, Hindocha S, Mafi R, Griffin M, Khan WS. Adult mesenchymal stem cells and cell surface characterization – a systematic review of the literature. Open Orthop J 2011;5(Suppl 2):253-260.

Majore I, Moretti P, Stahl F, Hass R, Kasper C. Growth and differentiation properties of mesenchymal stromal cell populations derived from whole human umbilical cord. Stem Cell Rev 2011;7(1):17-31.

Martin-Rendon E, Sweeney D, Lu F, Girdlestone J, Navarrete C, Watt SM. 5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies. Vox Sang 2008;95(2):137-148.

Maurer MH. Proteomic definitions of mesenchymal stem cells. Stem Cells Int 2011; DOI:10.4061/2011/7042562011.

Michalczyk K, Ziman M. Nestin structure and predicted function in cellular cytoskeletal organisation. Histol Histopathol 2005;20(2):665-671.

Mitchell KE, Weiss ML, Mitchel BM, Martin P, Davis D, Morales L, Helwig B, Beerenstrauch M, Abou-Easa K, Hildreth T, Troyer D, Medicetty S. Matrix cells from Wharton’s jelly form neurons and glia. Stem Cells 2003;21:50-60.

Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100(10):5807-5812.

Morikawa S, Mabuchi Y, Niibe K, Suzuki S, Nagoshi N, Sunabori T, Shimmura S, Nagai Y, Nakagawa T, Okano H, Matsuzaki Y. Development of mesenchymal stem cells partially originate from the neural crest. Biochem Biophys Res Commun 2009; 379(4):1114-1119.

Morsczeck C, Götz W, Schierholz J, Zeilhofer F, Kühn U, Möhl C, Sippel C, Hoffmann KH. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol 2005;24(2):155-165.

Morsczeck C, Schmalz G, Reichert TE, Völlner F, Galler K, Driemel O. Somatic stem cells for regenerative dentistry. Clin Oral Investig 2008;12(2):113-118.

Mosna F, Sensebé L, Krampera M. Human bone marrow and adipose tissue mesenchymal stem cells: A user’s guide. Stem Cells Dev 2010;19(10):1449-1470.

Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet neurology 2003;2(12):731–740.

Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrow-derived mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 2009;66(2):236-253.

Nabeshima Y, Hanaoka K, Hayasaka M, Esumi E, Li S, Nonaka I, Nabeshima Y. Myogenin gene disruption results in perinatal lethality because of severe muscle defect. Nature 1993;364(6437):532-535.

Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S, Muneta T, Ishikawa I. Stem cell properties of human periodontal ligament cells. J Periodontal Res 2006;41(4):303-310.

Nagoshi N, Shibata S, Kubota Y, Nakamura M, Nagai Y, Satoh E, Morikawa S, Okada Y, Mabuchi Y, Katoh H, Okada S, Fukuda K, Suda T, Matsuzaki Y, Toyama Y, Okano H. Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell 2008;2(4):392-403.

Nakamura S, Yamada Y, Katagiri W, Sugito T, Ito K, Ueda M. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod 2009;35(11):1536-1542.

Nakatsuka R, Nozaki T, Uemura Y, Matsuoka Y, Sasaki Y, Shinohara M, Ohura K,
Sonoda Y. 5-Aza-2′-deoxycytidine treatment induces skeletal myogenic differentiation of
mouse dental pulp stem cells. Arch Oral Biol 2010;55(5):350-357.

Niemeyer P, Kornacker M, Mehlhorn A, Seckinger A, Vohrer J, Schmal H, Kasten P, Eckstein V, Südkamp NP, Krause U. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro. Tissue Eng 2007;13(1):111-121

Niemeyer P, Fechner K, Milz S, Richter W, Suedkamp NP, Mehlhorn AT, Pearce S, Kasten P. Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia the influence of platelet-rich plasma. Biomaterials 2010;31(13):3572-3579.

Nimura A, Muneta T, Koga H, Mochizuki T, Suzuki K, Makino H, Umezawa A, Sekiya I. Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum. Arthritis Rheum 2008;58(2):501-510.

Nourbakhsh N, Soleimani M, Taghipour Z, Karbalaie K, Mousavi SB, Talebi A, Nadali F, Tanhaei S, Kiyani GA, Nematollahi M, Rabiei F, Mardani M, Bahramiyan H, Torabinejad M, Nasr-Esfahani MH, Baharvand H. Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells. Int J Dev Biol 2011;55(2):189-195.

Oberlender SA, Tuan RS. Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development 1994;120:177-187.

Paul A, Srivastava S, Chen G, Shum-Tim D, Prakash S. Functional Assessment of Adipose Stem Cells for Xenotransplantation Using Myocardial Infarction Immunocompetent Models: Comparison with Bone Marrow Stem Cells. Cell Biochem Biophys 2011;DOI: 10.1007/s12013-011-9323-0.

Peng Y, Kang Q, Cheng H, Li X, Sun MH, Jiang W, Luu HH, Park JY, Haydon RC, He TC. Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling. J Cell Biochem 2003;90(6):1149-1165.

Peng Y, Kang Q, Luo Q, Jiang W, Si W, Liu BA, Luu HH, Park JK, Li X, Luo J, Montag AG, Haydon RC, He TC. Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells. J Biol Chem 2004;279(31):32941-32949.

Perán M, Marchal JA, López E, Jiménez-Navarro M, Boulaiz H, Rodríguez-Serrano F, Carrillo E, Sánchez-Espin G, de Teresa E, Tosh D, Aranega A. Human cardiac tissue induces transdifferentiation of adult stem cells towards cardiomyocytes. Cytotherapy 2010;12(3):332-337.

Peters H, Bailing R. Teeth: Where and how to make them. Trends Genet 1999;15:59-65.

Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999;284:143–147.

Planat-Bénard V, Menard C, André M, Puceat M, Perez A, Garcia-Verdugo JM, Pénicaud L, Casteilla L. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 2004;94(2):223-229.

Pountos I, Jones E, Tzioupis C, McGonagle D, Giannoudis PV. Growing bone and cartilage. The role of mesenchymal stem cells. J Bone Joint Surg Br 2006;88(4):421-426.

Prockop DJ. Marrow Stromal Cells as Stem Cells for Nonhematopoietic Tissues. Science 1997; 276:71–74.

Puetzer JL, Petitte JN, Loboa EG. Comparative review of growth factors for induction of three-dimensional in vitro chondrogenesis in human mesenchymal stem cells isolated from bone marrow and adipose tissue. Tissue Eng Part B Rev 2010;16(4):435-444.

Quirici N, Scavullo C, de Girolamo L, Lopa S, Arrigoni E, Deliliers GL, Brini AT. Anti-L-NGFR and -CD34 monoclonal antibodies identify multipotent mesenchymal stem cells in human adipose tissue. Stem Cells Dev 2010;19(6):915-925.

Rangappa S, Entwistle JW, Wechsler AS, Kresh JY. Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg 2003a;126(1):124-132.

Rangappa S, Fen C, :Lee EH, Bongso A, Sim EK. Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Ann Thorac Surg 2003b;75(3):775-779.

Rastegar F, Shenaq D, Huang J, Zhang W, Zhang BQ, He BC, Chen L, Zuo GW, Luo Q, Shi Q, Wagner ER, Huang E, Gao Y, Gao JL, Kim SH, Zhou JZ, Bi Y, Su Y, Zhu G, Luo J, Luo X, Qin J, Reid RR, Luu HH, Haydon RC, Deng ZL, He TC. Mesenchymal stem cells: Molecular characteristics and clinical applications. World J Stem Cells 2010;2(4):67-80.

Rebelatto CK, Aguiar AM, Moretão MP, Senegaglia AC, Hansen P, Barchiki F, Oliveira J, Martins J, Kuligovski C, Mansur F, Christofis A, Amaral VF, Brofman PS, Goldenberg S, Nakao LS, Correa A. Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med 2008;233(7):901-913.

Rider DA, Dombrowski C, Sawyer AA, Ng GH, Leong D, Hutmacher DW, Nurcombe V, Cool SM. Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells. Stem Cells 2008;26(6):1598-1608.

Roche S, Delorme B, Oostendorp RA, Barbet R, Caton D, Noel D, Boumediene K, Papadaki HA, Cousin B, Crozet C, Milhavet O, Casteilla L, Hatzfeld J, Jorgensen C, Charbord P, Lehmann S. Comparative proteomic analysis of human mesenchymal and embryonic stem cells: towards the definition of a mesenchymal stem cell proteomic signature. Proteomics 2009;9(2):223-232.

Romanov YA, Darevskaya AN, Merzlikina NV, Buravkova LB. Mesenchymal stem cells from human bone marrow and adipose tissue: isolation, characterization, and differentiation potentialities. Bull Exp Biol Med 2005;140(1):138-143.

Rose RA, Jiang H, Wang X, Helke S, Tsoporis JN, Gong N, Keating SC, Parker TG, Backx PH, Keating A. Bone marrow-derived mesenchymal stromal cells express cardiac-specific markers, retain the stromal phenotype, and do not become functional cardiomyocytes in vitro. Stem Cells 2008;26(11):2884-2892.

Rosen ED. Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol 2000;16:145-171.

Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 1993;75(7):1351-1359.

Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum 2005;52(8):2521-2529.

Santa María L, Rojas CV, Minguell JJ. Signals from damaged but not undamaged skeletal muscle induce myogenic differentiation of rat bone-marrow-derived mesenchymal stem cells. Exp Cell Res 2004;300(2):418-426.

Schmitt B, Ringe J, Haupl T, Notter M, Manz R, Burmester GR, Sittinger M, Kaps C. BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. Differentiation 2003;71:567-577.

Scuteri A, Miloso M, Foudah D, Orciani M, Cavaletti G, Tredici G. Mesenchymal stem cells neuronal differentiation ability: a real perspective for nervous system repair? Curr Stem Cell Res Ther 2011;6(2):82-92.

Secco M, Moreira YB, Zucconi E, Vieira NM, Jazedje T, Muotri AR, Okamoto OK, Verjovski-Almeida S, Zatz M. Gene expression profile of mesenchymal stem cells from paired umbilical cord units: cord is different from blood. Stem Cell Rev 2009;5(4):387-401.

Segawa Y, Muneta T, Makino H, Nimura A, Mochizuki T, Ju YJ, Ezura Y, Umezawa A, Sekiya I. Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. J Orthop Res 2009;27(4):435-441.

Sekiya I, Colter DC, Prockop DJ. BMP-8 enhances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun 2001;284:411-418.

Sekiya I, Vuoristo JT, Larson BL, Prockop DJ. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA 2002;99:4397-4402.

Sekiya I, Larson BL, Vuoristo JT, Reger RL, Prockop DJ. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res 2005;320(2):269-276.

Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364(9429):149-155.

Shea CM, Edgar CM, Einhorn TA, Gerstenfeld LC. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis. J Cell Biochem 2003;90(6):1112-1127.

Shi S, Robey PG, Gronthos S. Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis. Bone 2001;29(6):532-539.

Shim SW, Jiang S, Wong P, Tan J, Chua YL, Tan YS, Sin YK, Lim CH, Chua T, Teh M, Liu TC, Sim E. Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun 2004;324(2):481-488.

Si W, Kang Q, Luu HH, Park JK, Luo Q, Song WX, Jiang W, Luo X, Li X, Yin H, Montag AG, Haydon RC, He TC. CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells. Mol Cell Biol 2006;26(8):2955-2964.

Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989;244(4912):1578-1580.

Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78(1):55-62.

Simmons PJ, Masinovsky B, Longenecker BM, Berenson R, Torok-Storb B, Gallatin WM. Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells. Blood 1992;80(2):388-395.

Singh P, YashRoy RC, Hoque M. Augmented bone-matrix formation and osteogenesis under magnetic field stimulation in vivo XRD, TEM and SEM investigations. Indian J Biochem Biophys 2006;43(3):167-172.

Smith CK 2nd, Janney MJ, Allen RE. Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells. J Cell Physiol 1994;159(2):379-385.

Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S, Shi S. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One 2006;1:e79.

Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 2008;34(2):166-171.

Stewart K, Walsh S, Screen J, Jefferiss CM, Chainey J, Jordan GR, Beresford JN. Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells. J Bone Miner Res 1999;14(8):1345-1356.

Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Nishikawa S. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 2007;129(7):1377-1388.

Tang N, Song WX, Luo J, Luo X, Chen J, Sharff KA, Bi Y, He BC, Huang JY, Zhu GH, Su YX, Jiang W, Tang M, He Y, Wang Y, Chen L, Zuo GW, Shen J, Pan X, Reid RR, Luu HH, Haydon RC, He TC. BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signalling. J Cell Mol Med 2009;13(8B):2448-2464.

Thesleff I, Mikkola M. The role of growth factors in tooth development. Int Rev Cytol 2002;217:93-135.

Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: current findings and future perspectives. Discov Med 2011;11(57):160-170.

Tominaga H, Maeda S, Miyoshi H, Miyazono K, Komiya S, Imamura T. Expression of osterix inhibits bone morphogenetic protein-induced chondrogenic differentiation of mesenchymal progenitor cells. J Bone Miner Metab 2009;27(1):36-45.

Tropel P, Platet N, Platel JC, Noël D, Albrieux M, Benabid AL, Berger F. Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells. Stem Cells 2006;24(12):2868-2876.

Troyer DL, Weiss ML. Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 2008;26:591-599.

Trzaska KA, Rameshwar P. Dopaminergic neuronal differentiation protocol for human mesenchymal stem cells. Methods Mol Biol 2011;698:295-303.

Tsai MS, Lee JL, Chang YJ, Hwang SM. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 2004;19:1450–1456.

Tsai MS, Hwang SM, Chen KD, Lee YS, Hsu LW, Chang YJ, Wang CN, Peng HH, Chang YL, Chao AS, Chang SD, Lee KD, Wang TH, Wang HS, Soong YK. Functional network analysis of the transcriptomes of mesenchymal stem cells derived from amniotic fluid, amniotic membrane, cord blood, and bone marrow. Stem Cells 2007;25(10):2511-2523.

Tuli R, Tuli S, Nandi S, Huang X, Manner PA, Hozack WJ, Danielson KG, Hall DJ, Tuan RS. Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogenactivated protein kinase and Wnt signaling cross-talk. J Biol Chem 2003;278:41227-41236.

Van Landuyt KB, Jones EA, McGonagle D, Luyten FP, Lories RJ. Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis. Arthritis Res Ther 2010;12(1):R15.

Vidal MA, Robinson SO, Lopez MJ, Paulsen DB, Borkhsenious O, Johnson JR, Moore RM, Gimble JM. Comparison of chondrogenic potential in equine mesenchymal stromal cells derived from adipose tissue and bone marrow. Vet Surg 2008; 37(8):713-724.

Vieira NM, Zucconi E, Bueno CR Jr, Secco M, Suzuki MF, Bartolini P, Vainzof M, Zatz M. Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice. Stem Cell Rev 2010;6(4):560-566.

Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W, Ho AD. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005;33(11):1402-1416.

Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, Goldberg VM. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am 1994;76;579-592.

Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995;18:1417–1426.

Walsh S, Jefferiss C, Stewart K, Jordan GR, Screen J, Beresford JN. Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1-4. Bone 2000;27(2):185-195.

Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, Fu YS, Lai MC, Chen CC. Mesenchymal stem cells in the Wharton’s Jelly of the human umbilical cord. Stem Cells 2004;22:1330-1337.

Wang L, Tran I, Sesharedy K, Weiss ML, Detamore MS. A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stem cells for cartilage tissue engineering. Tissue Eng 2009;15(8):2259-2266.

Wang Y, He W, Bian H, Liu C, Li S. Small molecule induction of neural-like cells from bone marrow-mesenchymal stem cells. J Cell Biochem 2011;DOI: 10.1002/jcb.24021.

Warejcka DJ, Harvey R, Taylor BJ, Young HE, Lucas PA. A population of cells isolated from rat heart capable of differentiating into several mesodermal phenotypes. J Surg Res 1996;62:233–242.

Weiss S, Hennig T, Bock R, Steck E, Richter W. Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells. J Cell Physiol 2010;223:84-93.

Winter A, Breit S, Parsch D, Benz K, Steck E, Hauner H, Weber RM, Ewerbeck V, Richter W. Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells. Arthritis Rheum 2003;48(2):418-429.

Wislet-Gendebien S, Hans G, Leprince P, Rigo JM, Moonen G, Rogister B. Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype. Stem Cells 2005;23(3):392-402.

Wongchuensoontorn C, Liebehenschel N, Schwarz U, Schmelzeisen R, Gutwald R, Ellis E 3rd, Sauerbier S. Application of a new chair-side method for the harvest of mesenchymal stem cells in a patient with nonunion of a fracture of the atrophic mandible–a case report. J Craniomaxillofac Surg 2009;37(3):155-161.

Woods A, Wang G, Beier F. Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions. J Cell Physiol 2007;213:1-8.

Xu W, Zhang X, Qian H, Zhu W, Sun X, Hu J, Zhou H, Chen Y. Mesenchymal stem cells from adult huan bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp Biol Med (Maywood) 2004;229(7):623-631.

Xu D, Gechtman Z, Hughes A, Collins A, Dodds R, Cui X, Jolliff e L, Higgins L, Murphy A, Farrell F. Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFbeta3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells. Growth Factors 2006;24:268-278.

Xu H, Miki K, Ishibashi S, Inoue J, Sun L, Endo S, Sekiya I, Muneta T, Inazawa J, Dezawa M, Mizusawa H. Transplantation of neuronal cells induced from human mesenchymal stem cells improves neurological functions after stroke without cell fusion. J Neurosci Res 2010;88(16):3598-3609.

Yablonka-Reuveni Z, Rivera AJ. Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat fibers. Dev Biol 1994;164(2):588-603.

Yamada Y, Nakamura S, Ito K, Sugito T, Yoshimi R, Nagasaka T, Ueda M. A feasibility of useful cell-based therapy by bone regeneration with deciduous tooth stem cells, dental pulp stem cells, or bone-marrow-derived mesenchymal stem cells for clinical study using tissue engineering technology. Tissue Eng Part A 2010;16(6):1891-1900.

Yamada Y, Ito K, Nakamura S, Ueda M, Nagasaka T. Promising cell-based therapy for bone regeneration using stem cells from deciduous teeth, dental pulp, and bone marrow. Cell Transplant 2011;20(7):1003-1013.

Yang Y, Li Y, Lv Y, Zhang S, Chen L, Bai C, Nan X, Yue W, Pei X. NRSF silencing induces neuronal differentiation of human mesenchymal stem cells. Exp Cell Res 2008;314(11-12):2257-2265.

Yang J, Song T, Wu P, Chen Y, Fan X, Chen H, Zhang J, Huang C. Differentiation potential of human mesenchymal stem cells derived from adipose tissue and bone marrow to sinus node-like cells. Mol Med Report 2012;5(1):108-113.

Yoo JU, Barthel TS, Nishimura K, Solchaga L, Caplan AI, Goldberg VM, Johnstone B. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80(12):1745-1757.

Yoon J, Min BG, Kim YH, Shim WJ, Ro YM, Lim DS. Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model. Acta Cardiol 2005a;60(3):277-284.

Yoon J, Shim WJ, Ro YM, Lim DS. Transdifferentiation of mesenchymal stem cells into cardiomyocytes by direct cell-to-cell contact with neonatal cardiomyocyte but not adult cardiomyocytes. Ann Hematol 2005b;84(11):715-721.

Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449-462.

Young HE, Mancini ML, Wright RP, Smith JC, Black AC Jr, Reagan CR, Lucas PA. Mesenchymal stem cells reside within the connective tissues of many organs. Dev Dyn 1995;202:137–144.

Yu G, Wu X, Dietrich MA, Polk P, Scott LK, Ptitsyn AA, Gimble JM. Yield and characterization of subcutaneous human adipose-derived stem cells by flow cytometric and adipogenic mRNA analyzes. Cytotherapy 2010;12(4):538-546.

Zhang YD, Chen Z, Song YQ, Liu C, Chen YP. Making a tooth: Growth factors, transcription factors, and stem cells. Cell Res 2005;15(5):301-316.

Zhang W, Zeng YS, Zhang XB, Wang JM, Zhang W, Chen SJ. Combination of adenoviral vector-mediated neurotrophin-3 gene transfer and retinoic acid promotes adult bone marrow cells to differentiate into neuronal phenotypes. Neurosci Lett 2006;408(2):98-103.

Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol 2002;174(1):11-20.

Zhu Y, Liu T, Song K, Fan X, Ma X, Cui Z. Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct 2008;26(6):664-675.

Zucconi E, Vieira NM, Bueno CR Jr, Secco M, Jazedje T, Costa Valadares M, Fussae Suzuki M, Bartolini P, Vainzof M, Zatz M. Preclinical studies with umbilical cord mesenchymal stromal cells in different animal models for muscular dystrophy. J Biomed Biotechnol 2011; DOI:10.1155/2011/715251.

Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7(2):211-228.

Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13(12):4279-4295.

Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2000;2(6):477-488.

Mesenchymal Stem Cell Web Site Publishes A Review Article Written By the Author of This Web Site


The mesenchymal stem cell web site has published my review that compares mesenchymal stem cells from different sources. The article is entitled “Comparisons of Mesenchymal Stem Cells from Bone Marrow and Other Sources,” It can be found at this link.

Polish Study Shows Stable Improvements Two Years After Heart Stem Cell Transplant


Stem cell treatments for heart attacks can improve heart function after a heart attack. This has been repeated shown in laboratory animals and human trials have also established the efficacy of bone marrow-based heart treatments. Despite these successes, there are some indications that the improvements wrought by bone marrow stem cell transplants into the heart are not stable, and the functional increases caused by it are transient.

To determine is functional improvements in the heart are transient or stable, Jaroslaw Kaspzak and his colleagues at the Medical University of Lodz, Poland have published a study in which they treated 60 heart attack patients and then tracked them for two years. Their study was published in the journal Kardiologia Polska, which, fortunately, is in English, since I do not read Polish.

In this study, 60 heart attack patients were treated with primary angioplasty and randomly assigned to two groups. The first group consisted of 40 patients who were treated with standard care, and bone marrow stem cell transplants.  The bone marrow cells were harvested 3-11 days after the heart attack. The bone marrow cells were administered to the heart by means of intracoronary catheters (over the wire balloon catheter) very near to the area of the infarct (the area in the heart damaged by the heart attack).  The second group of 20 patients were treated with standard care.  All patients were subjected to echocardiography before treatment and 1, 3, 6, 12, and 24 months after treatment.  Additionally, the percentages of patients who experienced subsequent heart attacks, admission to the hospital for heart failure, or revascularization, was tabulated two years after the treatment.

Just after the heart attack, the heart function of both groups was essentially the same.  The fraction of blood pumped from the heart (ejection fraction) in the treated group was 35% ± 6% and 33% ± 7% in the control group (normal is 55% – 70%).  The volume of blood left in the heart after it contracts (end systolic volume) was 95 milliliters ± 39 milliliters in the treated group and 99 milliliters ± 49 milliliters in the control group (normal is 50-60 milliliters).  The amount of blood in the heart after it fills (end diastolic volume) was 149 ± 48 milliliters in the treated group and 151 ± 65 milliliters in the control group (normal is 120-130 milliliters).  The end diastolic volume  (EDV) is a measure of the firmness of the heart walls.  A damaged heart is not as firm and its flaccid walls expand greatly and take up more volume, which puts further strain upon the heart.  Thus a DECREASE in the EDV is an indication of improvement in the heart.  Nevertheless, it is clear that before the treatment regimes were instigated, the average medical conditions of the two groups was essentially the same, at least when it comes to the heart.

The results of each treatment strategy are rather telling. The ejection fraction in the control group increased 3.7% one moth after the heart attack, 4.7% by 6 months, 4.8% at 12 months, and 4.7% at 24 months.  This this group saw its greatest increase six months after the heart attack and although this increase was stable, it was modest at best.  The bone marrow-treated group, however, saw an average ejection fraction increase of 7.1% after one month, 9.3% at 6 months, 11.0% after one year, and 10% after two years.  Thus the bone marrow-treated group not only showed a much faster and more robust increase in injection fraction, but an increase that was sustained two years after the procedure.  Also, the treated group saw half the percentage of deaths due to cardiac events (5%) than that observed in the control group (10%).  The percentage of hospitalizations for heart failure in the treated group (3%) was 20% of that seen in the control group (15%).  The rates of revasculaizations and new heart attacks was essentially the same in both groups.

This study joins other long-term studies that have demonstrated long-term improvements in heart attack patients treated with heart infusions with bone marrow-derived stem cells.  The REPAIR-AMI clinical trial, which examined 204 heart attack patients, showed stable, long-term benefits that lasted for at least two years for those patients who had been treated with infusions of bone marrow stem cells.  Other studies have not found no significant differences between heart attack patients treated with standard care and those who also received bone marrow infusions.  However, there are probable explanations for many of these failures.  The ASTAMI study that failed to show significant differences between the two groups not only transplanted a lower number of cells than this present study and the successful REPAIR-AMI study.  Secondly, the negative FINCELL study used patients whose average ejection fractions were 59% ± 11%.  Clinical studies that have tested bone marrow heart infusions have established that those patients with lower ejection fractions are helped the most by them.  This is the case of the negative HEBE study; the patients with the lowest ejection fraction showed the greatest improvements relative to the control group, but these improvements were swamped out by those with higher ejection fractions that were not helped nearly as much.  Third, the meta-analysis of Martin-Rendon showed that the best time period to treat heart attack patients was 4-7 days after the heart attack.  In the HEBE study, patients received bone marrow infusions 7 days after angioplasty.  How soon after the heart attack was the angioplasty performed?  This is not reported, probably because it varied from patient to patient.  Nevertheless, this places the treatment outside the optimum established by other experiments.

Thus, once again, we see that bone marrow treatments for hearts are safe, and effective, and they convey long-term benefits to patients who receive them.  Much work remains, since only some people consistently benefit from these treatments.  Why is this the case?  Only more work will tell.

Stem Cell Treatments for Hearing Loss?


In mammals, loss of hearing is irreversible because neurons in the cochlea and so-called “hair cells” do not regenerate. More than half of the population over the age of 60 suffers from severe hearing loss. Replacing cells in the inner is an important goal for regenerative medicine.

The ear consists of three main compartments. The outer ear consists of the cup-like structure on the sides of our heads called “pinnae,” and the opening to the middle called “external auditory meatus” (EAM). The EAM terminates at the eardrum. Behind the ear lies the middle ear, inside which is housed three small bones called the “auditory ossicles.” The auditory ossicles are attached to the eardrum and when the eardrum vibrates as a result of air pressure disturbances caused by sound waves traveling through the air, the ossicles vibrate with the eardrum and set up a series of vibrations on the other side of the middle ear where the ossicles are attached to the so-called oval window.

The vibrations that occur at the oval window are passed into the cochlea, which is derived from the Latin word for snail-shell.

This coiled structure contains two main compartments, one of which extends throughout the cochlea, and the other of which surrounds this central compartment.  The central compartment is called the scala media, and the surrounding compartments are the scala vestibuli (above) and the scala timpani (below).  The scala media contains an organ called the Organ of Corti, and this structure is responsible for producing the signals that are interpreted by the brain as hearing.

The organ of Corti consists of a series of cells with hair-like extensions that called “hair cells.” When the vibrations from the oval window are transmitted to the cochlea, the fluid in the scala vestibuli and scala timpani vibrates and these vibrations are transmitted into the scala media.  The vibrations of the scala media causes a membrane above the hair cells, called the “tectorial membrane” to vibrate and this vibrating tectorial membrane, into which the tips of the hair cells are embedded, moves the hair cell extensions back and forth.  The louder the sound, the greater the degree to which the tectorial membrane vibrates.  Also, the frequency of the sound varies the type of vibrations in the scala media and each hair cell releases neurotransmitters to the neurons that connect with them only when vibrations of the proper frequency activate them.  These activated hair cells release neurotransmitters to the connecting neurons and these neurons take those signals to the brain where they are interpreted and turned into sound sensations.

Sensoineural deafness results from the death of neurons that innervate the hair cells, or the hair cells themselves.  Replacing the hair cells or the connecting neurons is one of the main goals of regenerative medicine.  To that end, several labs have injected neural stem cells, embryonic stem cells or neural stem cells made from embryonic stem cells into the cochleas of deaf animals.  These experiments have shown that injected embryonic stem cells can survive when injected into the cochlea (see Hildebrand MS, et al., J Assoc Res Otolaryngol.2005 Dec;6(4):341-54), and injected cells can even differentiate into cell types that are specific for the cochlea (see Coleman B, et al., Cell Transplant.2006;15(5):369-80).  Inject neural stem cells made from embryonic stem cells can even extend axons that move into the cochlea and make contact with hair cells (Corrales CE,, et al. J Neurobiol. 2006 Nov;66(13):1489-500).  The difficulty with these cells is that they are not originally from the ear and might not differentiate fully into the tissue they are trying to repair.

What is a better cell type for repairing the inner ear?  Fetal ears contain a stem cell population that was identified in 2007 by Wei Chen in Marcelo Rivolta’s lab from the University of Sheffield (Chen et al., Hear Res. 2007 Nov;233(1-2):23-9).  Rivolta’s lab has also designed protocols for isolating and expanding these cells in vitro.  These fetal auditory stem cells form structures that resembled those found in organ of Corti.  The electrophysiological profiles of these cells also greatly resembled those observed in organ of Corti cells.  Also, the auditory stem cells expressed a great many of the genes found in developing inner ear cells when induced with various growth factors and small molecules (Chen W, et al., Stem Cells. 2009 May;27(5):1196-204).

These cell lines be used to cure deafness?  That is a different question, but they can certainly be used as a model system for drug screening, toxicity, and testing therapies to cure hearing loss.  Treating defects of the inner ear have many challenges, and while such cells are a first start, they represent the beginning of what might be a viable source of treatment for hearing loss.

There is also much to say about the fetal source of these cells.  Fetal cells were used to treat Parkinson’s disease and Huntington’s chorea.  The use of brain tissue from aborted fetuses is gruesome to say the least, and while these experiments did not cause the death of the pre-born baby, they represent an acceptance of the killing of unborn children that is execrable.  While the knowledge that has been gained from these experiments is certainly useful, it was gained over the bodies of innocent victims of children who were killed because they were an inconvenience.  This should disturb and sicken us.  The fact that it often doesn’t is a testimony of our moral deafness as a nation.

Bone Marrow Stem Cells Make the Blind (Lab Animals) See


There has been a great deal of discussion of embryonic stem cell-derived retinal pigment cells and the transplantation of these cells into the retinas of two human patients who subsequently showed improvements in their vision. One of these patients had a degenerative eye disease called “Stargardt’s macular dystrophy,” and the other had dry, age-related macular degeneration.

Stargardt Macular Dystrophy (SMD) is one of the main causes of eyesight loss in younger patients (affects 1/10,000 children), and retinal damage begins somewhere between the ages of 6 – 20. Visual impairment is usually not obvious to the patient until ages 30 – 40. Children with SMD usually notice that they have difficulty reading. They may also complain that they see gray, black or hazy spots in the center of their vision. Additionally, SDM patients take a longer time to adjust between light and dark environments.

Mutations in the ABCA4 gene seem to be responsible for most cases of SDM.  Defects in ABCA4 prevent the photoreceptors from disposing of toxic waste products that accumulate within build up in the disc space of the photoreceptors.  These toxic waste products are a consequence of housing light-absorbing pigments, and intense light exposure.  The pigment, all-trans retinal, binds to membrane lipids, and this forms a compound called NRPE (short for N retinylidene-phosphatidylethanoliamne, which is a mouth-full).  The protein encoded by ABCA4 moves NRPE into the cytoplasm of the photoreceptor cells, but if ABCA4 is not functional, NRPE accumulates in the disc space and binds more all-trans retinal to form a toxic sludge called “lipofuscin.”  Lipofuscin is taken up from the photoreceptors by the RPE cells and it kills them (see Koenekoop RK. The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review. Ophthal Genet. 2003;24(2):75–80).  Mutations in other genes (ELOVl4, PROM1, and CNGB3) also cause SDM.

Dry, age-related macular degeneration is associated with the formation of small yellow deposits in the retina known as “drusen.”  Drusen formation leads to a thinning and drying of the macula that eventually causes the macula to lose its function.  There is loss of central vision and the amount of vision loss is directly related to the amount of drusen that forms.  Early stages of age-related macular degeneration is associated with minimal visual impairment, but is characterized by large drusen and abnormalities in the macula.  Drusen accumulates near the basement membrane of the retinal pigment epithelium.  Almost everyone over the age of 50 has at least one small druse deposit in one or both eyes.  Only those eyes with large drusen deposits are at risk for late age-related macular degeneration.

All of this is to say that these diseases are progressive.  They have no cure and little can be done for treatment.  Secondly, people rarely get better.  However, both patients in this study showed quantifiable improvements.  The patient with age-related macular degeneration went from being able to see 21 letters in the visual acuity chart (20/500 vision for the patient, with 20/20 being perfect vision) to 28 letters (20/320).  This improvement remained stable after 6 weeks.  The patient with SMD was able to detect hand motions only, but after the stem cell injection, she could count fingers and see one letter in the eye chart by week 2, and was able to see five letters (20/800) after 4 weeks.  She also was able to see colors and contrast better and had better dark adaptation in the treated eye.

Now there are some caveats for this report.  First of all, the patient with SMD showed distinct structural improvements in the retina of the injected eye.  This patient also had distinct improvements in visual acuity.  However, the patient with dry, age-related macular dystrophy had no detectable structural improvements in the injected eye. The paper states, “Despite the lack of anatomical evidence, the patient with macular degeneration had functional improvements.”  Additionally, the non-injected eye also showed some visual improvements.  Note the words of the paper:  “Confounding these apparent functional gains in the study eye, we also detected mild visual function increases in the fellow eye of the patient with age-related macular degeneration during the postoperative period.”  Therefore, this experiment is highly preliminary and has equivocal results.  The SMD patient does show recognizable improvements, but this is only one patient.

While we are considering the efficacy of embryonic stem cells in the treatment of retinal degenerative diseases, a paper that was published in 2009 shows that bone marrow stem cells that have a cell surface marker celled “CD133” can become retinal pigment (RPE) cells.  This paper was published in the journal “Stem Cells,” and the principal author was Jeffrey Harris who did his work in the laboratory of Edward W. Scott at the University of Florida.  These cells were extracted from the bone marrow of mice and implanted into the retinas of albino mice.  Since the donor mice had pigmented skin and fur coats, the bone marrow cells were capable of making pigmented cells.  Once the CD133 cells were implanted, they survived and became pigmented.  When examined in postmortem sections, it was exceedingly clear that the transplanted CD133 cells expressed RPE-specific genes and assumed a RPE-like morphology.  Additionally, the implanted bone marrow cells also contributed functional recovery of retina.  A second set of experiments showed that human CD133 cells from umbilical cord could also integrate into mouse retinas and differentiate into RPEs.

This paper shows that embryonic stem cells are probably not necessary for retinal repair of RPE-based retinal degeneration.  Umbilical cord CD133 stem cells or bone marrow stem cells can differentiate into RPEs when transplanted into the retina.  While this paper does not address whether or not such differentiation occurs in human patients, such results definitely warrant Phase I studies. Thus once again, embryonic stem cells seem not be necessary.

CADUCEUS Clinical Trial Shows that Cardiosphere-Derived Stem Cells Can Regrow Heart Muscle After a Heart Attack


Cedars-Sinai Heart Institute is home to the CADUCEUS clinical trial. CADUCEUS stands for cardiosphere-derived autologous stem cells to reverse ventricular dysfunction. In this clinical trial, patients who had experienced a heart attack (and had left ventricular ejection fractions between 25% – 45%) were split into two groups. One group was given standard medical care for a heart attack patient and the other group was given standard care plus heart-based stem cells known as CDCs, which is short for “Cardiosphere-Derived Cells.” Patients assigned to receive CDC infusions of 12-25 million cells into the infarct-related artery 1.5 – 3 months after the heart attack.

The results 6 months after the stem cell infusion revealed that none of the patients in either group had died, developed tumors in their hearts or had experienced any major adverse heart-related event. Also Magnetic Resonance Imaging analysis of patients from both groups showed that those patients treated with CDCs displayed reductions in the mass of the heart scar, and increases in living, heart muscle mass. Additionally, the ability of the region of the heart that had experienced the heart attack contracted better in those patients who had received the CDC infusion. Also, the thickness of the wall of the heart was thicker in those patients who had received CDC infusions. Unfortunately, changes in other heart-specific functions such as EDV (end-diastolic volume), ESV (end-systolic volume), and LVEF (left ventricular ejection fraction) did not differ between the two groups by 6 months, which is difficult to reconcile with the structural changes in the hearts. .

Inventor of the procedures and technology used in this study, Eduardo Marbán, MD, PhD, who is also the director of the Cedars-Sinai Heart Institute, noted, “While the primary goal of our study was to verify safety, we also looked for evidence that the treatment might dissolve scar and regrow lost heart muscle. This has never been accomplished before, despite a decade of cell therapy trials for patients with heart attacks. Now we have done it. The effects are substantial, and surprisingly larger in humans than they were in animal tests.”

Shlomo Melmed, MD, dean of the Cedars-Sinai medical faculty and the Helene A. and Philip E. Hixon Chair in Investigative Medicine added, “These results signal an approaching paradigm shift in the care of heart attack patients. In the past, all we could do was to try to minimize heart damage by promptly opening up an occluded artery. Now, this study shows there is a regenerative therapy that may actually reverse the damage caused by a heart attack.”

An initial part of this study was conducted in 2009. In that study, Marbán and his colleagues used a patient’s own heart tissue to grow specialized heart stem cells. These specialized stem cells were injected back into the patient’s heart in an effort to repair and re-grow healthy muscle in a heart that had been injured by a heart attack. This experiment, at that time, was the first of its kind.

The results of that initial study were quite encouraging. The 25 patients, who participated in the study, had an average age of 57 and had suffered heart attacks that left them with damaged heart muscle. Each patient underwent extensive imaging scans to precisely locate the exact location and severity of the scars generated by the heart attack. Patients were treated at Cedars-Sinai Heart Institute and at Johns Hopkins Hospital in Baltimore.

Of these patients, eight received conventional medical care for heart attack survivors (prescription medicine, exercise recommendations and dietary advice) and were the control patients in this study. The remaining 17 patients were randomized to receive the stem cells underwent a minimally invasive heart biopsy, under local anesthesia that utilized a catheter inserted through a vein in the patient’s neck. From this catheter, doctors removed small pieces of heart tissue, about half the size of a raisin, that were taken to Marbán’s laboratory at Cedars-Sinai, where they were subjected to culture methods invented by Marbán to grow and expand the heart-based stem cells.

During a second, minimally invasive [catheter] procedure, the expanded heart-derived cells were reintroduced into the patient’s coronary arteries. Patients who received stem cell treatments experienced an average of 50 percent reduction in their heart attack scars 12 months after infusion while patients who received standard medical management did not experience shrinkage in the damaged tissue.

Marbán explained, “This discovery challenges the conventional wisdom that, once established, scar is permanent and that, once lost, healthy heart muscle cannot be restored.”

This phase I study definitely shows that the CDC infusion procedure is safe, which warrants the expansion of this procedure to a phase 2 study.

Severe Shoulder Rotator Cuff Tear treated with Stem Cells


Shoulder rotator-cuff tears are painful and usually require surgery. Can stem cells treat such a condition? Regenexx has attempted to do just that with surprising success.

One patient who is called JS had a particularly bad rotator cuff tear that included a retraction of the rotator cuff muscle. Stem cell treatments might not help heal this tear since the two ends of the tendon or muscle need to be surgically pulled together for the stem cells to heal the tendon. When the tendon experiences a retracted tear of the rotator cuff muscle the two sides of the tear pull back like a rubber band. The tendon bunches up on either side of the tear, and it is difficult to envision how stem cells might heal such a tear, since tears where the two ends of the tear are close together can be filled in with stem cells that are precisely applied to the tear.

All of this changed, however, with the treatment of a patient known as JS. He had a 1.5 cm retracted tear from a weight-lifting injury. After reluctantly agreeing to treat him with a mesenchymal stem cell application, they placed the shoulder in a splint to bring the two ends of the tear closer together. Then JS received a Regenexx-SD procedure that was followed up with a Regenexx-SCP procedure. The Regenexx-SD procedure takes mesenchymal and hematopoietic stem cells from a bone marrow aspiration and then mixes them with a preparation of platelets taken from peripheral blood. Regenexx-SCP treatment uses a specific stem cell population from bone marrow (CD66e+ cells) that are mixed with a platelet-rich mixture from blood serum and injected into joints.

The Regenexx physicians had low expectations of this procedure, but to their surprise, JS reported a 99% improvement over the three months. A follow-up ultrasound demonstrated excellent healing with some fill-in of the retracted gap.  For MRIs for this patient’s shoulder before and after the treatment, see here.

One year after the shoulder stem cell injections, the improvement due to precision injections of the patient’s own stem cells is nothing short of amazing. The large gap in the rotator cuff  is now healed.  For MRI images of JS’s shoulder one year after the Regenexx treatment, see here.  JS did require a specialized treatment and bracing protocol unique to the Regenexx procedures and developed by Dr. Hanson. This is an example of stem-based orthopedic surgery at its best.

Forbes blogs About the FDA v. Regenexx Stem Cell Lawsuit That May Shape Our Medical Future


Gergana Koleva blogs about public health for Forbes. She has written an excellent piece on the FDA’s lawsuit against Regenexx in their quest to regulate (read shunt down) a procedure that uses a patient’s own stem cells to treat ailing joints. In this lawsuit, the FDA justified their action by stating that cells are chemicals, just like drugs, and therefore, the have the right to regulate them. Ms. Koleva rightly notes that this lawsuit will change the face of medicine and medical innovation in this country. Read her blog article here.

Rats with Premature Birth-Type Brain Damage Show Neurologic Improvement After Stem Cell Transplants


Can stem cells transferred into the brains of newly-born babies with brain damage reverse brain damage? A study presented at the Society for Maternal-Fetal Medicine’s annual meeting in Dallas, Texas, researchers suggests that such a treatment might actually work. In this study, early transplantation of human placenta-derived mesenchymal stem cells into the lateral ventricles of neonatal rats with birth-related brain damage is feasible in this animal model. The transplanted donor cells survive and migrate within the recipient’s brain. Researchers designed this study so that the rat’s brain damage would mimic the type of brain injury observed in infants with very low birth weight.

Preterm delivery is one of the major causes of neonatal brain damage. Despite all efforts to prevent it, survivors of premature birth often suffer from some kind of injury to the brain. Survivors of preterm labor often display cognitive, behavioral, attention related and/or socialization deficits in twenty-five to fifty percent of cases; and major motor deficits in five to ten percent of cases.

Those infants with very low birth weights compose the majority of neonatal encephalopathy Such infants present with hypoxia-ischemia (low oxygen delivery to the tissues, which results in cell death and tissue damage) and inflammation. Approximately 63,000 infants are born in the United States with a very low birth weight (one to five percent of all live births). In order to understand the pathology of very premature infants, and if stem cells could ameliorate their conditions, this study, Early Intracranial Mesenchymal Stem Cell Therapy After a Perinatal Rat Brain Damage, was undertaken. This study investigated the neuroprotective effects of transplanted mesenchymal stem cells in recently born rats that had brain injuries that mimicked those found in infants with a very low birth weight.

One of the study’s authors, Martin Müller, MD, of the University of Bern, Obstetrics and Gynecology, Bern, Switzerland, said: “Stem cells are a promising source for transplant after a brain injury because they have the ability to divide throughout life and grow into any one of the body’s more than 200 cell types, which can contribute to the ability to renew and repair tissues. In our study, the donor cells survived, homed and migrated in the recipient brains and neurologic improvement was detected.”

Examination of the level of brain damaged after mesenchymal stem cell treatment indicated that stem cells exerted a neuroprotective effect on the brain. The transplanted cells survived in the brain, homed to damaged areas and migrated throughout the recipient brains. Furthermore, a combination of mesenchymal stem cells and erythropoietin (the signaling molecule made by the kidneys to signal to the bone marrow to make more red blood cells) might work even better.

While this work is still ongoing, it shows that such stem treatments are feasible and exert some positive effects.

Osiris’ Prochymal Mesenchymal Stem Cell Formulation is Safe for Diabetes Treatments


The biotechnology company called Osiris Therapeutics, Inc. has developed an adult mesenchymal stem cell formulation it calls “Prochymal.” Osiris scientists have been busy subjecting Prochymal to a battery of clinical trials that include testing Prochymal as a treatment for chronic obstructive pulmonary disease, Crohn’s disease, myocardial infarction, and acute graft-versus-host disease. Now Osiris is in the process of testing Prochymal as a treatment for newly diagnosed diabetes mellitus.

This clinical trial transferred mesenchymal stem cells from unrelated adult donors into 63 pediatric and adult type diabetics to determine if such a transfer can slow the progression of this debilitating disease. Patients will randomly receive either the stem cells or a placebo. Thus far, no patients who have received the mesenchymal stem cell infusion have shown any adverse reactions, despite receiving the cells from unrelated donors and without any drugs to suppress the immune system. Additionally, no significant differences in insulin levels were observed between the placebo and the experimental group after one year of receiving the mesenchymal stem cell infusion. However, patients who had received Prochymal showed fewer severely low blood glucose concentrations hypoglycemic events) than those who had been given the placebo. The test is still ongoing, and all patients will be observed for another year.

The rationale behind this trial resides in the unique ability of mesenchymal stem cells to down-regulate the immune response. Because type 1 diabetes typically results from the patient’s immune system attacking and destroying the insulin-secreting beta cells found in the pancreatic islets, an influx of mesenchymal stem cells might be able to decelerate the destruction of the beta cells. This suppression of beta cell destruction might lead to the regeneration of the beta cells, since several stem cell populations in the pancreas and pancreatic ducts can differentiate into beta cells. Since, Prochymal is specifically designed to control inflammation, promote tissue regeneration and prevent the formation of scar tissue; it is a prime candidate agent to reduce the loss of beta cells at the onset of type 1 diabetes.

Jay Skyler, professor and medicine and deputy researcher of the Diabetes Research Institute at the University Of Miami Miller School Of Medicine commented, “This groundbreaking study in an important first step in the use of stem cells to potentially alter the course of type 1 diabetes. The ability to safely use stem cells from unrelated donors is an important finding of this study and provides new possibilities for further development and stem cell therapies for type 1 diabetes.”

Exercise Triggers Muscle Stem Cells


New findings from researchers from the University of Illinois showed that adult stem cells in muscle are responsive to exercise. This discovery might provide a link between exercise and muscle health, and could provide the impetus for therapeutic techniques that use muscle-specific stem cells to heal injured muscles and prevent or restore muscle loss with age.

Mesenchymal stem cells (MSCs) in skeletal muscles have been known to be important for muscle repair in response to injury. Experiments that demonstrate the roles of mesenchymal stem cells in muscle repair have use chemical-induced injuries that initiate damage muscle tissue and inflammation. However, exercise also stresses muscle, and a research group led by kinesiology and community health professor Marni Boppart investigated whether MSCs also responded to exercise-induced stress.

According to Boppart, “Since exercise can induce some injury as part of the remodeling process following mechanical strain, we wondered if MSC accumulation was a natural response to exercise and whether these cells contributed to the beneficial regeneration and growth process that occurs post-exercise.”

Boppart’s group found that muscle-based MSCs respond to mechanical strain. In fact, mice subjected to vigorous exercise showed robust accumulation after exercise. They also found that MSCs do not directly contribute to new muscle fibers, but, instead, they release growth factors that spur other cells in muscle to fuse and generate new muscle.

Boppart’s research group isolated muscle-based MSCs after the mice exercised, and then they stained the MSCs with a fluorescent marker and injected them into other mice to see how they coordinated with other muscle-building cells. In addition to examining MSCs in vivo, Boppart’s laboratory examined the response of MSCs to strain on different substrates. They discovered that MSC response is very sensitive to the mechanical environment, indicating that conditions under which muscles are strained affects the activity of the cells.

Boppart added, “We’ve identified an adult stem cell in muscle that may provide the basis for muscle health with exercise and enhanced muscle healing with rehabilitation/movement therapy. The fact that MSCs in muscle have the potential to release high concentrations of growth factor into the circulatory system during exercise also makes us wonder if they provide a critical link between enhanced whole-body health and participation in routine physical activity.”

Since, preliminary data suggest MSCs become deficient in muscle with age; the group hopes to determine if these cells contribute to the decline in muscle mass over a person’s lifetime. The team hopes to develop a combinatorial therapy that utilizes molecular and stem-cell-based strategies to prevent age-related muscle loss.

A New Hybrid Molecule Directs Mesenchymal Stem Cells To Increase Bone Formation and Bone Strength


Osteoporosis is a disease that affects bone and results from aging or a lack of estrogen. Osteoporotic bone is less dense than normal bone, and the loss of bone density leads a tendency for bones to fracture easily. In particular, the bones of the wrist, hip, or back can fracture and fortunately, bone scans can help diagnose osteoporosis earlier and earlier.

Typically, osteoporosis is treated by prescribing a group of drugs collectively known as the “bisphosphonates.” These drugs have a common mode of action that includes one of the two cells involved in bone remodeling and healing. Cells called “osteoblasts” act as bone-building cells. Osteoblasts come from bone marrow (the squishy stuff inside your long bones), and they make new bone called “osteoid” that consists of a protein called “collagen” and a few other proteins. Then they deposit calcium and other minerals onto the protein matrix. After filling a cavity with bone, the osteoblasts flatten and line the cavity where they regulate the movement of calcium into and from the bone. Some of the osteoblasts become trapped in the bone while it is being deposited and they extend long extensions and become known as “osteocytes.” Osteocytes monitor the bone health and signal when there are breaks in the bone.

Osteoblasts

The second cell involved in bone remodeling is the osteoclast. Osteoclasts are large cells with many nuclei that dissolve existing bone. When a bone is broken, the osteocytes signal to each other and recruit osteoclasts to the site of the bone break. Osteoclasts dissolve the broken bone, and this gives room to the osteoblasts so that they can deposit new bone. The activities of both cell types are essential for bone healing and remodeling. The activities of these two cell types are also very carefully regulated.

When osteoblast activity is too high, a disease called “osteopetrosis” ensues, and this disease squeezes out the bone marrow and prevents the synthesis of enough blood cells. When osteoclast activity is too high, osteoporosis ensues, and bone density decreases so that fractures are a genuine possibility. Bisphosphonates bind to the surface of osteoclasts and prevent them from destroying bone. However, since both osteoclasts and osteoblasts are required for proper bone health, bisphosphonates essentially cause bone deposition to come to a stand-still. For this reason, some people experience increased fractures on bisphosphonates. What is needed is a treatment that can reverse the thinning of the bones and increase bone density.

A very interesting study led by scientists at the UC Davis Heath System examined a mouse model of osteoporosis to test the efficacy of a new treatment that can potentially increase bone density. If the results of this study are confirmed by further work, it could revolutionize osteoporosis treatments. The UC Davis team developed a novel technique to enhance bone growth by injecting a specific molecule into the bloodstream that guides mesenchymal stem cells to bone surfaces. Once there, these stem cells differentiate into osteoblasts, which promote bone growth.

Wei Yao, the principal investigator and lead author of the study said: “There are many stem cells, even in elderly people, but they do not readily migrate to bone. Finding a molecule that attaches to stem cells and guides them to the targets we need is a real breakthrough.”

Even though there is a great deal of research to develop stem cell-based treatments for many conditions and injuries that range from peripheral artery disease and macular degeneration to blood disorders, skin wounds and diseased organs, directing stem cells to travel and adhere to the surface of bone for bone formation has been among the elusive goals in regenerative medicine. To accomplish this, Yao and others used a unique hybrid molecule, LLP2A-alendronate that consists of two parts: the LLP2A part that attaches to mesenchymal stem cells in the bone marrow, and a second part that consists of the bone-homing bisphosphonate-class drug, alendronate (trade name – Fosamax). LLP2A-alendronate was injected into the bloodstream, and it bound to the cell surfaces of mesenchymal stem cells in the bone marrow and directed those cells to the surfaces of bone, where the stem cells carried out their natural bone-formation and repair functions.

The study shows that stem-cell-binding molecules can be exploited to direct stem cells to therapeutic sites inside an animal. One author even said. It represents a very important step in making this type of stem cell therapy a reality.

Twelve weeks after the LLP2A-alendronate was injected into mice, bone mass in the femur (thigh bone) and vertebrae (in the spine) increased and bone strength improved compared to control mice who did not receive LLP2A-alendronate. The treated mice were older mice that normally showed a particular degree of bone loss, but with this treatment, they had improved bone formation, as did those that were models for menopause.

Even though alendronate is commonly prescribed to women with osteoporosis to reduce the risk of fracture, it was used in this study because it goes directly to the bone surface, where it slows the rate of bone breakdown. The alendronate dose in this experiment was very low and was, therefore, unlikely to have inhibited LLP2A’s therapeutic effect.

Co-investigator on the study and director of the UC Davis Musculoskeletal Diseases of Aging Research Group, Nancy Lane, noted: “For the first time, we may have potentially found a way to direct a person’s own stem cells to the bone surface where they can regenerate bone. This technique could become a revolutionary new therapy for osteoporosis as well as for other conditions that require new bone formation.”

Mesenchymal stem cells from bone marrow induce new bone remodeling, which thicken and strengthen bone. The potential use of this stem cell therapy is not limited to treating osteoporosis, since it may prove invaluable for other disorders and conditions that could benefit from enhanced bone rebuilding, which includes bone fractures, bone infections or cancer treatments.

Jan Nolta, professor of internal medicine, an author of the study and director of the UC Davis Institute for Regenerative Cures opined, “These results are very promising for translating into human therapy. We have shown this potential therapy is effective in rodents, and our goal now is to move it into clinical trials.”

Paper citation: “Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass;” Min Guan, Wei Yao, Nancy E Lane et al.; Nature Medicine, 2012; DOI: 10.1038/nm.2665.

Whitehead Scientists Discover Critical Role Played By One Enzyme In Embryonic Stem Cell Differentiation


Cells use gene expression programs to respond to external stimuli and maintain their present form and identity. Genes are stretches of DNA that encode a protein or RNA. Gene expression requires DNA sequences directly attached to the gene, and these sequences are called the “promoter” of the gene. The promoter is the binding site for the enzyme that executes the first step of gene expression. That enzyme is called “RNA polymerase.” RNA polymerase binds to the promoter and initiates the synthesis of an RNA copy of the gene, and process by which an RNA copy of the gene is synthesized rom the DNA template is called “Transcription.”

If the gene encodes a protein, the RNA is processed and sent from the nucleus of the cell to the cytoplasm, where protein/RNA complexes called “ribosomes” use the sequence in the RNA to make proteins that have amino acid sequences. Some genes, however, encode RNA molecules that are not used to direct the synthesis of protein, but are used for some other purpose.

Whatever the case, cells have a great deal of DNA in their nuclei. Almost every human cell, for example, contains so much DNA that if the DNA in one human cell was laid out end-to-end, it would stretch to a length of at least 1 meter. To pack all that DNA into the nucleus of a cell, the DNA is wound into a tight complex of DNA and proteins that is collectively called “chromatin.” Chromatin consists of DNA wound around proteins called histones, in ways that resemble the way thread is wound around a spool. These little histone spools are then wound into spirals that are then wound into a rosette of fibers. It is exceedingly for RNA polymerase to transcribe genes when they are wound into chromatin. How then are genes expressed? It turns out that particular proteins modify chromatin and cause it to loosen up so that RNA polymerase can access it.

Histone modifying proteins include those that encourage the formation of chromatin and tend to shut gene expression off (histone deacetylase, Polycomb-group proteins), and those that loosen chromatin and encourage gene expression (histone acetyl-transferases, histone methyltransferases). Therefore, we might expect to see such enzymes playing an important role in stem cell differentiation.

Therefore, we should not be surprised that stem cells researchers at the Whitehead Institute have discovered that a specific chromatin enzyme called lysine-specific demethylase 1 (LSD1) plays as embryonic stem cells differentiate into other cell types. Cell differentiation requires two key steps: 1) the genes active in the initial cell type must be deactivated; and 2) those genes important for the establishment of the new cell type must be activated. If the switch is not flawless, a transitioning cell may die or be driven to divide uncontrollably. Interestingly, LSD1 was known to be critical to development, but little was known about the key role it plays during differentiation, when cell-specific gene expression systems are switched on or off.

Paper author, Steve Bilodeau, who is also a postdoctoral research fellow in the laboratory of Whitehead Member, Richard Young, said; “We knew that cells express a new set of genes when the operating switch occurs. But this study shows it is also essential to shut off genes that were active in the prior cell state. If you don’t, the new cell is corrupted.”

Bilodeau and Warren Whyte, a Young lab graduate student and co-author, redefined LSD1’s role and described a previously unknown mechanism for silencing genes. They examined embryonic stem cell gene expression during differentiation and concentrated their efforts on those genes that must be shut off during differentiation. Whyte and Bilodeau found LSD1 was located on the promoters of those genes that had to be repressed in order for differentiation to occur. LSD1 was also found near DNA sequences called “enhancers,” which are associated with promoters and increase the ability of the promoters to activate gene expression.

What is LDS doing at the promoter and enhancer? When LSD1 receives the signal that the stem cell is going to differentiate, it transitions into an active conformation and silences those genes. Specifically, LSD1 hamstrings the ability of the enhancers of those genes to activate gene expression. With their enhancers rendered nonfunctional, transcription of these genes is silenced. While this occurs, other mechanisms switch on those genes necessary for the adoption of the new cell type.

Whyte added: “This reveals the critical function of LSD1 in cell differentiation. The enzyme decommissions the stem cell enhancers, thus allowing the new cell to function entirely within the parameters of the new operating system.”

Although this work focuses on one enzyme’s job in normal cells, Young sees broader implications, since LSD1 is a member of a class of molecules that regulate both gene activity and chromosome structure. Therefore, these findings about LSD1 could provide insights into how related regulators function. Similarly, understanding how a mechanism operates in normal cells provides a solid foundation for teasing apart what is going wrong in abnormal cells.

Young summed it up this way: This new knowledge brings us one important step closer to understanding defective operating systems in diseases such as cancer. And this may give us a new angle on drug development for these diseases.”

This work was published in “Enhancer decommissioning by LSD1 during embryonic stem cell differentiation;” Warren A. Whyte, Steve Bilodeau et al.; Nature, 2012; DOI: 10.1038/nature10805.