New Stem Cell Found in the Brain

A new type of stem cell has been discovered in the brain by research scientists at Lund University in Sweden. These cells have the ability to proliferate in culture and renew themselves, but the can also differentiate into several different types of brain-specific cell types. They can also form new brain cells and scientists hope that they develop methods to use these cells to design treatments that can potentially repair disease and injury in the brain.

This new stem cells was found when workers in the laboratory of Patrik Brundin analyzed biopsies from the brain. These tissues had stem cells located around small blood vessels in the brain. While the physiological function of these cells are still presently unclear, their ability to form several different cell types in the brain suggests that they might help rebuild the brain after specific traumas or help remodel the brain in response to learning and adaptation to new conditions.

Patrik Brundin, M.D., Ph.D., who is the Jay Van Andel Endowed Chair in Parkinson’s Research at Van Andel Research Institute (VARI), Head of the Neuronal Survival Unit at Lund University and senior author of the study, said “A similar cell type has been identified in several other organs where it can promote regeneration of muscle, bone, cartilage and adipose tissue.”

Such blood vessel-associated cells are usually a population of mesenchymal stem cells, and in other organs, these cells contribute to repair and wound healing. Brundin and his colleagues think that these curative properties might also apply to these cells in the brain. The next step is to try to control and enhance stem cell-mediated healing properties, and then manipulate these cells so that they can be used for certain therapies in the brain.

“Our findings show that the cell capacity is much larger than we originally thought, and that these cells are very versatile,” said Gesine Paul-Visse, Ph.D., Associate Professor of Neuroscience at Lund University and the study’s primary author. “Most interesting is their ability to form neuronal cells, but they can also be developed for other cell types. The results contribute to better understanding of how brain cell plasticity works and opens up new opportunities to exploit these very features.”

This study, was published in the journal PLoS ONE, and is of interest to a broad spectrum of brain research. Future possible therapeutic targets range from neurodegenerative diseases to stroke.

“We hope that our findings may lead to a new and better understanding of the brain’s own repair mechanisms,” said Dr. Paul-Visse. “Ultimately the goal is to strengthen these mechanisms and develop new treatments that can repair the diseased brain.”

Cardiosphere-Derived Stem Cells Improve Function in the Infarcted Rat Heart for 16 Weeks

Stem cells in the heart were first identified in 2003 and since then there has been a great deal of excitement about their potential for use in regenerative therapy. Two clinical trials have been conducted with heart-specific stem cells, SCIPIO and CADUCEUS. Both of these clinical trials were very successful, and further trials might, hopefully, bring this work to the operating room. In the meantime, further work with cardiac stem cells in animals models is necessary in order to clarify the mechanism by which these cells improve the function of an ailing heart.

In light of these findings, Carolyn Carr in Kieran Clarke’s lab at the University of Oxford has published an interesting paper in collaboration with Georgina Ellison at the University La Sapienza in Rome. This paper used magnetic resonance imaging (MRI) to examine rats hearts that had suffered a heart attack, and then had been implanted with cardiosphere-derived stem cells (CDCs). The study showed that CDCs not only improve the function of the heart, but do so for an extended period of time.

Cardiospheres are small balls of cells that have been grown from stem cells that are harvested from the heart. The cells are easily isolated from the upper chambers of the heart (atria; see Messina et al., Circulation Research 95 (2004): 911-24), and when the cells grow, they beat in synchrony and strongly attach to each other. The strong attachment of the cells for each other causes them to grow into small balls of cells that can be implanted into a living heart.

When the CDCs are grown in culture, they generate a mixed population of cells that include progenitor cells that express a gene called “c-kit,” and heart-specific mesenchymal cells that express a surface proteins called “CD90.” Additionally, there are other cells in cultured cardiac cells called explant derived cells (EDCs), which are round and refractory. There is a fair amount of debate as to the nature of these EDCs. Three main papers have demonstrated that EDCs neither form heart muscle nor survive when they are implanted into a living heart (see Shenje LT,et al., Lineage tracing of cardiac explant derived cells. PLoS One. 3(4), 2008:e1929; & Li Z, et al., Imaging survival and function of transplanted cardiac resident stem cells. J Am Coll Cardiol. 53(14), 2009 :1229-40; & Andersen DC,et al., Murine “cardiospheres” are not a source of stem cells with cardiomyogenic potential, Stem Cells. 27(7) 2009, 1571-81). However, other papers have established that EDCs and CDCs can grow in culture and renew themselves (Davis et al., PLoS ONE 4 (2009): e7195; & Chimenti et al., Circulation Research 106 (2010): 971-80; & Davis et al., Journal of Molecular and Cellular Cardiology 49 (2010): 312-21).  Other studies have even shown that implantation of EDCs can improve the function of the heart after a heart attack (Shintani, et al., Journal of Molecular and Cell Cardiology 47 (2009): 288-295).

If you are confused by all these conflicting data, join the club because I am too.  The clinical studies say that CDCs work to fix a heart, but these animals studies leave us asking if the whole thing is not just a ponzi scheme.  This shows us why more animal studies are necessary.  Just because something works, does not mean that we know how it works.

In the Carr study, CDCs were isolated from rats and cultured.  The cultured tissue yielded CDCs and EDCs that grew well in culture.  The CDCs were then labeled with magnetic microspheres to make them easier to detect after transplantation.  The CDCs were able to differentiate into heart muscle in culture as determined by their expression of proteins and genes that are specific to heart muscle.  Then the rats were given heart attacks, and seven of them received CDC infusions and another seven rats did not.  All rats underwent perfusion of the heart, and two days after perfusion, all rats were given CDCs in the tail vein (4 x 10[6] cells).  MRI was used to view the heart and assess its function, and to view the CDCs in the heart.

EDCs in the hands of these researchers not only grew and renewed themselves in culture, but they also formed cardiospheres.  Most of the cells were mesenchymal stem cells, but others were fibroblasts and a few others were stem cells (3%).  Cardiosphere-derived cells expressed several heart-specific genes.  It was also clear that labeling the cells with iron microspheres did not affect the cells.

MRI of the heart showed that the untreated hearts were in functional decline, but those that had been treated with CDCs had better functional readings, an a much smaller scar, and had less problems with heart wall that did not move.  MRI also showed that the CDCs had integrated into the heart muscle and were a part of the heart muscle wall.  They did not have the maturity of adult heart muscle cells in that they were poorly connected with the neighboring cells.  However, they did not die but were maintained in the heart for at least 16 weeks. Blood vessel density was also increased in the hearts of the rats that had received the CDC implantations.

Thus, this CDC population, which far more CD90-expressing mesenchymal cells (30%) and fewer c-Kit cells (12%) homed to the site of heart damage and retained for at least 16 weeks.  Furthermore, a large proportion of CDCs formed heart muscle, reduced scarring and increased blood vessel synthesis.  These features improved the function of the rat hearts and prevented them from deteriorating further.  These cells also survived in the heart and did not die.  This shows that the EDCs can differentiate into heart muscle cells and preserve long-term function in the infarcted heart.  Therefore, we have evidence that the cells in cardiac stem cells can contribute to a damaged heart and help regenerate dead heart muscle.

Eye Stem Cell Researcher on Whose Work the Human Clinical Trials Were Based Faked Data

A former Oregon Health Sciences University (OHSU) eye researcher, Peter Francis, has received a reprimand from the U.S. Office of Research Integrity (ORI). The reason for the reprimand is that an institutional investigation found Francis guilty of research misconduct. Specifically, Francis faked data for a grant application by writing about an experiment that he never did. Francis left the university at the end of the investigation, but now faces two years of supervised research and also any of his future proposals for federal research funding will be subjected to intense scrutiny.

The National Institutes of Health of NIH contains a division called the National Eye Institute, which funds a large proportion of the eye-based research in the United States. In a grant to the National Eye Institute, Francis claimed to have performed experiments that had not been done. Furthermore, he used that information to apply for more than one grant, according to a statement from the ORI.

John Dahlberg, the director of ORI’s division of investigative oversight commented, “The pressures to succeed are difficult, making it even more difficult to get funding.” Dahlberg also said that fabrication of results and data is “not uncommon,” but he also ominously noted that such an offense can be considered and prosecuted as a felony.

In the suspicious grant proposals, Francis claimed to have injected retinal pigment epithelial cells that were made from rhesus monkey embryonic stem cells into rats that had been genetically conditioned to retinal degeneration. The grant observed that those rats that had received the injected eye cells had better-preserved photoreceptor cells in comparison to rats that had received injections into their retinas that did not contain any cells. During the ORI investigation, Francis admitted that the experiment described in the grant had not been done before the submission of either grant proposal.

Francis had won the National Research prize for “Best Up-and-Coming Medical Research in the UK” in 2002 for his research into the genetic basis of congenital cataracts. More recently, the Foundation for Fighting Blindness and Research to Prevent Blindness awarded Francis a career development award. Francis also worked with Advanced Cell Technologies in Santa Monica, CA, which is one of the premier companies when it comes to developing cell-based therapies for retinal disease.

A search of the bio-informational site PubMed shows that Francis has published more than 75 articles. These include articles in prestigious journals such as Public Library of Science One, New England Journal of Medicine, and the Journal of the American Medical Association. The investigation found no misconduct with his published work while at OHSU’s Casey Eye Institute.

OHSU published a statement that appeared at the blog Retraction Watch. According to this statement, OHSU “takes research integrity matters very seriously,” and placed Francis on leave when his research projects were questioned. The university’s Scientific Integrity Committee performed the investigation and reported the results to the ORI. According to this published statement, at the end of the investigation, Francis decided to leave the university.

Francis has also entered into a voluntary settlement agreement with the Department of Health and Human Services, in which the NIH is housed. Effective March 29, 2012, for the next two years, all of Francis’ federally-funded research projects will be supervised by whatever institute decides to employ him. That institution must also verify that all results or methods that he submits in grant proposals are accurately reported. He will also have to recuse himself from serving in any advisory capacity to the Public Health Service on peer review and board committees or as a consultant.

NIH still recognizes the importance of the research currently being done in his former lab and has determined that a new principal investigator should be appointed to continue the work.

T Cells from Engineered Stem Cells Clear HIV from Infected Mice

A research team at UCLA has published a proof-of-principal study that demonstrates that human stem cells can be genetically engineered to create HIV-fighting cells. Their study was published on April 12, 2012 in the open journal PLoS Pathogens. This paper shows for the first time that engineering stem cells to form immune cells that specifically target HIV is effective in suppressing the virus in living tissues in an animal model. Lead investigator Scott G. Kitchen, an assistant professor of medicine in the division of hematology and oncology at the David Geffen School of Medicine at UCLA and a member of the UCLA AIDS Institute said: “We believe that this study lays the groundwork for the potential use of this type of approach in combating HIV infection in infected individuals, in hopes of eradicating the virus from the body.”

In previous research, this research group took a special group of immune cells known as CD8 cytotoxic T lymphocytes from an HIV-infected individual. CD8-positive T cells specifically attack virus-infected cells and destroy them so that they do not anymore virus. After they collected CD8 T cells from HIV-infected individuals, they grew them in culture. Next they established that these cells could attack and destroy HIV-infected cells in culture. The next step was to determine if these same cells could attack HIV-infected cells in a living organism.

When CD8 cells engage an infected cell, they use a molecule on their surfaces called the “T cell Receptor” (TCR). The TCR is an unusual protein that is encoded by a gene complex that consists of many copies of different versions of various regions of the TCR. During the development of the T cell one gene from each of these copies is chosen and spliced together with one copy from each of the other regions. The result is a TCR molecule that is unique to the T cell that makes it. These TCRs are able to bind to foreign substances and when they do, the T cell becomes activated. In the case of CD8 cytotoxic T cells, the binding of foreign substances on the surfaces of cells tell the cells that something dangerous is afoot inside the cell. Therefore, it secretes toxic chemicals that kill the cell.

In previous research carried out by the UCLA team, they isolated CD8 cytotoxic T lymphocytes from an HIV-infected individual and identified the genes from the TCR. Since the TCR guides the T cell in recognizing and killing HIV-infected cells, they reasoned that by making more of the T cells that recognize HIV-infected cells they could potentially provide HIV-infected animals with a way to clear the virus from the cell. The problem in HIV-infected individuals is that CD8 cells that are specific for HIV-infected cells do not exist in great enough quantities to clear the virus from the body.

To solve this problem, the researchers cloned the receptor and used this to genetically engineer human blood stem cells. They then placed the engineered stem cells into human thymus tissue that had been implanted in mice. Now the engineered T cells were observed interacting in a living organism. The engineered stem cells developed into a large population of mature, multi-functional HIV-specific CD8 cells that were able to specifically target HIV-infected cells with HIV proteins on their surfaces. Interestingly, the research group found that HIV-specific T cell receptors have to be matched to an individual in much the same way an organ is matched to a transplant patient.

In the current study, the UCLA group similarly engineered human blood stem cells and discovered that they can form mature T cells that can attack HIV in tissues where the virus resides and replicates. To show this they used the humanized mouse. This animal is a rodent with a human immune system. In these animals, HIV infection closely resembles the disease and its progression in humans.

Two-six weeks after introducing their engineered blood stem cells into the peripheral blood of the mouse, they found that the number of CD4 “helper” T cells — which become depleted as a result of HIV infection — increased and levels of HIV in the blood decreased. CD4 cells or T-helper cells are white blood cells that play a vital role in the immune system. These results indicated that the engineered cells were capable of developing and migrating to the organs to fight infection there.

There is, however, a potential weakness with this study: Human immune cells reconstituted at a lower level in the humanized mice than they would in humans, and as a result, the mice’s immune systems were mostly, though not completely, reconstructed. Because of this, HIV may be slower to accumulate mutations in the mice than in human hosts. Thus the use of multiple, engineered T cell receptors may be one way to adjust for the higher potential for HIV mutation in humans.

Kitchen sounded this optimistic note: “We believe that this is the first step in developing a more aggressive approach in correcting the defects in the human T cell responses that allow HIV to persist in infected people.”

Lipitor potentiates the healing capacity of mesenchymal stem cells in rats that have had heart attacks

Mesenchymal stem cells from bone marrow, fat, and other tissues show remarkable abilities to induce healing of various damaged tissues. Several experiments in laboratory animals have established the ability of these cells to improve heart function after a heart attack, and these results have also been confirmed by many clinical trials that have transplanted bone marrow-derived mesenchymal stem cells into the hearts of human patients after a heart attack.

The difficulty with such treatments is that the transplanted mesenchymal stem cells (MSCs) find themselves in a hostile environment that tends to cause them to die. Several enterprising scientists have tried to mitigate this problem by pre-conditioning the MSCs or by genetically engineering them to survive in a tough milieu such as the infarcted heart. These experimented have resulted in greater MSC survival and better recovery of heart function after a heart attack. A recent clinical trial has also shown that ischemia-treated MSCs show increased ability to improve heart function after a heart attack.

Into this fray comes a very interesting paper in the journal Acta Biochimica et Biophysica Sina that uses the anti-cholesterol drug atorvastatin (Lipitor) to increase the survival of implanted MSCs from fat. In this paper, Anping Cai and Dongdan Zheng at the First Affiliated Hospital of Sun Yat-sen University in Guangzhou, China, in the laboratory of Weiyi Mai extracted rat MSCs from fat and then cultured them to convert them into heart muscle cells. This culture system involves co-culturing fat MSCs with heart muscle cells. Previous work has shown that when MSCs are co-cultured with MSCs, the MSCs tend to start expressing heart muscle-specific genes and even spread out and start of look and act like heart muscle cells. By using antibodies to heart muscle-specific proteins, Cai and Zheng and co-workers showed that most of the cultured MSCs had, in fact, converted to heart muscle cells. These cells also beat in culture, attached to other heart muscle cells and beat in synchrony with them.

Next, they took Sprague-Dawley rats and used surgical procedures to give these rats heart attacks. Fourteen days later, the rats were divided into five groups: 1) the first groups were operated on, but they were not given heart attacks (Sham operated); 2) a group to which one million heart muscle-like MSCs were transplanted into the damaged heart; 3) a group to which one million heart muscle-like MSCs were transplanted plus Lipitor (10 mg/kg/day) 4) a group to which Lipitor was injected into the heart; 5) a group to which Lipitor and MSCs that had not been differentiated into heart muscle cells was injected into the heart. The rats were kept for four weeks and then the animals were sacrificed and their hearts were examined.

The results are rather interesting. The hearts were examined for the degree of inflammation in them. Hearts that have experienced a heart attack are full of inflammatory cells and the damaging chemicals made by them. The animals that had been given Lipitor had substantially less inflammation in their hearts. Also, the MSCs that had been converted into heart muscle cells showed the greatest degree of survival (35%), but the undifferentiated MSCs showed the highest amount of heart muscle-specific gene expression.

When heart function was examined in the five groups, the sham group showed the highest heart function, but all four other groups showed large decrease in heart function after the heart attack (no surprise there). However, all the groups showed some improvement except for the group that was not give any Lipitor. The Lipitor-only group improved after two weeks, but these improvements disappeared after two more weeks. The two groups implanted with Lipitor and MSCs showed the best improvements. Of these two groups, the rats implanted with Lipitor and differentiated MSCs showed the most improvements in heart function. While they were not the same as the sham-operated rats, their functional parameters were reasonably close, which indicates that under the direction of the differentiated MSCs, the hearts of these rats had experienced generous healing.

Thus MSCs can survive and heal the hearts of heart attack patients, but in order to help them survive in the hostile environment of the infarcted heart, drugs like Lipitor can help them survive. Lipitor seems to do this by decreasing inflammation in the damaged heart, which allows the MSCs to work their healing magic in the heart.

Pluripotency Genes Play Distinctly Different Roles in Mouse and Human Stem Cells

In 2000, scientists began identifying and characterizing proteins that help drive cells into the unique properties of embryonic stem cells. In August 2006, Shinya Yamanaka and colleagues at Kyoto University used four of these genes, all of which encoded transcription factors, Oct3/4, Sox2, c-Myc, and Klf4, to reprogram mouse skin cells to stem cells that exhibited most but not all of the properties of embryonic stem cells. The following year, Yamanaka’s team and James Thompson’s team at the University of Wisconsin–Madison concomitantly made induced pluripotent stem cells using Oct3/4, Sox2, Lin28, and Nanog.

Since this time, researchers have defined the function of these genes in detail in mouse embryonic stem cells (mESCs). Underneath this research was the assumption that the function of these genes in mESCs was the same in human cells. New research, however, has seriously challenged this notion.

Natalia Ivanova and her colleagues at Yale University in New Haven, CT, planned to study other genes that might be involved in maintaining pluripotency in human ESCs (hESCs). They used gene silencing to define the function of individual genes in hESCs. When they first silenced three of the genes that encode traditional pluripotency factors (Nanog, Oct4, and Sox2) they were very surprised to find that the stem cells did not act as they had expected. In mouse ESCs, Nanog, Oct4, and Sox2 bind the same locations on the genome and act cooperatively to maintain stem cell self-renewal and pluripotency. When any one of those factors is unable to perform its function, mESCs differentiate into extraembryonic tissues, such as placenta.

However, when they silenced each of the three factors in three different hESC lines, Ivanova and her team identified fundamentally different roles for these proteins in hESCs. First, the three factors prevent hESCs from transforming into non-embryonic tissues. Specifically, Nanog appears to prevent cells from becoming neuroectoderm, which is a tissue that eventually becomes the nervous system. Sox2 prevents cells from becoming mesoderm, which forms connective tissue and muscle.

Oct4 has varying roles depending on the presence of a protein called BMP4. In the absence of BMP4, inactivated Oct4 induces ectoderm, the outer layer of an embryo that forms the nervous system and skin, but in the presence of BMP4, it specifies extraembryonic cell fates. Also, Ivanova found that the trio does not function as a complex. Instead, Sox2 is the “odd man out,” since silencing Sox2 does not prevent hESCs from maintaining pluripotency in hESCs since Sox3, a related protein, compensates for Sox2’s absence. “It just shows you that in human [ESCs], repression by these three factors works in a completely different way” than in mouse ESCs, said Ivanova.

The findings could have a major impact on embryonic stem cell research. Mouse ESCs are traditionally easier to grow in culture than hESCs. One can grow sufficient mESCs for use in an experiment in two days, while it may take two months to grow enough hESCs. By understanding how human ESCs are regulated by these factors may help scientists fine-tune and speed up the expansion of hESCs in culture.

Also, because Nanog and Oct4 appear to be involved in the differentiation of the ectoderm, scientists may be able to use this knowledge to come up with new ways to inhibit these factors to improve differentiation of hESCs into neurons, which are quite valuable in a number of medical and scientific applications.

For their next project, the Yale team plans to get back to their original investigation of additional factors involved in hESC pluripotency. “Some other genes may be contributing to the regulation of self-renewal and differentiation,” said Ivanova. “We’re going to try to look at what these other players might be, to find out what else regulates this process.”