Scar-less Healing in the Fetus

In early fetal development, skin wounds undergo regeneration and healing without scar formation. Unfortunately, this wound healing mechanism later disappears, but by studying the fetal stem cells capable of this scarless wound healing, researchers may be able to apply these mechanisms to develop cell-based approaches able to minimize scarring in adult wounds.

Michael Longaker, Peter Lorenz, and co-authors from Stanford University School of Medicine and John A. Burns School of Medicine, University of Hawaii, Honolulu, describe a new stem cell that has been identified in fetal skin and blood that may have a role in scarless wound healing. In the article “The Role of Stem Cells During Scarless Skin Wound Healing,” the authors propose future directions for research to characterize the differences in wound healing mechanisms between fetal and adult skin-specific stem cells.

“This work comes from the pioneers in the field and delineates the opportunities towards scarless healing in adults,” says Editor-in-Chief Chandan K. Sen, PhD, Professor of Surgery and Director of the Comprehensive Wound Center and the Center for Regenerative Medicine and Cell-Based Therapies at The Ohio State University Wexner Medical Center, Columbus, OH.

Restoring Muscle Strength in Aging Muscle

Unfortunately, muscle tone and strength decrease as we age. You can work out at the gym all you want. Eventually the relentless march and deterioration of age catches up with even the most avid athlete. However, a Stanford University group believes that they might have discovered why this happens and new cell targets to help reverse it.

According to Helen Blau (the doyen of muscle research), over time, stem cells that help repair damaged muscle cells after injury are less able to do so. This explains why regaining strength and recovering from a muscle injury gets more difficult with age. Blau and her team published their results in the journal Nature Medicine.

Fortunately, Blau’s study also suggests a way to make older muscle stem cells function more like younger ones. The caveat is that research in mice often doesn’t translate to humans. Therefore more work is necessary in order to determine if this technique could ever be used in people.

“In the past, it’s been thought that muscle stem cells themselves don’t change with age, and that any loss of function is primarily due to external factors in the cells’ environment,” study senior author Helen Blau, director of Stanford’s Baxter Laboratory for Stem Cell Biology, said in a university news release.

“However, when we isolated stem cells from older mice, we found that they exhibit profound changes with age,” said Blau, a professor of microbiology and immunology at the university. “Two-thirds of the cells are dysfunctional when compared to those from younger mice, and the defect persists even when transplanted into young muscles.”

The research also revealed, however, that there is a defect specific to old muscle stem cells that can be corrected, which allowed scientists to rejuvenate these stem cells.

“Most exciting is that we also discovered a way to overcome the defect,” Blau said. “As a result, we have a new therapeutic target that could one day be used to help elderly human patients repair muscle damage.”

The muscle stem cells in 2-year-old mice are the equivalent of those found in 80-years-old humans. In the course of their study, Blau and her team found that many muscle stem cells from these mice had increased activity in a certain biological pathway (p38α and p38β mitogen-activated kinase pathways, for those who are interested) that inhibits the production of the stem cells.

Drugs that block this pathway in old stem cells, however, allowed the aged stem cells to make a larger number of new cells that could effectively repair muscle damage.

According to Blau: “In mice, we can take cells from an old animal, treat them for seven days — during which time their numbers expand as much as 60-fold — and then return them to injured muscles in old animals to facilitate their repair.”

Once the mice received their rejuvenated muscle stem cells, the researchers tested their muscle strength with assistance from co-author Scott Delp, a professor in the School of Engineering, who has developed a way to measure muscle strength in animals that underwent stem cell therapy for muscle injuries.

Study lead author Benjamin Cosgrove, a postdoctoral scholar at the university, said: “We were able to show that transplantation of the old, treated muscle stem cell population repaired the damage and restored strength to injured muscles of old mice. Two months after transplantation, these muscles exhibited forces equivalent to young, uninjured muscles. This was the most encouraging finding of all.”

The study’s authors said they plan to continue their research to determine if people could benefit from this technique.

“If we could isolate the stem cells from an elderly person, expose them in culture to the proper conditions to rejuvenate them and transfer them back into a site of muscle injury, we may be able to use the person’s own cells to aid recovery from trauma or to prevent localized muscle atrophy and weakness due to broken bones,” Blau said.

“This really opens a whole new avenue to enhance the repair of specific muscles in the elderly, especially after an injury,” she said. “Our data pave the way for such a stem cell therapy.”

Training Stem Cells to Differentiate Properly

Pluripotent stem cells have the ability to differentiate into a whole host of adult cell types. Unfortunately this ability to differentiate into any adult cell type also comes with it the tendency to form tumors. Controlling stem cell differentiation requires that you give a little “push” in the right direction. What is the nature of that push? It varies from stem cell to stem cell and it also depends on what type of cell you want you stem cells to make. Therefore, pluripotent stem cell differentiation is sometimes a matter of art as much as a matter of science.

A research group at Stanford University School of Medicine have designed an experimental protocol that uses the signals in the body to direct the differentiation of stem cells to a desired end.

Stanford University professor Michael Longaker, who is also the director of the Institute for Stem Cell Biology and Regenerative Medicine at Stanford University, explained it this way: “Before we can use these cells, we have to differentiate or ‘coach,’ them down a specific developmental pathway.” Longaker continued: “But there’s always a question as to exactly how to do that, and how many developmental doors we have to close before we can use the cells. In this study, we found that, with appropriate environmental cues, we could let the body do the work.”

Allowing the patient’s body to direct differentiation of pluripotent stem cells could potentially speed approval of stem cell-based treatments by the US Food and Drug Administration (FDA). If Longeker’s protocol pans out, it could eliminate long period of extended laboratory manipulation in order to force stem cells to differentiate into the desired cell type.

“Once we identify the key proteins and signals coaching the tissue within the body, we can try to mimic then when we use the stem cells,” said Longaker. “Just as the shape of water is determined by its container, cells respond to external cues. For example, in the future, if you want to replace a failing liver, you could put cells in a scaffold or microenvironment that strongly promotes liver cell differentiation and place the cell-seeded scaffold into the liver to let them differentiate in the optimal macroenvironment.”

Longaker does not work on liver, but bone. As a pedatric plastic and reconstructive surgeon who specializes in craniofacial malformations, finding ways to coax pluripotent stem cells to make bone is his research Holy Grail. “Imagin being able to treat children and adults who require craniofacial skeletal reconstruction, not with surgery, but with stem cells,” opined Longaker.

In this experiment, Longaker and his colleagues removed a four-millimeter circle of bone taken from the skulls of anesthetized mice and implanted a tiny, artificial scaffold coated with a bone-promoting protein called BMP-2 (bone morphogen protein-2) that was seeded with one million human pluripotent stem cells.

According to Longaker, these implants formed bone and repaired the defect in the skulls of the mice even the original stem cells were not differentiated when added to the wound. These human stem cells made human bone that was then replaced by mouse bone as time progressed. This shows that the repair was physiologically normal.

This bone growth was stimulated by the presence of BMP-2 and the microenvironment that induced the stem cells to differentiate into bone-making cells that made normal bone.

In this experiment, Longaker and his group used human embryonic stem cells and induced pluripotent stem cells and both stem types seemed to work equally well at repairing the skull defect.

Teratomas (tumors made by pluripotent stem cells) were observed but only rarely (two of the 42 animals that received the stem cell implants developed tumors). Interestingly, the few teratomas that formed developed in two laboratory animals that received embryonic stem cell implants and not induced pluripotent stem cell implants. This is surprising, since most stem cells researchers consider induced pluripotent stem cells to be more tumorigenic than embryonic stem cells. Standard tests of these stem cells (implantation under the kidneys of immunodeficient mice) showed that they did produce teratomas under these conditions.

Longaker commented: “We still have work to do to completely eliminate teratoma formation, but we are highly encouraged.” Longaker also thinks that by combining this technique with other strategies, he and his group might be able to completely prevent teratoma formation. For example, including other cell types that can act as shepherds for the stem cells as they differentiate into the desired cell type can also increase differentiation into a desired cell type.

Longaker said, “I want to see how broadly applicable this technique may be.” He was referring to tissues that do not heal well. For example, cartilage heals very poorly if at all. Longaker wonders if you could “add some cells that can form replacement tissue in this macroenvironment while you’re already looking at the joint.”

Protein Induction of Pluripotent Stem Cells Made More Efficient

Clinicians and stem cells scientists have been hopeful but also quite cautious about the use of induced pluripotent stem cells iPSCs in human treatments. One of the primary concerns in the use of viral vectors that insert themselves into the genome of the cells they infect. Such insertions can create activating mutations or insertional inactivation mutations that can transform cells into tumors.

However, scientists at Stanford University School of Medicine have designed a safer way to make iPSCs that is also very efficient. This method is an extension of a protocol that has already been tried; treating the cells with recombinant proteins that can pass through the cell membrane and transform the cells into iPSCs without the use of viruses. Unfortunately, this protocol has proven to be rather inefficient relative to methods that use genetically engineered viruses.

The Stanford researchers discovered that viruses were not simply burrowing into cells to deposit genes. According to John Cooke, MD, PhD, professor of medicine and associate director of the Stanford Cardiovascular Institute and senior author of this work: “It had been thought that the virus served simply as a Trojan horse to deliver the genes into the cell. Now we know that the virus causes the cell to loosen its chromatin and make the DNA available for the changes necessary for it to revert to the pluripotent state.”

The derivation of iPSCs does not require the destruction of embryos. and therefore, offer an ethical alternative to embryonic stem cells (ESCs). Instead of using embryos, iPSCs are made from adult cells that have been genetically engineered to overexpress four different genes (Oct4, Sox2, Klf4 and c-Myc). These four genes are heavily expressed in ESCs and by transiently overexpressing them in adult cells, the adult cells revert to an ESC-like state.

The derivation of iPSCs from adult cells was discovered by Shinya Yamanaka and his colleagues, and Yamanaka won the Nobel Prize for this achievement.

The research of Cooke and his colleagues, however, provides an important clue as to how this reversion to the embryonic state occurs. Cooke noted, “We found that when a cell is exposed to a pathogen, it changes to adapt or defend itself against a challenge. Part of this innate immunity includes increasing access to its DNA, which is normally tightly packaged. This allows the cell to reach into its genetic toolbox and take out what it needs to survive.”

It is this loosening of the structure of DNA in adult cells that allows the pluripotency-inducing proteins to modify the expression pattern of the cell and transform it into an ESC-like cell.

This type of response to viral infections that causes the DNA of cells to loosen up has been termed “transflammation” by Cooke and his team. They think that this finding could easily simplify and increase the efficiency of iPSC derivation.

Cooke’s laboratory initially tried to increase the efficiency of cell-permeable proteins that can reprogram adult cells into iPSCs. These proteins can bind to their target sequences on DNA and can also enter the nucleus when they pass into the cell. Why were these proteins so inefficient when compared to viral-based techniques?

To answer this question, Cooke’s lab examined the gene expression patterns of cells treated with iPSC-inducing viruses or iPSC-transforming proteins. They discovered that the gene expression patterns differed extensively. This led Cooke to hypothesize the virus itself was causing some sort of change in the adult cells that was necessary for iPSC derivation.

To test this hypothesis, they repeated the experiment with recombinant proteins but also concomitantly treated the cells with an unrelated virus. This dramatically increased the rates of pluripotency transformation. The increased rate of transformation was also linked to a signaling pathway called the toll-like receptor-3 (TLR-3) pathway.

Toll-like receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific molecular patterns normally found on microbial components. Each TLR recognizes a different set of microbial-specific molecules, and TLR-3 binds to double-stranded RNA molecules. Therefore, these cells activate those pathways that are normally turned when they are infected by viruses.

According to Cooke, “These proteins are non-integrating, and so we don’t have to worry about any viral-induced damage to the host genome.” Cooke also pointed out that cell-permeable proteins can allow the researchers to exert greater amounts of control over the reprogramming process. This, essentially could speed the use of iPSCs in human therapies. Cooke continued: “Now that we understand that the cell assumes greater plasticity when challenged by a pathogen, we can theoretically use this information to further manipulate the cells to induce direct reprogramming.”

Therefore, to sum up, the elimination of TLR3 reduces the efficiency and yield of human iPSC generation, but if TLR3 is activated, it enhances human iPSC generation by cell permeant peptides. Also, TLR3 activation enables changes to the structure of DNA (epigenetic changes), and these changes promote an open chromatin state that makes iPSC generation much more efficient.

Stanford study finds Induced pluripotent stem cells match embryonic stem cells in modeling human disease

Investigators from Stanford University School of Medicine have shown that induced Pluripotent Stem cells (iPSCs), which are made from adult cells through genetic engineering techniques, are a possible alternative to human embryonic stem cells when it comes to modeling those defects caused by a particular genetic condition. The example used in this study was Marfan syndrome, and in this study, iPSCs modeled the disease as well as embryonic stem cells (ESCs). Thus, iPSCs could be used to examine the molecular aspects of Marfan on a personalized basis. Embryonic stem cells, on the other hand, can’t do this because their genetic contents are those of the donated embryo are not the same as the patient’s.

Marfan syndrome is an inherited connective-tissue disorder that occurs in one in 10,000 to one in 20,000 individuals. It results from a large number of defects in one gene called “fibrillarin.” People with Marfan syndrome tend to be very tall and thin, and also tend to suffer from osteopenia, or poor bone mineralization. Medical experts have speculated that Abraham Lincoln, for example, suffered from this disorder. Marfan can also profoundly affect the eyes and cardiovascular system.

This proof-of-principle study, with regards to the utility of iPSCs also has more universal significance; it advances the credibility of using iPSCs to model a broad range of human diseases. iPSCs, unlike ESCs, are easily obtained from virtually anyone and possess a genetic background identical to the patient from which they were derived. Moreover, they carry none of the ethical controversy associated with the necessity of destroying embryos.

“Our in vitro findings strongly point to the underlying mechanisms that may explain the clinical manifestations of Marfan syndrome,” said Michael Longaker, MD, professor of surgery and senior author of the study, which will be published online Dec. 12 in Proceedings of the National Academy of Sciences. Longaker is the Dean P. and Louise Mitchell Professor in the School of Medicine and co-director of the school’s Institute for Stem Cell Biology and Regenerative Medicine. The study’s first author is Natalina Quarto, PhD, a senior research scientist in Longaker’s laboratory.

In this study, both iPSCs and ESCs, and embryonic stem cells that carried a mutation that causes Marfan syndrome showed impaired ability to form bone, and all too readily formed cartilage. These aberrations mirror the most prominent clinical manifestation of the disease.

iPSCs were discovered in 2006, and are derived from fully differentiated tissues such as the skin. However, they harbor the same capacity as embryonic stem cells; namely to differentiate into all the tissues of the body, and replicate for indefinite periods in a cell culture dish. Because iPSCs offer an ethically uncomplicated alternative to ESCs, IPSCs have fueled the hope that they can replace ESCs in scientists’ efforts to analyze, in a dish, those cellular defects ultimately responsible for diseases ranging from diabetes to Parkinson’s and even such complex conditions as cardiovascular disease and autism.

One hope for iPSCs is to be able to differentiate them in a dish into tissues of interest and then study these cells and their characteristics. This would help scientists better understand diseases in a patient-specific way, which would be impossible to do with ESCs unless ESCs were made from donated human eggs that were modified by cloning procedures. Cloning human embryos to the blastocyst stage has yet to occur, which makes this option technically impossible at the present time.

While scientists want to us iPSCs to develop therapeutic applications for regenerative medicine. This strategy, however, is technically more difficult, since scientists will have to develop the capacity first to repair genetic defects within cells before they can be used for regenerative medicine. iPSCs in theory might be a better bet because they are derived from patients’ own cells and, therefore, are less likely to provoke graft rejection than similar tissues produced using a donor embryo’s ESCs.

Unfortunately, several studies have reported subtle differences between iPSCs and ESCs, and these differences imply that the two cell types may not be equivalent. Stem cell experts have wondered whether these differences may render iPSCs inadequate substitutes for ESCs in modeling disease states, but this Stanford study suggests otherwise.