Firefly gene reporter allows scientists to track the exact fate of transplanted stem cells

Researchers from the University of Central Florida’s College of Medicine’s Burnett School of Biomedical Sciences (BSBS) and the Gazes Cardiac Research Institute at the Medical University of South Carolina have used firefly luciferase to track, in real-time, the differentiation of transplanted cardiac embryonic stem cells. They expressed the firefly luciferase reporter gene (luc) in a mouse embryonic stem (mES) cell line that constitutively expresses the enhanced yellow fluorescent protein (EYFP). Researchers were able to follow differentiation and proliferation of transplanted cardiac stem cells both in vivo and post-mortem with traditional histological assays. Steven N. Ebert, an associate professor at Burnett School of Biomedical Sciences, said, “Cardiac muscle cells are the holy grail in the cardiac world. We were able to clearly demonstrate that the stem cells were becoming heart muscle cells, and we could see that not only in histological sections, as is traditionally done; the firefly activity let us see them glow in vivo.”
With previous stem cell therapies, researchers could not monitor the activity of transplanted cells once they had been inserted into the subject. However, this dual reporter system allows researchers to observe the functionality of the cells and then even exactly document exactly where the transplanted cells wound up through histological examinations.
Ebert’s team previously attempted to track mES cells by loading the cells with super paramagnetic microparticle beads and using magnetic resonance imaging (MRI). Unfortunately even though this technique was able to determine the location of the cells and provide high-resolution images, but the beads migrated out of the target cells. Additionally, this method did not provide any new insights into the function of the cells. Ebert admitted, “Fundamentally, there’s a lot that we don’t know about how stem cells behave once they’ve been put back into the heart and become stimulated under natural conditions. An impetus behind the firefly strategy was to give us a way to see if the cells were actually becoming heart muscle by observing the functional activity.”
Although the current paper only documents the incorporation and differentiation of basic cardiac embryonic stem cells, Ebert says the reporter system can be adapted for a variety of clinical uses. His lab is testing the mES dual-reporter cell line to different application. The team is also developing new lines for more specialized investigation, such as examining the rehabilitation differences for damaged cardiac cells that produce adrenaline and those that do not.
Ebert concluded, “We’d like to put [the cardiac stem cells] into disease models where there’s actual damage to the heart and see if they can regenerate some of the cardiac muscle that has been lost. There’s a lot of potential in the field and a lot we don’t know yet, so there’s a lot more investigation to keep us busy for a while.”

Harvard stem cell researcher retracts two papers

Harvard Medical School stem researcher Amy Wagers, who works at the Joslin Diabetes Center has issued a retraction notice for a 2010 paper, and has also issued a “statement of concern” in which she intends to review a second paper from 2008. One of her former postdoctoral fellows, Shane Mayack, was the first author on both of these papers, and she maintains the validity of the results reported in these papers.

On October 13, 2010, Wagers and two other authors retracted their January 2010 paper “Systemic signals regulate ageing and rejuvenation of blood stem cell niches.” This paper reported data that they interpreted to mean that stem cell aging processes might be reversible but the notice references “serious concerns” with the data that led to that hypothesis, which prompted the retraction.  Wagers has even issued a statement that upon reviewing the data from the Nature paper, she immediately notified Nature, JDC, and HMS that she and her colleagues have begun to repeat the experiments to test their validity.

A point of concern for Wager is the appearance of very similar figures in both papers; Figure S3b from the Nature paper and Figure 6c from the Blood paper chart the frequency of blood stem cells but result from very different protocols.  A day later, Wagers published a notice of concern in the journal Blood that stated that a 2008 paper that she coauthored with Mayack is also under review for possible misreporting of the data.  No further information has yet been published concerning the study, “Osteolineage niche cells initiate hematopoietic stem cell mobilization,” which features a figure that is strikingly similar to one published in the retracted Nature paper.

Although Mayack has not personally commented on the retractions, her lawyer issued a statement saying that although Mayack realizes the data presentation was improperly handled, she believes the underlying research remains conclusive. Accordingly, Mayack did not sign the retraction notice. Further examination is ongoing to determine if the conclusions presented in either paper are still viable.

At this point it is hard to say, but it seems possible that this is just a mix-up.  Hopefully, work from Wager’s lab will determine if the conclusions are justified.  Academic misconduct is a problem is science these days.  The most notorious case is that of South Korean researcher Professor Woo Suk Hwang,  However it is exceedingly hard to say if this case falls under this category.  My feeling is that the jury is still out on this one.  It is better to give the researchers the benefit of the doubt, given the information in hand.  As things develop, there will be more to say.

The Sept4 Gene Prevents Stem Cells From Turning Cancerous

Stem cells, those prodigious precursors of all the tissues in our body, can make almost anything, given the right circumstances, but unfortunately that can also cause cancer sometimes. However research from Rockefeller University has shown that having too many stem cells, or stem cells that live too long, can increase the odds of developing cancer. By identifying a mechanism that regulates programmed cell death in precursor cells for blood, or the stem cells that make all blood-based cells (hematopoietic stem cells), these researchers have connected the death of such cells to a later susceptibility to develop tumors in mice. This work also provides evidence of the potentially carcinogenic downside to stem cell treatments, and suggests that there is a balance between stem cells’ regenerative power and their potentially lethality.
Research associate Maria Garcia-Fernandez and head of the Strang Laboratory of Apoptosis and Cancer Biology, Hermann Steller, and colleagues explored the activity of a gene called Sept4. Sept4 encodes a protein called ARTS that increases programmed cell death, or apoptosis, by antagonizing other proteins that prevent cell death. ARTS was originally discovered by Sarit Larisch while visiting Rockefeller, and is lacking in human leukemia and other cancers, which suggests that it suppresses tumors. To study the role of ARTS, the experimenters bred a line of mice genetically engineered to lack the Sept4 gene.
For several years, Garcia-Fernandez studied cells that lacked ARTS. He goal was to look for signs of trouble relating to cell death. However, in mature B and T cells, she could not find any. Then when she examined these cells at earlier and earlier times in their development she found crucial differences between the stem cells that gave birth to the progenitor cells that eventually became the mature B and T cells and the stem cells themselves. Newborn ARTS-deprived mice had about twice as many hematopoietic stem cells as their normal, ARTS-endowed peers. Furthermore, these stem cells were extraordinary in their ability to survive experimentally induced mutations. “The increase in the number of hematopoietic progenitor and stem cells in Sept4-deficient mice brings with it the possibility of accelerating the accumulation of mutations in stem cells,” says Garcia-Fernandez. “They have more stem cells with enhanced resistance to apoptosis. In the end, that leads to more cells accumulating mutations that cannot be eliminated.”
In fact the ARTS-deprived mice developed spontaneous tumors at about twice the rate of their controls. Herman Steller said, “We make a connection between apoptosis, stem cells and cancer that has not been made in this way before: this pathway is critically important in stem cell death and in reducing tumor risk…. The work supports the idea that the stem cell is the seed of the tumor and that the transition from a normal stem cell to a cancer stem cell involves increased resistance to apoptosis.”
ARTS interferes with molecules called inhibitor of apoptosis proteins (IAPs), which prevent cells from undergoing programmed cell death. When cells accumulate too much damage to work properly, programmed cell death ensues, and the damaged cell is replaced by a new cell. Programmed cell death is also called “apoptosis.” By inhibiting these IAPs, under the right circumstances ARTS helps to take the brakes off the process of apoptosis that normally permits the cell to die on schedule. Pharmaceutical companies are working to develop small molecule IAP antagonists, but this research is the first to show that inactivating a natural IAP antagonist actually causes tumors to grow, according to Steller. It also suggests that premature silencing of the Sept4/ARTS pathway at the stem cell level may herald cancer to come.
“This work not only defines the role of the ARTS gene in the underlying mechanism of mammalian tumor cell resistance to programmed cell death, but also links this gene to another hallmark of cancer, stem and progenitor cell proliferation,” said Marion Zatz, who oversees cell death grants, including Steller’s, at the NIH’s National Institute of General Medical Sciences. “The identification of the ARTS gene and its role in cancer cell death provides a potential target for new therapeutic approaches.”

Reduction of teratoma formation in embryonic stem cells

Tumor formation is an ongoing problem with embryonic stem cell treatments. The pluipotency of these cells and their ability to replicate themselves indefinitely that makes them so promising also gives them the ability to form tumors. These tumors, called teratomas, are mixtures of different types of tissues, and depending on where they are located, can be benign or rather dangerous. However, a new study performed at the University of California, San Diego (UCSD) shows that researchers have managed to reduce the size and number of them formed after implantation, which is an important first step to eradicating them completely.

Yang Xu, a biology professor at UCSD, said “To eliminate the residue of undifferentiated embryonic stem cells during the differentiation, it is important to elucidate the pathways that are important to maintain the self-renewal of embryonic stem cells.”

Xu and his colleagues identified a new signaling pathway that undifferentiated cells use for propagation. It depends upon the phosphorylation of the transcription factor called Nanog. It turns out that this phosphorylation (attachment of a phosphate group to a protein) is crucial for cell replication. By inhibiting this phosphorylation with small-molecules, they observed a stark decrease in resulting teratomas after transplantation. Apparently, Xu and colleagues observed a 70% reduction in the mass of teratomas, and also, there was a lack of self-renewing stem cells in those teratomas that did form.

Xu hopes to eventually eliminate teratoma altogether, which will make embryonic stem cells safer for use in human applications. See “Phosphorylation stabilizes Nanog by promoting its interaction with Pin1,” which was published on the PNAS website on July 6.

Stem Cells Used to Model Infant Birth Defect

One of the powerful uses of stem cells is their ability to model diseases. One recent report shows how stem cells can provide such a use.

Do you remember those strawberry-like birthmarks you have? They’re called hemangiomas. For the most part they are quite harmless. However, a stark minority of hemangiomas (~10%) can cause trouble if they occur within particular tissues. Hemangiomas consist of knots of blood vessels and if they form in the eyes, they can produce vision trouble, and if they form in lungs, they can cause circulation defects in the lungs.  Worse still, hemangiomas can continue growing and become tumors.

Traditionally, treatment of damaging hemangiomas has been with steroid drugs. There are problems with such treatments though. Steroids have many undesirable side effects, and they don’t always work. Even more troubling is the fact that the means by which steroids work are a complete mystery.

Now, workers at Children’s Hospital Boston recently shown that hemangiomas that form during development come from stem cells. Additionally, by growing these stem cells in the laboratory, scientists have been able to use them to understand how steroids treat hemangiomas (New England Journal of Medicine, March 18).

Hemangiomas are tangles of blood vessels that originate from specialized stem cells called hemangioma stem cells.  These cells can overgrow and produce little knobs of tissue.  Steroids work by shutting down the ability of hemangioma stem cells to grow.  In order to grow, hemangioma stem cells make a growth factor called vascular endothelial growth factor-A (VEGF-A).  This self-made growth factor increases the growth of the hemangioma stem cells, and steroids shut down the production of VEGF-A, thus inhibiting their growth.

Why is this exciting?  It turns out that VEGF-A is rather well understood, and there are other tools for inhibiting VEGF-A signaling.  This means that much safer drugs are available to treat hemangiomas.  Furthermore, not all hemangiomas respond to steroids (~30%).  This work suggests that hemangioma stem cells that form the hemangiomas may harbor mutations that causes them to overgrow and form knots of blood vessels.  Some of these mutant stem cells respond to steroids and some do not.  Some of the screening methods applied in this study may tell pathologists if steroid treatment will help or not.

By using stem cells from the tumor and manipulating them in the laboratory, scientists were able to learn basic things about common tumors and often manifest themselves as birthmarks.  This is hopefully only one of many different kinds of diseases that will be modeled through experiments on stem cells.

Reprogramming for Stem Cells

Regenerative medicine possesses tremendous potential. At the center of regenerative medicine is stem cells. How we derive stem cell lines is a central concern of this blog, but I remain convinced that embryonic stem cells do not represent the future of regenerative medicine. My reasons are manifold, but one of my greatest concerns is that embryonic stem cells (ESCs) require the death of human embryos. Human embryos are young human persons at the earliest stages of life. Destroying them is killing an innocent person. There has to be a better way.

Induced pluripotent stem cells (iPSCs) provide one possible alternative to ESCs, and while these cells show tremendous promise, they have their share of problems. While many of the safety concerns with these cells have been nicely addressed, others remain. Is there an even better way?

Hopefully the answer is “yes.” As it turns out, it is possible to reprogram cells to form another cell type without taking them through an embryonic-like stage. This strategy is called reprogramming, and it has been used by Doug Melton and co-workers in his lab at Harvard University to make insulin-making beta cells from other types of pancreas cells that do not normally make insulin (Qiao Zhou, et al., Nature 455, 627-632).  Likewise, the steroid dexamethasone can convert pancreatic cells into liver cells.

Now other researchers have found that small molecules can reprogram cells to become another cell type.  Small molecules can cause unwanted side effects, but James Chen, a chemical biologist at Stanford University School of Medicine, says they “are more in our comfort zone in terms of clinical therapies.”  Chen also said, “Chemists can synthesize and derivatize them, there are standard methods for determining compound pharmacokinetics, and the path to FDA [Food & Drug Administration] approval is well established.”

Researchers also favor small molecules because they have more control over dosage and delivery time with them than they do with genetic techniques.

In 2007, Sheng Ding, chemical biologist at Scripps Research Institute, reported the first small molecule that could substitute for one of the four reprogramming transcription-factor genes. Researchers continue to identify small molecules that can replace one, two, or three of the four reprogramming factors. Among the newest transcription-factor gene stand-ins are molecules such as the lactam kenpaullone from Peter Schultz’s laboratory at Scripps and the heterocyclic RepSox from Lee Rubin and Doug Melton at Harvard Stem Cell Institute (Proc. Nat. Acad. Sci. USA 2009, 106, 8912; Cell Stem Cell 2009, 5, 491).

Reprogramming might be able to do great things.  Out bodies are filled with stem cells.  We just need to know how to manipulate them.