Reducing the Incidence of a Deadly Side Effect of Bone Marrow Transplants in Mice

Bone marrow transplants save the lives of leukemia, but they have one risky drawback and that is “graft-versus-host disease.” Graft-versus-host disease (GVHD) results when immune cells in the donor’s bone marrow attack the tissues and cells of the recipient’s body as foreign. Almost half of bone marrow transplant recipients develop graft-versus-host disease (GVHD), the main organs affected are the skin, liver and gut. Obviously, finding a way to quell or even prevent GVHD would be a boon for bone marrow transplantations.

By utilizing a mouse model, researchers at Washington University School of Medicine in St. Louis have managed to reduce the risk of GVHD from bone marrow transplants. Since bone marrow transplants are the only available curative treatment when leukemia returns, decreasing the risk of GVHD is the first step to improving the prognosis of leukemia patients.

The main strategy behind decreasing the effects of GVHD is to direct immune cells from the donor’s bone marrow away from healthy tissue and lead them to their intended purpose, which is to kill cancer cells.

“This is the first example of reducing graft-versus-host disease not by killing the T-cells, but simply by altering how they circulate and traffic,” says John F. DiPersio, MD, PhD, the Virginia E. and Sam J. Golman Professor of Medicine at Barnes-Jewish Hospital and Washington University School of Medicine. “Donor T-cells do good things in terms of eliminating the recipient’s leukemia, but they can also attack normal tissues leading to death in a number of patients. The goal is to minimize graft-versus-host disease, while maintaining the therapeutic graft-versus-leukemia effect.”

By working in a mouse model, Jaebok Choi, PhD, research assistant professor of medicine, showed that if he eliminated or blocked a particular protein known as the interferon gamma receptor on donor T-cells, these cell were unable to migrate to critical organs such as the intestines. However, these same T-cells were still capable of killing leukemia cells.

“The fact that blocking the interferon gamma receptor can redirect donor T-cells away from the gastrointestinal tract, at least in mice, is very exciting because graft-versus-host disease in the gut results in most of the deaths after stem cell transplant,” DiPersio says. “People can tolerate graft-versus-host disease of the skin. But in the GI tract, it causes relentless diarrhea and severe infections due to gut bacteria leaking into the blood, which can result in severe toxicity, reduction in the quality of life or even death in some patients.”

Interferon gamma has, for some time, been known to play a vital role in inflammation. The signal transduction pathway that works downstream of the receptor is just now being better understood. It is this signal transduction pathway downstream of the receptor that is responsible for activating the T-cells so that they cause GVHD. The signaling cascade initiated when interferon gamma binds its receptor activates molecules known as JAK kinases, followed by another protein called “STAT,” and finally a protein called CXCR3. CXCR3 mediates the trafficking of donor T-cells to the GI tract and other target organs.

Deleting the interferon gamma receptor from donor T-cells steers them away from target organs. This, however, leads to a second question: “Could the same result be observed by inhibiting some of the other molecules that act downstream of the interferon gamma receptor?” To address this question, Choi knocked out CXCR3 and discovered that such a knock out reduced graft-versus-host disease, but did not completely wipe it out.

“There are probably additional downstream targets of interferon gamma receptor signaling other than JAKs, STATs and CXCR3 that are responsible for T-cell trafficking to the GI tract and other target organs,” DiPersio says. “We’re trying to figure out what those are.”

This worked beautifully in mice, but could it work in humans? To make these data more relevant to human biology, Choi and DiPersio used drugs known to block JAK kinases in human cells. These drugs are presently approved by the Food and Drug Administration to treat myelofibrosis, which is a pre-leukemic condition in which bone marrow is replaced with fibrous tissue. Ruxolitinib and pacritinib are two such drugs and Choi and DiPersio showed that treating mice with either of these two drugs could mimic the protective effect of deleting the interferon gamma receptor. The JAK inhibitors definitely redirect the donor T-cells away from target organs and reducing graft-versus-host disease in leukemic mice. Unfortunately, they have yet to determine if these drugs preserve the anti-leukemia effect of these T-cells.

“The proof-of-principle behind these experiments is the exciting part,” DiPersio says. “If you can change where the T-cells go as opposed to killing them, you prevent the life-threatening complications and maintain the clinical benefit of the transplant.”

A Return to 2004

My favorite bioethics blogger Wesley Smith has just been given a new gig at National Review called the Human Exceptionalism Blog.

This recent post examines an article in Fortune magazine that bemoans the lack of success with translating embryonic stem cell research into cures. It must be due to a lack of funding, right?

Smith puts the kibosh on that one pretty fast. Read it here.

Losing Your Skin to Gain Your Life

Regenerative medicine can find tips in the oddest corners of biology, and today’s tip is no different. African spiny mice (Acomys) are very unusual critters among mammals. Apparently some people like to keep them as pets, but other types of mammals find the mice rather tasty. If a predator gets its teeth into the mouse, pieces of the spiny mouse’s hide rip off in the mouth of the attacker, and the rodent runs free to live another day. Disgusting? The mouse gets the best end of the deal because its skin grows back almost as good as new. This discovery provides a viable model for the study of regeneration in a mammal, and since humans are mammals, such studies can apply to human regeneration.

Some lizards, salamanders, sea cucumbers, crustaceans, and other types of arthropods (insects, crabs, lobsters, and so on) have the ability to lose a body part to avoid capture and then replace it. Unfortunately, until now, this ability was completely unknown in mammals.

Developmental biologist Ashley Seifert of the University of Florida in Gainesville and his colleagues had their interest piqued when they heard rumors about an African mouse that could grow back its skin. The African spiny mouse is a rodent with stiff hairs on its back that resemble the spines of a hedgehog. Since the animal is kept as a pet by some people, owners have noticed that the animal has a tendency to lose patches of skin when handled.

Seifert and his colleagues were able to find the spiny mouse in the wild in parts of Kenya. When they took the animals into the laboratory, they discovered that spiny mice have skin that is 20 times weaker than the skin of standard laboratory mice. The weakness of the spiny mouse’s skin might result from the very large hair follicles. Further work with the spiny mouse showed that the animal’s wounds heal faster than those of standard laboratory mice. In addition to increase speed of healing, the skin heals without forming scar tissue, and even replaces the hair follicles after an injury.

The photograph comes from Ashley W. Seifert, Stephen G. Kiama, Megan G. Seifert, Jacob R. Goheen, Todd M. Palmer & Malcolm Maden, (2012) Skin shedding and tissue regeneration in African spiny mice (Acomys) Nature 489: 561–565.

How much skin can this animal replace at a time? Seifert’s team showed that African spiny mice can survive the loss of 60% of the skin off their backs and still regenerate it and bounce back. Elly Tanaka, who works as a developmental biologist at the Technical University of Dresden in Germany, but was not involved with the work, said: “It seems remarkable that an animal can lose so much skin and heal the skin so well that it looks normal,” Tanaka has a point. There are a few species of lizard that can shed their skin in a hurry as an escape response. For example, Anguis fragilis, a legless lizard can shed patches of its skin to escape a predator. In this case, however, only the top layer of skin falls off and the lower layers of skin regrow the upper layers. In the case of the African spiny mouse, however, the animals lose their entire skin with all its layers. Nothing remains but bare muscles, which makes the replacement of the skin a much more daunting process. “It’s a nice example that shows the maximum capacity of what the mammalian skin can do in terms of healing,” Tanaka says.

Seifert and his team investigated the mechanism of regeneration in spiny mice by punching holes in their ears. They discovered that regeneration in the ears mimicked limb regeneration in newts. Upon injury, the mouse’s body generates a population of embryonic-like cells that aggregate underneath the layer of cells that first cover the wound. These embryonic-like cells divide and differentiate into the different cell types that will re-form the ear tissue.

“It is truly exciting to discover that these mammals are capable of losing and regrowing complex tissue in such an efficient manner,” says Tara Maginnis, an evolutionary biologist at the University of Portland in Oregon who was not involved with the work. “It’s another ‘gripping’ example of how organisms can evolve alternative, adaptive traits not by inventing new structures or pathways but by modifying existing structures” such as the skin, she says.

Regeneration is not unheard of mammals, since rabbits also have the ability to replace bit of their ears is they go missing, and deer can regrow their antlers. However the ability to regrow large swaths of e lost skin is unique among mammals. In the words of University of Utah molecular biologist Shannon Odelberg, this constitutes a “surprising example of mammalian regeneration.” Odelberg continued: “This discovery puts scientists one step closer to being able to unravel the molecular bases of regeneration in vertebrates and possibly translating these discoveries into therapies.”

Let’s think about this for a while. These mice almost certainly have a genome that is not all that different from that of other mice. The differences are due to the way those genes are regulated. If we could truly understand how these mice get their cells to revert to an embryonic-like state and spread out to heal wounds, we could offer therapies to burn patients that could repair over 60% of their skin. Think of it. This would be nothing short of biblical.

This nicely illustrated why basic scientific research is so important, and why is must continue to occur. Model systems matter.

Pea-Sized Telescopic Implant Restores Vision in Patient with Advanced Macular Degeneration

Eye surgeons at my alma mater, UC Davis Medical Center, have managed to successfully implant a new telescope-type implant in the eye of a patient who suffers from end-stage age-related macular degeneration (AMD). AMD is the most advanced form of the macular degeneration and is a leading cause of blindness in older Americans.

These telescope implants were approved by the Food and Drug Administration in 2010, and it is the only medical/surgical option available that can restore at least a portion of the patient’s vision. Eye doctors at the UC Davis Health System’s Eye Center collaborated with the Society for the Blind in this procedure. The UC Davis Health System’s Eye Center is one of the few medical centers in California and the whole nation to offer this innovative procedure.

Mark Mannis, professor and chair of ophthalmology and vision sciences and director of the Eye Center at UC Davis Health System, explained: “Macular degeneration damages the retina and causes a blind spot in a person’s central field of vision. The telescopic implant restores vision by projecting images onto an undamaged portion of the retina, which makes it possible for patients to again see people’s faces and the details of objects located directly in front of them.”

It is presently unclear what causes dry macular degeneration. It clearly forms as the eye ages. Macular degeneration consists of massive die offs of the cells in a particular part of the retina called the “macula.” The macula contains millions of light-sensing cells that provide sharp, detailed central vision, and it is also the most light-sensitive part of the retina. The retina quickly turns light into electrical signals and then sends these electrical signals to the brain through the optic nerve. The brain translates the electrical signals into images. If the macula is damaged, fine points in these images become unclear, fuzzy, spot-ridden, or simply black.

In May 2012, UC Davis cornea specialists Mannis and Jennifer Li implanted the miniature telescope, which is smaller than a pea, in the left eye of a macular degeneration patient names Virginia Bane, who is 89 years old and is from the California town of Pollock Pines, which is near Sacramento. Mrs.Bane is an artist who loves to paint, but has not painted for four years because her eyesight does not allow her to see well enough to do so. Mrs.Bane is the first in Northern California and among the first 50 individuals in the nation to receive this implant.

“I can see better than ever now,” Bane said. “Colors are more vibrant, beautiful and natural, and I can read large print with my glasses. I haven’t been able to read for the past seven years. I look forward to being able to paint again.”

Optometrists from the Society for the Blind and UC Davis occupational therapists have been working with Mrs. Bane to help to learn how to use her implant to its full extent.

“Virginia’s vision will keep getting better and better over time as she retrains her brain how to see. She basically uses her left eye with the telescopic implant to see details, such as using a microwave keypad and reading a book,” said Richard Van Buskirk, who works as an optometrist with the Society for the Blind in Sacramento who specializes in treating patients with low vision. “Her untreated right eye provides peripheral vision, which helps with mobility, such as walking or navigating within her home. Ultimately, her brain will automatically make the shift, using the capability of each eye as needed.”

Retina specialists from UC Davis who treat macular degeneration and other eye disorders associated with the back part of the eye coordinate the treatment program with optometrists who specialize in caring for patients with low vision. Patients are extensively screened before they can participate in this program and undergo medical, visual and functional evaluations to determine if they are good candidates for the procedure.  However, there is a simulator that can show patients what their eyesight might resemble if they were to receive the implant.  This simulator determines if the procedure will actually help the patient see better.

Most candidates for this procedure have very advanced, untreatable eye diseases and include end-stage, age-related macular degeneration (dry form).  All patients must have a disease that is stable and severely impairs vision. Candidates must also be at least 75 years old and have adequate peripheral vision in the eye that will not receive the implant and have no other ocular diseases, such as glaucoma.

A Better Way to Make Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are made from adult cells that have been genetically engineered so that they revert to the stem cell state.  Unfortunately, iPSC production is rather inefficient and it is a long procedure that eats up time.  Are there ways to speed this whole thing up?

Stem cell researchers at Sanford-Burnham have used an unusual strategy to save time and increase the efficiency of iPSC derivation; they used “kinase inhibitors.”  Kinases are enzymes that attach phosphate groups to other molecules.  Placing phosphates on proteins often significantly regulates their activity.  Some proteins are activated when phosphates are attached and others are turned off when phosphates are attached.  As it turns out, kinases are front and center when it comes to the control of cell division.  The scientists at Sanford-Burnham (at UCSD) used chemicals that block the activity of kinases, and this helps generate many more iPSCs than the standard method of iPSC derivation.  This new capability will hopefully facilitate and activate research in many fields that use iPSCs, which includes those who study human diseases and develop new treatments.

Tariq Rana, Ph.D., program director in Sanford-Burnham’s Sanford Children’s Health Research Center and senior author of the study, said: “Generating iPSCs depends on the regulation of communication networks within cells.  So, when you start manipulating which genes are turned on or off in cells to create pluripotent stem cells, you are probably activating a large number of kinases. Since many of these active kinases are likely inhibiting the conversion to iPSCs, it made sense to us that adding inhibitors might lower the barrier.”

Kinase expert, Tony Hunter, Ph.D., who is professor in the Molecular and Cell Biology Laboratory at the Salk Institute for Biological Studies and director of the Salk Institute Cancer Center, commented: “The identification of small molecules that improve the efficiency of generating iPSCs is an important step forward in being able to use these cells therapeutically. Tariq Rana’s exciting new work has uncovered a class of protein kinase inhibitors that override the normal barriers to efficient iPSC formation, and these inhibitors should prove useful in generating iPSCs from new sources for experimental and ultimately therapeutic purposes.” Hunter was not directly involved with this study.

A graduate student in Rana’s laboratory, Zhonghan Li, was assigned the task of kinase inhibitors that might speed up iPSC-derivation.  Li had some help from some very enterprising scinetists at the Conrad Prebys Center for Chemical Genomics.  Conrad Prebys Center for Chemical Genomics is the Sanford-Burnham’s drug discovery facility, and this center provided Li with a collection of more than 240 chemical compounds that are known to inhibit kinases. Li used the brute-force method of biochemistry by painstakingly adding each chemical, one-by-one, to his cells and then waited to see what happened.  Fortunately, several kinase inhibitors produced many more iPSCs than the untreated cells.  In fact, in some cases, there were so many iPSCs that they outgrew the dish that housed them.  The most potent inhibitors targeted three kinases in particular: AurkA, P38, and IP3K.

Rana and Li worked with the staff in Sanford-Burnham’s genomics, bioinformatics, animal modeling, and histology core facilities.  These valuable resources and their expertise are available to all Sanford-Burnham scientists and also the scientific community at large.  Rana and Li further confirmed the specificity of their findings and even nailed down the mechanism behind one inhibitor’s beneficial actions.

“We found that manipulating the activity of these kinases can substantially increase cellular reprogramming efficiency,” Rana said. “But what’s more, we’ve also provided new insights into the molecular mechanism of reprogramming and revealed new functions for these kinases. We hope these findings will encourage further efforts to screen for small molecules that might prove useful in iPSC-based therapies.”


Enhanced Migration of Cardiac Stem Cells by Overexpression of Stromal Cell Factor-1alpha

The heart has its own reservoir of stem cells. These cardiac stem cells (CSCs) can be easily harvested from the heart, grow in culture, and then re-injected into heart after a heart attack to improve the structure and function of the heart.

However, what would it take to simply get CSCs to move from their present location to the damaged area without any invasive procedures? A chemical called “stromal cell factor-1alpha (SDF-1alpha) attracts stem cells to it. Stem cells have a receptor on their surfaces (CXCR4 for those who are interested), and when those receptors bind SDF-1alpha, stem cells move towards the source of SDF-1alpha. Therefore, some enterprising scientists from the Third Military Medical Hospital University, from Chongquing, China have overexpressed SDF-1alpha in the damaged region of the heart. Their results have shown that not only do CSCs move to the site of injury, but the size of the infarct shrinks without surgery.

In their first experiment, Kui Wang and co-workers from the laboratory of Lan Huang isolated heart tissues from mice and the stem cells migrated onto the culture plates. When these migrating cells were isolated, they expressed genes that are common the stem cells (c-kit and sca-1). These stem cells also expressed a whole host of heart muscle-specific genes, which confirms that they are definitely heart-based stem cells.

In the next experiment, Huang’s group exposed these isolated CSCs to cells that were expressing SDF-1alpha. The CSCs migrated toward the SDF-1alpha source with abandon. Also, they discovered some inhibitors that prevent CSC migration. These inhibitors prevent activation of a pathway called the PI3K pathway. This shows that the activation of PI3K pathway is the reason the stem cells become activated and move toward SDF-1alpha.

The final experiment involved inducing heart attacks in mice and then engineering the damaged portions of the heart to express SDF-1alpha with viruses. In response to this, CSCs migrated to the damaged area and made new heart muscle. The damaged area in the heart shrunk and the hearts functioned more effectively. Unsurprisingly, the inhibitors that prevented CSC migration in the test-tube assays, also prevented CSC migration to the site of damage in mice. Thus, CSC migration is also dependent on the activation of the PI3K pathway.

This paper shows that the PI3K pathway is the main way cells get their signal to migrate. Secondly, it shows that engineering damaged sites in the heart can recruit CSCs to the site and stimulate their healing activities. While this is an animal study, it sets a template for future clinical studies in heart attack patients.

Salk and UCSD Scientists Identify Core Epigenetic Signature In iPSCs

Scientists from the Salk Institute at the University of California, San Diego have managed to characterize a unique molecular feature of induced pluripotent stem cells (iPSCs) that seems to earmark them. This earmark is a kind of signature of iPSCs, and it identifies them as reprogrammed cells. iPSCs show a great deal of promise in regenerative medicine because of their ability to differentiate into a wide range of body tissues, although there are safety concerns with these cells.

The Salk scientists report that there is a consistent molecular difference between embryonic stem cells and iPSCs. These findings could potentially help overcome some of the safety problems that must be solved if iPSCs are to be used in regenerative medicine.

Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory and the senior author on the paper: “We believe that iPSCs hold a great potential for the treatment of human patients. Yet we must thoroughly understand the molecular mechanisms governing their safety profile in order to be confident of their function in the human body. With the discovery of these small, yet apparent, epigenetic differences, we believe that we are now one step closer to that goal.”

Embryonic stem cells (ESCs) possess a characteristic known as “pluripotency.” Pluripotency refers to the ability of these cells to differentiate into nearly any cell type in the adult human body. The pluripotency of ESCs makes them a candidate for therapeutic uses, but once ESCs differentiate into mature into specific cell types and then transplanted into a patient, they may elicit immune responses, potentially causing the patient to reject the cells.

To overcome this problem, scientists in Japan and the United States found ways to use genetic engineering techniques to revert mature, adult cells, into a pluripotent state. Thus were born “induced pluripotent stem cells” (iPSCs), which could be developed from the patient’s own adult cells, and would theoretically carry no risk of immune rejection.

Soon after iPSCs were discovered, however, scientists found that iPSCs had molecular differences from embryonic stem cells. In particular, iPSCs had “epigenetic changes.” Epigenetic changes are changes in those chemical modifications in DNA that can alter genetic activity. At certain points in the genomes of iPSCs, there are different patterns of methyl groups attached to the bases of DNA in comparison to the genomes of ESCs. At first, it seemed that these epigenetic changes occurred randomly.

Izpisua Belmonte and his colleagues worked on iPSCs in order to understand more about these differences. Were these epigenetic changes really random, or was there a discernible pattern?

Previous studies had primarily analyzed iPSCs derived from only one mature type of cells; a connective tissue cell called a fibroblast. However, the Salk and UCSD researchers examined 17 iPSC lines derived from six different mature cell types in order to determine if there were any commonalities. Their experiments revealed that even though there were hundreds of unpredictable changes, there were some epigenetic changes that were consistent across the cell types: the same nine genes were associated with these common changes in all iPSCs.

“We knew there were differences between iPSCs and ESCs,” said Sergio Ruiz, first author of the paper, “We now have an identifying mark for what they are.”

The therapeutic significance of these nine genes awaits further investigation, but the current importance of this study is that it gives stem cell researchers a new and more precise understanding of iPSCs, and their epigenetic signatures.

See: “Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells;” Sergio Ruiz et al.; Proceedings of the National Academy of Sciences, 2012; DOI: 10.1073/pnas.1202352109