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

Regenexx-C Hip Treatment Failure

Dr. Centeno at the Regenexx blog reports on a patient with arthritis of the hip who was deemed a poor candidate for a stem cell treatment, but went through with it anyway. The treatment failed and the patient ended up getting a hip replacement anyway.

Centeno’s entry on this case shows us that stem cells are not magic bullets. Sometimes they work and sometimes they do not. Furthermore they are not a one size fits all kind of treatment. Some patients are good candidates for stem cell treatments and some aren’t. Patients must be considered on a case-by-case basis.

Centeno has seen many successes with treating arthritic hips with his stem cell treatments. However, just as drugs and surgery are not magic bullets, neither are stem cells. Therefore, we should not expect them to be.   They are powerful tools for healing, but they are not the equivalent of magic.

Autophagy – When Cells Take The Garbage Out

How do cells deal with junk? What would junk be to a cell? Sometimes proteins become unfolded or degraded, and such proteins are nonfunctional, which is to say that they become junk. Also, fats can become oxidized as can membrane lipids, and thus become junk. Sugars and degrade and become junk. Therefore, there are many ways that cells can acquire junk.

To deal with the junk, cells have a process known as autophagy, which is derived from two Greek words that mean “self” and “eat.” Autophagy is a vitally important process, because the breakdown of autophagy generates a lot of trouble for the cell. In fact, some diseases characterized by degeneration of the nervous system are characterized by a breakdown or overwhelming of autophagy.

As you might guess, autophagy is a highly regulated process. It consists of the formation of double- or multi-membrane vesicles called autophagosomes that enclose portions of the inside of the cells that are then delivered for degradation following fusion with lysosomes, which are the garbage disposals of the cell. If the cell revs up autophagy, then most of the proteins that are prone to clumping are quickly degraded. Such proteins are genetically linked to protein misfolding disorders (PMDs), and PMDs include diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Also, in cells, the accumulation of mutant proteins that tend to clump increases autophagy in cells and acts to detoxify the effects of these proteins.

Autophagy impairment and Alzheimer’s disease. (a) Schematic representation of the general steps of the autophagy pathway. (b) Ablation of the essential autophagy regulator Atg7 in neurons triggers spontaneous neurodegeneration, leading to the accumulation of ubiquitin-positive inclusions in a p62-dependent manner. Aggregate formation also involves the recruitment of partially phosphorylated Tau and active GSK-3β, resembling a ‘pre-tangle’ non-amyloidogenic state. (c) Speculative model: in Alzheimer’s disease (AD), defects in the autophagy pathway due to genetic mutations, environmental factors and/or aging may contribute to the accumulation of abnormal protein aggregates and possible phospho-Tau on a pre-tangle state. For example, mutations in Presenilin-1 alter the pH of the lysosome, thus decreasing autophagy activity, which may enhance neurodegeneration and further accumulation of amyloid-β. Since during aging autophagy activity decreases over time, we speculate that autophagy impairment, together with other factors related to AD (genetic and environmental), may lead to a progressive flow from a pre-tangle to a neurofibrillary stage. This progression may be linked to the cognitive deficits associated with AD and tauopathies.

These facts about autophagy suggest that it could be a good target for disease intervention. Also, many PMDs result from specific defects in particular steps of the autophagy process. Therefore, understanding this process is crucial for developing treatments to these diseases.

In the cell of people with Alzheimer’s disease (AD), there is a large accumulation of vesicles in the degenerating neurons. In a mouse model of AD, several studies have identified specific defects in the autophagy process. In a fascinating paper from the laboratory of Ralph Nixon, a protein called “presenilin-1” (PS1), regulates lysosomal function. In order for lysosomes to work, they must pumps hydrogen ions into their interiors. This acidifies the lysosome and activates the enzymes inside it. PS1, according to the Nixon paper targets the protein that pumps the hydrogen ions into the lysosome interior (v-ATPase, see Lee JH, et al., Cell. 2010 141(7):1146-58). PS1 is central to AD in humans because mutations in the gene that encodes PS1 lead to inherited forms of AD that tend to develop rather early in life. Autophagy requires PS1 and mutations in PS1 disrupt it. In the AD mouse, if genetic strategies are used to restore lysosomal function, the degeneration of the nervous system was halted prevented neuropathology and cognitive deficits of an AD mouse model (Yang DS, et al., Brain 2011, 134:258-277). Thus restoring lysosomal function in AD mice significantly decreases the accumulation of amyloid protein in the brain. This places autophagy impairment as an early feature in the pathology of AD.

Autophagy decreases as we age, and this is the main reason that our risk for developing PMDs increases as we age. Likewise, stimulating autophagy with drugs like rapamycin or resveratrol, or by means of caloric restriction, can cause anti-aging effects. In the mouse, inhibition of autophagy through genetic means causes their neurons to undergo spontaneous degeneration, not unlike what is observed in PMDs. The symptoms are also uncannily similar to other types of neurodegenerative diseases: death of neurons, motor dysfunction, and premature death (Komatsu M, et al., Nature 2006, 441:880-884 & Komatsu M, et al., Nature 2006, 441:880-884).

These data are the reason why introducing normal cells that can ingest and destroy aggregated proteins can possibly help patients with PMDs. Thus even though stem cells may not be able to rewire the damaged brain of an AD patient, by designing stem cells that can clean up the brain and improve the autophagy of the brain, we might be able to improve the clinical outcomes of AD patients and other patients with PMDs.

Stable Culture of Mouse Cranial Neural Crest Cells Established

During development, the nervous system forms from a tube that begins as deep indentations on the presumptive back of the embryo. This deep groove brings two folds of tissue very close together – so close that they fuse to form a tube called the neural tube. When the ends of the folds fuse together, cells stream from these folds and move down into the body of the embryo. These migrating cells are called :neural crest” cells, and they form a very wide variety of cell types. The cells inside the adrenal glands, the pigment-bearing cells of the skin, the ganglia on the digestive system, the flat bones of the skull, the heart valve cushions in the heart and some of the vessels of the heart, corneal cells of the eye, and other cells and tissues are all formed from the neural crest cells. Consequently, there is a great deal of interest in neural crest cells for the purposes of regenerative medicine. These cells have the capacity to regenerate a fair number of tissues, and there is a great desire to learn more about these cells.

In particular, the neural crest cells in the head of the embryos give rise to a very wide range of head-specific tissues and there is a great deal in interest in learning more about them. To that end, Robert Maxson and his colleagues at the University of Southern California, in collaboration with Marianne Bronner (a former professor of mine at University California, Irvine), but now a California Institute of Technology, have made and characterized a new stable cranial neural crest cell line from mouse called O9-1.

To isolate cranial neural crest cells, mouse embryos that were genetically engineered to expressed a glowing protein in neural crest cells were dissected and the glowing cells were cultured. Apparently, these cells grow readily in culture and can be passaged indefinitely.

In culture, however, these O9-1 cells still keep their developmental potency. When induced in culture, these cells could form bone-making cells (osteoblasts), cartilage-making cells (chondrocytes), smooth muscle cells, glial cells (cells that support neurons).

Gene expression studies showed that O9-1 cells expressed a gaggle of genes associated with neural crest cells 9AP-2alpha, Twist1, and Snail1). They also expressed some proteins that are considered to be the earmarks of genuine stem cells (CD44 and Sca-1). O9-1 cells maintained their ability to differentiate into many different cells types and still expressed all these interesting genes, even after growing in culture for 17-22 transfers.

To truly determine what kind of cells were represented by O9-1 cells, Maxson and his co-workers used a whole-genome expression profiling experiment. If such an experiment sounds like a lot of work, it is because it is. Because neural crest from different levels of the embryo have different developmental capacities, they often express different genes in combination with the neural crest-specific genes they express. First, O9-1 cells did not express genes commonly found in stable adult cells (no surprise there). However, they also did not express genes commonly found in neurons. This shows that these cells cannot be from the trunk because trunk neural crest, which tend to make nerves rather readily. Also, Thomas and others made trunk neural crest cell lines and these cells expression smooth muscle, glial and neuronal cell markers despite being passaged many times (see Thomas, et al., Human Molecular Genetics 17, 3411-25). Thus O9-1 cells make neural crest-specific genes, but they are almost certainly not trunk neural crest cells.

Instead, O9-1 cells expressed many genes at levels most consistent with neural crest cells from the head. Also, then O9-1 cells were injected into mouse embryos, they went to the head and contributed to the formation of the head. This pretty well establishes the identity of O9-1 cells as cranial neural crest cells.

Having such cells to study and manipulate is very useful. Because neural crest cells differentiate into a wide variety of cell types, abnormalities in neural crest development underlie a whole host of human diseases. Discerning the biochemical signals that drive neural crest cells to differentiate into one cell type or another is key to unlocking the potential of these cell types for regenerative medicine and the role of the cells in the pathology of many different types of developmental disorders.

Federal Court Rules in Favor of Federal Funding for Embryonic Stem Cell Research Legal

On August 24, 2012, a three-judge panel ruled that the government had properly interpreted a law that bans the use of federal funds research that destroys human embryos. Many legal observers, however, opined that this ruling will not end the controversy over this issue, and one of the judges on the three-judge panel importuned Congress to clarify what the government could and could not do with respect to human embryos.

This ruling upholds the dismissal of the case by the US Court of Appeals for the District of Columbia Circuit. The case, Sherley v. Sebellius, 11-5241, sought to prevent the US Department of Health and Human Services from using federal money to fund human embryonic stem cell research. The plaintiffs contended that funding human embryonic stem cell research would violate the Dickey-Wicker Amendment, which was passed as a rider to other legislation in 1996.

Predictably, biotechnology companies interested in embryonic stem cell-based treatments and other more academic embryonic stem cell researchers were quite pleased with the ruling. For example, Gary Rabin of Advanced Cell Technology said: “This court ruling should be of considerable benefit to our embryonic stem cell-based clinical programs. It effectively removes major speed bumps for the National Institutes of Health in terms of approving the several stem cells lines that we have submitted for their consideration for funding. We expect that a number of our embryonic stem cell lines will be approved for funding in coming months.”

The plaintiffs in this case were: a) James L. Sherley, who is a former a former member of the MIT faculty, but presently works as a senior scientist at the Boston Biomedical Research Institute; b) Theresa Deisher, who is the founder, managing member, and research and development director of AVM Biotechnology; and Nightlight Christian Adoptions, which is a non-profit, licensed adoption agency dedicated to protecting and finding adoptive parents for human embryos conceived through in vitro fertilization; c) the Christian Medical Association, a non-profit association of doctors dedicated to improving ethical standards of health care in the United States and abroad.

The main argument put forward by the plaintiffs in this lawsuit is that the present NIH Guidelines for the funding of embryonic stem cell projects violate existing federal law that bans the use of federal funds for the destruction of human embryos. Because the NIH created and sent its guidelines with a preconceived determination to fund human embryonic stem cell research and without considering scientifically and ethically superior alternatives, the guidelines are invalid regulations that violate the federal Administrative Procedure Act and, therefore, should be struck down.

With respect to the merits of the plaintiff’s case, it seems rather obvious, to me at least, that the NIH guidelines violate federal law. The Dickey-Wicker amendment was sponsored by former Congressman Jay Dickey (RAK) and Senator Roger Wicker (R-MS) who was then a member of the House of Representatives)], and applied to every Health and Human Services (“HHS”) appropriations bill governing the National Institutes of Health (NIH) since 1995. The bill states, “None of the funds made available by this Act may be used for . . . research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero under 45 C.F.R. § 46.204(b) and section 498(b) of the Public Health Service Act (42 U.S.C. § 289g(b).” Even for lawyer-speak, that seems pretty clear to me. Embryonic stem cell research includes the derivation of new embryonic stem cell lines from human embryos.  The derivation process destroys the embryo, which is precisely the thing that the Dickey-Wicker amendment prohibits the NIH from funding with federal money.

However, the federal judges did not see it that way for one reason:  funding for embryonic stem cell research also includes work on already established embryonic stem cell lines.  These lines are already in existence that the decision to kill the embryos has already occurred.  Therefore, the judges thought that the Dickey-Wicker amendment was simply not specific enough on the matter to merit stopping all funding for embryonic stem cell work.

While this argument has merit, it dodges the fact that embryo-destroying research will be funded by the NIH because of this ruling and some of this research will include laboratories that destroy embryos to make embryonic stem cells.  This is clear, since several sources have noted the increase in embryonic stem cell lines approved for NIH funding.  For example the Nature Medicine blog had this to say:  “Since the beginning of the month, the NIH has quietly kept adding ES cell lines to its registry, bringing the total tally up to 128.”  Thus the NIH funding policy has definitely led to the destruction of more embryos.

Where did this legal battle originate?  It began in 1994, during the presidency of Bill Clinton.  Before 1994, there was a ban on human embryo experimentation.  The Clinton Administration took steps to reverse this ban, and allow embryo-destructive research on donated embryos left over from fertility clinics while still prohibiting research that created its own embryos for research purposes.;  This policy shift was in response to the recommendation of an advisory committee known as the Human Embryo Research Panel (“HERP”).  This was an ad hoc committee formed to address the question of embryo research.  The phrase “ad hoc” is a Latin phae that means “for this.”  It simply means that this committee was formed for to address this particular question.  In testimony before the House Appropriations Committee, NIH Director Varmus stated that NIH would have funded six out of nine applications for grants involving embryo-related research “if the NIH had been able to proceed according to the recommendations and the President’s directive.”  Varmus also stated that he “firmly agree[d]” with particular sections of the HERP report, and further told the Committee that NIH was currently deciding whether to go forward with funding embryo-destructive research on donated human embryos.

However, before NIH was able to approve any grants that funded embryo-destructive research, Congress passed the Dickey-Wicker Amendment for the first time.  Opponents of the amendment argued that prohibiting federal funding of embryos research would push such research into the dark recesses of private industry where it would not be properly regulated.  Also, the understanding of the amendment was universal throughout Congress: it would prohibit ALL federal funding of embryo-destructive research.  Democratic senator from California, Barbara Boxer, understood the Dickey-Wicker Amendment as creating “a total prohibition of Federal funding for human embryo research,” and Republican Congressman John Porter understood the amendment in this way as well.  In fact Porter tried to pass an alternative rider to the Dickey-Wicker Amendment that would have prohibited federal funding for only creation of new embryos, but not other types of embryo-destructive research with donated embryos, but his rider was defeated.

Even more significantly, the NIH understood the amendment that way from 1996-1999 when they enforced it.  DNA research with DNA from human embryos that did not necessarily kill the embryos was prohibited from receiving federal funding under the NIH’s understanding of the Dickey-Wicker Amendment.  In a 1996 letter (October 10)  to Georgetown University Medical School researcher Mark Hughes who was using federally funded equipment to conduct tests on DNA derived from embryos, NIH “clarif[ied] . . .the NIH position on embryo research.” The agency explained that “analysis from DNA derived from a human embryo” violated the federal prohibition on research involving embryos and that NIH equipment “may not be used for embryo work of any kind.”

However, four years later, the NIH altered its position and issue Guidelines authorizing the funding of human embryonic stem cell research (65 Fed. Reg. 51976, Aug. 25, 2000).  Before the 2000 Guidelines were published, then-HHS General Counsel Harriet S. Rabb issued a memorandum on January 15, 1999, that supported the National Institutes of Health (NIH) claim that the Dickey-Wicker Amendment ought to be re-interpreted to ban federal funding of the derivation of embryonic stem cells – the procedure by which human embryos are destroyed to harvest their embryonic stem cells – but not research utilizing the derived embryonic stem cells.  This is in direct contradiction to the clear understanding of the Dickey-Wicker amendment put forward by Congress.

To remedy this reinterpretation of the Dickey-Wicker Amendment (DWA), seven senators sent a letter to then Secretary of Human Health and Services, Donna Shalala.  In that letter they stated that “Congress never intended for the National Institutes of Health to give incentives for the killing of human embryos for the purpose of stem cell research.”  The warnings of the senators and the comments from many dissenting parties were ignored by the NIH.

When President Bush took office, he rescinded the Clinton rules and upheld the original interpretation of the DWA.  This ended when Barak Obama became president in 2008.  President Obama revoked President Bush’s Executive Order 13435 (June 22, 2007) and ordered NIH “support and conduct responsible, scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent permitted by law.”

This was the reason for the lawsuit filed by Sherley and his co-plaintiffs.  They wanted the Federal government to simply recognize the law as it was originally written and interpreted.  Unfortunately, the judges ignored all that and went with their own shtick.  This is sad, and it should be remedied, but for now the situation seems to be that Congress’ laws do not mean what they originally say.

Icelandic Cancer Patient Receives a Completely New Windpipe Made From His Own Stem Cells

Andemariam Beyene, an African cancer patient from Eritrea had made his peace with his life, and talked about his impending death. Doctors found a golf ball-sized tumor growing in his windpipe, and despite several treatments with radiation and surgery to remove it, it kept growing. He was in pain, and out of options.

As a last resort, he went to the Karolinska Instute in Stockholm, Sweden to visit Dr. Paolo Macchiarini, who wanted to make a new windpipe for Mr Beyene from his own cells and spun plastic. This seemed like the medical equivalent of a “Hail Mary” pass in football, but it seemed worth a shot.

There is a precedent for such a procedure, since between 2008 and 2011, Dr. Macchiarini fitted nine people with new tracheas. These tracheas were built from the patient’s own stem cells that were grown on scaffolds made from tracheas that had been completely stripped of all their cells. These “decellularized” scaffolds work well, but they have one large drawback: .they required the existence of a pre-existing trachea from a human or an animal. Therefore, for Mr. Beyene, Dr. Macchiarini used a different procedure that built the scaffold from scratch out of plastic.

For this procedure, a mold of Mr. Beyene’s trachea was made from porous, fibrous plastic. This mold was then seeded with bone marrow stem cells and grown in a bioreactor. The bioreactor contained culture medium that turned much like a rotisserie. After one day and a half of growth in the bioreactor, the trachea was implanted into Mr. Beyene’s body.

Even though this procedure had been successful performed in pigs, it had never been tried on a human. Mr. Beyene was convinced by Dr. Macchiarini to give it a try. Now, the 39-year old African who lives in Iceland is back with his wife and children, getting to know people he thought he would never know. His strength is improving every day and can even run a little.

The synthetic scaffold used for Mr. Beyene’s windpipe was made by scientists at University College London. This mold was made to exact specifications so that it would perfectly fit inside Mr. Beyene’s chest. Then stem cells from his bone marrow were cultured in the laboratory and dripped with a pipette over the scaffold, in the same way that you baste a turkey. Then this whole thing is grown in a bioreactor that circulated fresh culture medium at regular intervals while spinning the scaffold and the cells. Macchiarini is quite sure that the original cells that are seeded onto the scaffold are dead. He suspects that the dying stem cells leave a host of chemicals in their stead that summon other stem cells from the bone marrow to come and seed the scaffold.

The windpipe is lined with several different types of cells. Some of them secrete mucus, which serves as a kind of adhesive tape for dust particles and microorganisms that are inhaled. The mucus is then moved to the top of the throat by a host of cells with hair-like extensions that move back and forth like oars on a Viking boat. At the top of the throat, the mucus is swallowed and destroyed in the stomach. Also, the windpipe is well endowed with blood vessels to feed the tissues oxygen and nutrients from the bloodstream.

Mr. Beyene’s windpipe was found to contain some mucus-producing cells at fives months after the surgery. At follow-up, it was also clear that his windpipe bled when nicked. Mr. Beyene hopes to return to Eritrea some day, but for now he will stay in Iceland, since he is close to Stockholm for his regular check-ups. He had some scar tissue from the implant that was impeding his breathing, but that has been removed. He still needs regular check-ups, but he is doing well.

Five months after implanting Mr Beyene with his engineered windpipe, Dr. Macchiarini implanted an American patient named Christopher Lyles with a tissue engineered windpipe made from a plastic scaffold and his own stem cells. Mr. Lyles’ windpipe was made with an improved type of scaffold fabricated from smaller plastic fibers. Mr. Lyles returned home to Maryland, by died in March of 2012. While the cause of death has not been released, Dr. Macchiarini has been told that his implant was not the cause of his death and that up until the time of his death, it had been functioning normally. Dr. Macchiarini has also implanted engineered windpipes into two Russian patients who have been discharged fro the hospital and are doing well.

While these procedures are remarkable feats of stem cell biology, tissue engineering and medical intervention, we must admit that these techniques are still experimental and are hugely expensive (half a million dollars per procedure). Dr. Macchiarini’s dream it to some day design drugs that induce the stem cells to build a new trachea within the patient from the inside out. This way, no surgery is required and the patient would have a rebuilt windpipe.

FDA Approves Clinical Trial that Used Cord Blood to Treat Autism

The Sutter Neuroscience Institute in Sacramento, California has announced its collaboration with the Cord Blood Registry, the world’s largest stem cell bank in what promises to be the first FDA-approved clinical trial to assess the use of a child’s own cord blood stem cells to treat selected patients with autism. This placebo-controlled study is the first of its kind and will evaluate the efficacy of cord blood stem cells to help improve language and behavior in autistic children.

The Centers for Disease Control (CDC) places the frequency of autism in the US as one in 88, but for boys, the rate is even higher (1 in 54). Autism, today, is part of a series of conditions that are collectively defined as autism spectrum disorders (ASPs). ASPs include individuals with very different symptoms, and include everything from autistic disorder (also known as classic autism, Asperger syndrome and pervasive developmental disorder not otherwise specified (also known as atypical autism). These conditions are thought to have multiple risk factors that include genetic, environmental and immunological components.

With regard to this study, Michael Chez, M.D., director of Pediatric Neurology with the Sutter Neuroscience and principal study investigator of this clinical trial said: “This is the start of a new age of research in stem cell therapies for chronic diseases such as autism, and a natural step to determine whether patients receive some benefit from an infusion of their own cord blood stem cells. I will focus on a select portion of children diagnosed with autism who have no obvious cause for the condition, such as known genetic syndromes or brain injury.”

This clinical study will enroll 30 children between the ages of two and seven who have been diagnosed with autism, and meet all the criteria for inclusion in the study. Over the course of this clinical trial, all enrolled participants will receive two infusions over the course of 13 months. One of the infusions will contain the child’s own cord blood stem cells, and the other infusion will contain a placebo. The participants and the lead investigators will not know the content of each infusion. To ensure the highest quality and consistency in cord blood stem cell processing, storage and release for infusion, Cord Blood Registry is the only family stem cell bank that provides umbilical cord blood units from clients for the study.

A newborn’s umbilical cord blood contains several unique populations of stem cells. Scientists and physicians have been used for more than 20 years in medical practice to treat certain cancers, blood diseases and immune disorders. When patients undergo a stem cell transplant for such conditions, the umbilical cord blood stem cells effectively rebuild the blood and immune systems.

According to Dr. Chez, “A focus of my research has been the complex relationship between a child’s immune system and central nervous system. We have evidence to suggest that certain children with autism have dysfunctional immune systems that may be damaging or delaying the development of the nervous system. Cord blood stem cells may offer ways to modulate or repair the immune systems of these patients which would also improve language and some behavior in children who have no obvious reason to have become autistic. The study is similar to other FDA-approved clinical trials looking at cord blood stem cells as a therapy for cerebral palsy.”

Heather Brown, vice president of scientific & medical affairs at Cord Blood Registry, said: “It’s exciting to partner with thought-leading medical researchers and clinicians, like Dr. Chez, who are pursuing a scientifically-sound approach in evaluating new therapeutic uses for cord blood stem cells for conditions that currently have no cures. Families who made the decision to bank their stem cells to cover the unknowns and what ifs in life are gaining access to this and other important clinical trials while playing an important role in the advancement of science.”

A co-investigator of the study is Michael Carroll, M.D., who is the medical director of the Blood and Marrow Transplantation and Hematological Malignancies Program at Sutter Medical Center, Sacramento. According to Dr. Carroll, “There is a vast amount of unchartered territory when it comes to how stem cell therapies may help patients living with these conditions. I’ve seen how stem cell therapy has changed my field of medicine and how I care for my blood cancer patients. I am eager to see how our work can open new doors for patients and families dealing with autism.”

Bone Marrow Stem Cells Can Become Kidney Stem Cells and Heal Acute Renal Injury.

Patients with failing kidneys often suffer from chronic kidney disease or end-stage renal disease. These two conditions are associated with a substantial amount of suffering and death, and current treatments from chronic kidney disease and end-stage renal disease do virtually nothing to halt the progression of these diseases.

Fortunately there has been a respectable amount of recent work on kidney regeneration after kidney injury, but these new discoveries have not led to therapeutic advances. The shortage of kidneys for transplantation and the structural complexity of the kidney have slowed the development of therapeutic strategies for the kidney.

Stem cell-based therapy for damaged kidneys is a distinct possibility for several reasons. First, kidneys do seem to possess resident stem cells and extra renal stem cells also seem to reside in the kidney. Several studies have confirmed the presence of cells in the kidneys that possess stem cell-specific proteins (Sca-1, c-Kit, and CD133). When isolated and tested in the laboratory, these renal stem cells can differentiate, proliferate, and eventually reline denuded renal tubules, and thereby restore the structural and functional integrity of the kidney (See Yeagy BA, Cherqui S, Pediatr Nephrol 2011, 26:1427–1434; Parikh CR, et al., Ann Clin Biochem 2010, 47:301–312; Lee P-T, et al., Stem Cells 2010, 28:573–584; Bussolati B, et al., Am J Pathol 2005, 166:545–555; Dekel B, et al., J Am Soc Nephrol 2006, 17:3300–3314; Gupta S, et al., J Am Soc Nephrol 2006, 17:3028–3040; Lazzeri E, et al., J Am Soc Nephrol 2007, 18:3128–3138; and Kitamura S, et al., FASEB J 2005, 19:1789–1797). Unfortunately, the exact role of renal stem cells and their functional limitations and physiological niche are all subjects that are still being investigated.

Other work has shown that bone marrow stem cells can contribute to kidney repair after kidney injury (see Park HC, et al., Am J Physiol Renal Physiol 2010, 298:F1254–F1262; Cheng Z, et al., Mol Ther 2008, 16:571–579; and Qian H, et al., Int J Mol Med 2008, 22:325–332). It is unclear however, if bone marrow stem cells can trans-differentiate into renal stem cells.

To this end, a Chinese group has examined if bone marrow stem cells can actually trans-differentiate into renal stem cells after acute kidney injury. This work resulted from collaboration between the laboratories of Yong Xu at the Urology department at the Second Hospital of Tianjin Medical University, in Tianjin, China, and Zongjin Li at the School of Medicine at Nankai University in Tianjin, China.

In this study, workers from Xu’s and Li’s laboratories transplanted bone marrow stem cells from mice that expressed a glowing protein in their cells into mice that had been subjected to radiation. Radiation treatment wipes out the bone marrow of the mouse, and the transplantation reconstitutes the bone marrow. Therefore, the mice that were treated with radiation now have bone marrow stem cells that glow in the dark and anywhere those cells go, they will be traceable.

Once it was clear that the irradiated mice that had received the bone marrow transplantations had normal blood work (5 weeks later), their kidneys were subjected to acute damage by being deprived of sufficient blood flow for a short period of time. Four weeks later, the kidneys of these animals were examined in order to determine if the transplanted bone marrow stem cells had migrated to the kidneys to help heal them. A second experiment utilized a small protein called a “cytokine,” which acts as a powerful signal to stem cells. This particular cytokine, granulocyte colony stimulating factor (G-CSF), mobilizes stem cells from bone marrow such that the bone marrow stem cells move from their comfortable, leisurely existence to the bloodstream where they can go to help heal other tissues. By giving some of the transplanted mice doses of G-CSF, Xu and Li and their co-workers were able to determine if the bone marrow stem cells moved from the bone marrow to the kidney to take up residence in the kidney as the new renal stem cell population.

The results clearly showed that bone marrow stem cells moved from the bone marrow to the kidney to participate in kidney healing. However, it did not end there. These same labeled, glowing bone marrow stem cells expressed the proteins normally found in resident renal stem cells. While these bone marrow stem cells only constituted a small proportion of the renal stem cell population, they were clearly a part of the Sca-1+ or c-Kit+ renal progenitor cell population. Secondly, treatment with G-CSF almost doubled the frequency of bone marrow-derived renal stem cells in the kidney. G-CSF treatment also increased the capillary density in the injured kidney, which is significant, because bone marrow stem cells are rich in a population of blood vessel-making stem cells. Furthermore, the new blood vessels all glowed in the dark, which shows that they were made by the bone marrow-derived stem cells that moved to the kidney are contributed to the resident renal stem cell population that participated in kidney repair.

Thus, these data in this study establish that stem cells from bone marrow can trans-differentiate into cells that share many of the properties of renal resident stem cells. Furthermore, mobilization of these stem cells with cytokines like G-CSF mobilization can enhance the healing effects of these cells and might provide the basis for a new therapeutic strategy for end-stage renal disease or chronic kidney disease.

Using Cultured Limbal Cells from Cadavers to Heal Blindness

Stem cell-based therapies have been available for the eye for several years. In particular, diseases of the outermost layer of the eye, the cornea, can be treated with “limbal cell” transplantations.

The human eye is more or less spherical, but is rather asymmetrical. Our eyes are also one inch in diameter. The eye consists of a front and rear compartment. The front compartment consists of the iris, which is pigmented, the cornea, which is transparent, the pupil, which is the black, round opening in the iris that lets light in, the sclera or white part of the eye, and the conjunctiva, which is an invisible, clear layer of tissue that covers the front of the eye, which the exception of the cornea. Just behind the iris and the pupil lies the lens, which focuses the light on the back of the eye. Most of the eye is filled with a clear gel called vitreous.

The rear compartment is filled with vitreous humor, which is a liquid that is also rich in a slippery, acidic carbohydrate called hyaluronic acid and several types of proteins. The back of the eye is covered with special light-sensing cells that are collectively called the retina. The retina converts the energy from the visible spectra of light into electrical impulses, and behind the eye, the optic nerve carries these impulses to the brain. The macula is a small, sensitive area within the retina that gives central vision. It is located in the center of the retina and contains the fovea, a small depression or pit at the center of the macula that gives the clearest vision.

If we now focus on the cornea, we will see that there is a ring of tissue that connects the cornea and the sclera known as the limbus. The limbus possesses a stem cell population that replenishes the cornea and also serves as a barrier for the cells on the conjunctiva. If this stem cell population is damaged or depleted, then conjunctival cells invade the cornea, and disrupt its unique structure. The cornea is transparent and allows light to pass through it unperturbed. The conjunctiva, however, is translucent, and filled with blood vessels. If the cornea becomes invaded with conjunctiva cells, it will cloud over and vision will be obscured.

To fix this problem, scientists and eye surgeons have experimented with limbal stem cell transplants. The most successful forms of transplantation use limbal cells from one eye that are transplanted into the other eye. This procedure, however, has a few drawbacks. The removal of limbal cells from one eye can compromise the integrity of the donor eye. Secondly, the patient is now left with two eyes that are healing rom surgery.

A second procedure uses limbal cells from cadavers. This procedure provides a better solution, but the availability of the tissue is a problem. To solve the problem of insufficiency of tissue, several labs have tried to culture the limbal cells and grow them to larger quantities.

A paper in the British Journal of Ophthalmology by Basu and others from the Prasad Eye Institute in Hyderbad, India has examined many patients who received limbal cell transplantations from cadavers. They followed 28 eyes from 21 patients who suffered from limbal stem cell deficiency (LSCD). While this disease is relatively rare, it prevents patients from receiving limbal cell grafts from their own eyes, since both eyes are deficient for limbal stem cells. These patients were treated between 2001 and 2010, and all limbal cells were cultured in the laboratory first and then transplanted 10-14 days after their removal from the cadaver.

Each patient was followed up after surgery for about 5 years, and 71.4% of all patients showed eyes that were stable and clear. The eye sight in the treated eye improved to 20/60 or better in 19 of the 28 treated eyes. There were no serious ocular complications in any patients.

This paper shows that transplantation of cultured limbal cells from cadavers successfully restores the surface of the eye and improves vision in patients with blindness as a result of LSCD. This same technique can also be applied to patients with other types of eye surface disorders. Limbal stem cell transplantations seem to keep improving and they will hopefully become rather routine.

Skin Cells Reprogrammed into Blood Vessel Cells

Induced pluripotent stem cells result from the conversion of adult cells into embryonic-like stem cells by means of genetic engineering techniques. In a nutshell, four specific genes (Oct4, Sox2, and c-Myc and Klf4, or Lin 28 and Nanog; see Takahashi K, Yamanaka S Cell 126:663–676 and Yu J, et al. Science 318:1917–1920) are introduced into adult cells by means of viral vectors (retroviral or adenoviral, see Maherali N, et al. Cell Stem Cell 1:55–70, Okita K, Ichisaka T, Yamanaka S Nature 448:313–317, and Stadtfeld M, et al., Science 322:945–949), plasmids (see Chang CW, et al. Stem Cells 27:1042–104 and Sommer CA, et al. Stem Cells 28:64–74), purified recombinant proteins (Zhou H, et al. Cell Stem Cell 4:381–384), or modified RNA molecules (see Warren L, et al. Cell Stem Cell 7:618–630; Desponts C, Ding S Methods Mol Biol 636:207–218, and Li W, Ding S Trends Pharmacol Sci 31:36–45).

Even though iPSCs have the ability to differentiate into a whole host of cell types, they are limited by their ability to form tumors and the mutations induced by the reprogramming process. Therefore, scientists have been trying to find a way to skip the embryonic-like state when it comes to making cells for therapeutic purposes. Therefore, one paper describes the production of “partial induced pluripotent stem” (PiPS) cells that do not cause tumors in laboratory animals and can still be differentiated into different cell types.

This report was published in the Proceedings of the National Academy of Sciences USA on August 21, 2012. The scientists involved in this work were from the Cardiovascular Division of King’s College London British Heart Foundation Center, UK.

To make PiPS cells, human skin fibroblasts were reprogrammed by means of a plasmid that drove the expression of Oct4, Sox2, Klf4 and c-Myc for 4 days only. This converted the fibroblasts into PiPS cells but not iPSCs. They also found that these PiPS cells could be differentiated into blood vessel cells (endothelial cells or ECs). In fact, PiPS cells differentiated into ECs very readily. The PiPS cell-derived expressed the major proteins normally found in blood vessels, and they no longer expressed any of the genes associated with pluripotency. The ECs also made blood vessels when grown under the right culture conditions (Matrigel plugs), and when injected into laboratory animals, they also made blood vessels.

The next group of experiments examined the mechanisms by which cells become ECs. There is a protein in cells called SETSIP (stands for “similar to SET translocation protein”) that is known to play some role in inducing cells to become ECs. Workers from the laboratory of Qingbo Xu showed that when PiPS cells are treated with a growth factor called VEGF (vascular endothelial growth factor), SETSIP moves into the nucleus and induces the expression of a protein that is specifically expressed on the surface of ECs (VE-cadherin). In fact, when SETSIP expression was decreased with small molecules, no the PiPs cells were completely unable to make blood vessels.

The PiPS cells could even be induced to form pure cultures of ECs. The group went the next step and implanted their PiPS cell-derived ECs into mice that had blocked blood vessels in their hind limbs. The transplanted ECs made blood vessels in the mice and prevented the hind limbs from experiencing damage from a lack of oxygen.  Also, further examination of the implanted cells showed that they indeed did form blood vessels that looked and functioned like normal blood vessels and expressed all the genes  of blood vessels.  See the figure below.

PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. PiPS-ECs, fibroblasts, or medium control (no cells) were injected i.m. into adductors of an ischemic model of SCID mice. (A) Representative color laser Doppler images of superficial blood flow (BF) in lower limbs taken 2 wk after ischemia induction. (B) Line graph shows the time course of postischemic foot BF recovery (calculated as the ratio between ischemic foot BF and contralateral foot BF) in mice given medium as control, fibroblasts, and PiPS-ECs. Statistical analysis showed significantly higher foot BF recovery for PiPS-ECs in comparison with both “no cells” control and fibroblasts at weeks 1 and 2 [data are means ± SEM (n = 6)]. Week 1: **P < 0.01, PiPS-EC vs. “no cells” control; **P < 0.01, PiPS-EC vs. fibroblasts. Week 2: **P < 0.01, PiPS-EC vs “no cell” control; *P < 0.05 PiPS-EC vs. fibroblasts. No significant differences were detected when fibroblasts were compared with “no cells” control. (C) Sections of adductors muscles were stained with CD31 antibody, and capillary density was expressed as the capillary number per mm2 [D; data are means ± SEM (n = 3); *P < 0.05]. (Scale bar, 100 μm.) (E) PiPS-ECs displayed an enhanced engraftment ability compared with fibroblasts when stained and quantified with a human-specific CD31 antibody at six randomly selected microscopic fields (at ×100) [F; data are means ± SEM (n = 3); *P < 0.05]. (Scale bar, 50 μm.)
Thus, this British research group has managed to make stem cells from adult cells that can be differentiated into blood vessels, without the risk of causing tumors. In the concluding words of the authors: “PiPS cells can be a useful cell source for regenerating damaged tissue and vascular engineering ex vivo.” We hope to see safety studies and progression of these cells to clinical trials soon.

See Andriana Margariti, et al., Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels. PNAS August 21, 2012 vol. 109 no. 34 13793-13798.

Embryonic Stem Cell-Derived Nerve Cells Restore Hearing to Deaf Animals

In a remarkable study, a research team from the University of Sheffield in England has improved the hearing of deaf animals by using embryonic stem cells. This result should certainly give new hope to those who suffer from hearing disorders.

In this study, an uncommon for of deafness that affects perhaps less than 1% and no more than 15% of all hearing-impaired patients, was treated. Even though this treatment would not benefit all cases of hearing impairment, the strategy developed in this study could be expanded to apply to other cases of deafness. Since these results are strictly pre-clinical in nature, it will be years before human patients might benefit from them.

This work used gerbils are a model system and the results were reported in the international journal Nature. The research team was led by Dr. Marcelo Rivolta and the scientists in his laboratory.

To induce deafness in the gerbils, the scientists ablated (killed off) those nerve cells that transmit auditory information from the ear to the brain. The nerve cells are called “spiral ganglia neurons” or SGNs, and if a patient suffers damage to the SGNs, they will not be able to receive a cochlear implant to restore their hearing. Therefore, this experiment attempted to replace these SGN cells in order the restore hearing.

Rivolta’s group used human embryonic stem cell (hESC) lines H7, H14, and Shef1 and treated them with two growth factors, FGF3 and FGF10. The combination of these two growth factors induced the expression of a whole host of genes normally found in SGN cells (for example, Pax8 & Sox2). These treatments converted the hESCs into otic neural progenitors (ONPs).

In order to destroy the SGN cells in the ears of gerbils, Rivolta and others used a drug called ouabain. This drug, when injected into the inner ear, will destroy the SGN cells and make the animals completely deaf. In the next experiment, Rivolta et al. transplanted the immature nerve cells into the ears of 18 gerbils. One ear received the transplantation, while the other ear was kept as is as a control.

10 weeks later, they used electrophysiology tests to measure the response of the brain to sound. Of the 18 gerbils transplanted with the hESC-derived ONPs, the animals had recovered their hearing by an average of 46%. The recovery differed from animal to animal, but it ranged from modest recovery to almost complete in others.

All animal subjects were kept on anti-rejection medications to prevent rejecting the implanted human cells. In order to prevent tissue rejection in human patients, either induced pluripotent stem cells should be used, or hESCs that match the tissue types in the patient.

Rivolta’s team is also in the process of making immature versions of a second kind of inner-ear cell, that is, the “hair cell” that detects the auditory vibrations in the cochlea. The induction of ONPs from hESCs tends to produce two types cells: ONPs and otic epithelial progenitors (OEPs), which are the precursors of cochlear “hair cells.” Since damage to hair cells is far more common in cases of hearing loss, implantation of such cells should be able to treat far more cases of hearing loss. Unfortunately, this has not yet been tested in animals, according to Rivolta.

Yehoash Raphael of the University of Michigan, who didn’t participate in the work, said it’s possible the stem cell transplants worked by stimulating the gerbils’ own few remaining nerve cells, rather than creating new ones. But either way, “this is a big step forward in use of stem cells for treating deafness.”

Likewise, Lawrence Lustig of the University of California, San Francisco, said, “It’s a dynamite study (and) a significant leap forward.”

Grafting Fat-Based Stem Cells Treats Cutaneous Radiation Syndrome.

If you are exposed to high local doses of radiation, your skin will burn and undergo very slow healing. Your skin will also experience a great deal of cell death, and high levels of cell death within a tissue cause a condition called necrosis. High levels of local radiation, which cause painful necrosis, slow healing and a delayed outcome, are characteristics of “cutaneous radiation syndrome.”

Recently, work on cutaneous therapeutic management of patients with cutaneous radiation syndrome has strongly suggested that such burn patients would benefit from stem cell treatments with mesenchymal stem cells. A paper from the laboratory of Michel Drouet, who is a member of the Radiology Department at the Institut de Recherche Biomédicale des Armées in La Tronche, France has examined such a treatment strategy in pigs.

Diane Riccobono and her colleagues compared the effectiveness of an animal’s own stem cells with the effectiveness of borrowed stem cells from another unrelated animal. They used minipigs for these experiments, and the animals were exposed to about 50 Grays of radiation, which is about the radiation dose someone would receive for radiation therapy. The animals were divided into three groups.

One group was engrafted with their own fat-based mesenchymal stem cell (5 animals in this group). The second group was engrafted with fat-based mesenchymal stem cells from another animals (5 animals in this group too). Animals received fat-based mesenchymal stem cells four times after receiving their dose of radiation. A third group consisting of eight animals received culture media but no cells.

All the pigs were examined and scored according to the severity of their wounds. The control animals showed local inflammatory that led to persistent painful necrosis. Since this display is very similar to what is observed in human patients with cutaneous radiation syndrome, it gave Riccobono and her colleagues a great deal of confidence that this animal model nicely mimics the clinical progression of this disease in human patients. Also, the clinical outcome was not significantly different in the animals treated with fat-based mesenchymal stem cells from another unrelated animal. These animals showed skin healing without necrosis, and the animals suffered from uncontrollable pain, much like the controls. However, in the animals engrafted with fat-based stem cells from their own bodies, the radiation wounds healed without necrosis. Furthermore, healing also did not progress to uncontrollable pain.

This study seems to show that stem cell grafting with fat-based stem cells a patient’s own body improves healing in patients with cutaneous radiation syndrome. However, fat-based mesenchymal stem cells from unrelated animals do not facilitate such healing. Can manipulation of allogeneic stem cells improve their therapeutic potential? Only further work will tell.

See Riccobono D, Agay D, Scherthan H, Forcheron F, Vivier M, Ballester B, Meineke V, Drouet M.., Application of adipocyte-derived stem cells in treatment of cutaneous radiation syndrome. Health Phys. 2012 Aug;103(2):120-6.

Newly Identified Stem Cell Population In Skin Is Responsible for Wound Healing

BRUSSELS, Belgium, September 3, 2012 – Skin researchers from the Universitй Libre de Bruxelles, Belgium have discovered a new stem cell population in skin that is responsible for tissue repair.

Our skin protects our bodies from the environment and its toxins, hard knocks, and extremes of temperature, pressure and so on. Consequently, the skin is subject to constant replacement and dead cells are sloughed off and replaced throughout our lifetimes.

However, the number of cells generated by the skin must exactly replace those that are lost. Different theories have been proposed to explain how this delicate balance is maintained.

In this new study, Prof. Cйdric Blanpain and his colleagues have collaborated with Prof. Benjamin Simons at the University of Cambridge, U.K. to show that a new population of stem cells in the skin give rise to a population of progenitor cells that are involved in the daily maintenance of the upper layers of the skin (epidermis). In fact, these stem cells are the major contributor during wound healing.

Blanpain and others used a novel genetic lineage tracing protocol to fluorescently mark two distinct skin cell populations, and follow their survival and contribution to the maintenance of the epidermis over time. These labeling experiments demonstrated the existence of two types of dividing cells. One cell population showed very long-term survival potential while the other population is progressively lost over time.

With Benjamin D. Simons, Blanpain and his lab devised a mathematical model of their lineage tracing analysis. The model suggested that skin, particularly the epidermis, contains a population of stem cells that divide very slowly that give rise to very fast dividing progenitor cells that ensure the daily maintenance of the skin epidermis. Cell proliferation patterns confirmed the existence of slow cycling stem cells. Furthermore, gene profiling experiments showed that the stem and the progenitor cells are characterized by distinct patterns of gene expression.

By assessing the contribution of these two cell populations during wound healing, they showed that only the skin stem cells were capable of extensive tissue regeneration and undergo major expansion during this repair process. The progenitors, on the other hand, did not expand significantly, but provided a short-lived contribution to the wound healing response.

These data resolve a long-standing debate regarding the cell populations that contribute to wound healing in the skin. Apparently, these epidermal stem cells are the main players during wound healing.

“It was amazing to see these long trails of cells coming from a single stem cell located at a very long distance from the wound to repair the epidermis,” said Dr.  Blanpain, who was the senior author of the study.

Thus the slow-dividing stem cells promote tissue repair and more the more rapidly dividing progenitors ensure the daily maintenance of the epidermis.

Interestingly, similar populations of slow cycling stem cells that can be rapidly mobilized in case of sudden need have been observed in other tissues, such as the blood, muscle and hair follicle. The division between rapidly cycling progenitors and slow cycling stem cells seems to be relatively conserved across the different tissues.

Of course, these findings may have important implications in regenerative medicine; in particular for skin repair in severely burnt patients or in chronic wounds.

Genome-Wide Scan Show Extra Copies of Sox2 in Deadly Lung Cancers

Researchers from several cancer research laboratories, including laboratories from Johns Hopkins, Genentech, the University of Texas Southwestern Medical Center and the University of Colorado Cancer Center have collaborated in a remarkable series of whole genome scans of lung cancers that reveal some potentially troubling results for the use of induced pluripotent stem cells.

Genome-wide scans use next generation sequencing to sequence the entire genome of cells. Such sequencing has dropped the price of DNA sequencing dramatically and also greatly accelerated the speed at which whole genome can be sequenced. Also, advances in computation all rapid comparison of the whole genome of cells allows rapid identification of mutations that might contribute to diseases.

These scientists used whole genome scans to examine very aggressive types of lung cancers, known as small cell lung cancer. Small cell lung cancer exhibits aggressive behavior, with rapid growth, and early spread to distant sites.

Among the mutations found in small cell lung cancers, these scientists, including those at the Johns Hopkins Kimmel Cancer Center, found an alteration in a gene called SOX2; a gene that is very active during early embryonic development.

Charles Rudin said: “Small cell lung cancers are very aggressive. Most are found late, when the cancer has spread and typical survival is less than a year after diagnosis. Our genomic studies may help identify genetic pathways responsible for the disease and give us new ideas on developing drugs to treat it.”

In this study, the research groups scanned the coding regions in the entire genomes of 63 small cell lung cancers. They also included 42 cancer samples whose sequenced genomes were matched with from the patients’ normal cells.

The scientists found an increase in the copy number of the SOX2 gene in about 27 percent of all screened small cell lung cancer samples. These copy number increases caused overproduction of proteins made by the SOX2 gene, and this plays a role in driving the abnormal cell growth observed in small cell lung cancer. Therefore, SOX2 offers a potential new target for those scientists working to develop new drugs to combat this intractable cancer. “SOX2 is an important clue in finding new ways to treat small cell lung cancer,” says Rudin. “We may be able to link a patient’s outcome to this gene and develop a drug to target it or other genes it regulates.” Rudin says his team will further explore the function of SOX2 and how to target it.

The SOX2 protein forms a complex with other proteins that bind to DNA and controls when and how genes are expressed. Sox2 is one of the four genes used by scientists to convert adult cells into an embryonic state. This study also seems to sound a caution to the use of induced pluripotent stem cells (iPSCs) in a clinical setting. iPSCs are made by overexpressing Sox2 and three other genes in adult cells. Overexpression of these genes de-differentiates adult cells to an embryonic state, but the activated growth also causes increased chances of acquiring mutations.

This study shows that acquisition of extra copies of Sox2 can increase the aggressive behavior of small cell lung cancers. However, iPSCs are made by endowing them with extra copies of the Sox2 gene. Also, the iPSC-derivation procedure tends to cause them to possess extra copies of particular regions of the genome too. Therefore, iPSCs would seem to be at risk for more aggressive, tumor-like behavior in the first place, and this would also seem to make iPSCs much more risky as treatment options than other options.

Having said all that, I must point out that induced pluripotent stem cell lines differ tremendously from one line to another, and clearly some iPSC lines have a greater tendency to cause tumors than others. Therefore, this finding is almost certainly not fatal to iPSCs. It just means that if iPSCs are going to be used for clinical purposes, they must be rigorously tested and evaluated for safety before they are used.

This study was published online Sept. 2 in Nature Genetics.