Josh Brahm runs the Life Report podcast and is one of the nicest guys on the planet. Josh invited me to his podcast at the end of last year to talk about my book and stem cell research in general. The editing of that exchange has become available.
Embryonic stem cells have been the gold standard for pluripotent stem cells. Pluripotent means capable of differentiating into one of many cell types in the adult body. Ever since James Thomson isolated the first human embryonic stem cell lines in 1998, scientists have dreamed of using embryonic stem cells to treat diseases in human patients.
However, deriving human embryonic stem cell lines requires the destruction or molestation of a human embryo, the smallest, youngest, and most vulnerable member of our community. In 2006, Shinya Yamanaka and his colleges used genetic engineering techniques to make induced pluripotent stem (iPS) cells, which are very similar to embryonic stem cells in many ways. Unfortunately, the derivation of iPSCs introduces mutations into the cells.
Now, researchers from Brigham and Women’s Hospital (BWH), in Boston, in collaboration with the RIKEN Center for Developmental Biology in Japan, have demonstrated that any mature adult cell has the potential to be converted into the equivalent of an embryonic stem cell. Published in the January 30, 2014 issue of the journal Nature, this research team demonstrated in a preclinical model, a novel and unique way to reprogram cells. They called this phenomenon stimulus-triggered acquisition of pluripotency (STAP). Importantly, this process does not require the introduction of new outside DNA, which is required for the reprogramming process that produces iPSCs.
“It may not be necessary to create an embryo to acquire embryonic stem cells. Our research findings demonstrate that creation of an autologous pluripotent stem cell – a stem cell from an individual that has the potential to be used for a therapeutic purpose – without an embryo, is possible. The fate of adult cells can be drastically converted by exposing mature cells to an external stress or injury. This finding has the potential to reduce the need to utilize both embryonic stem cells and DNA-manipulated iPS cells,” said senior author Charles Vacanti, MD, chairman of the Department of Anesthesiology, Perioperative and Pain Medicine and Director of the Laboratory for Tissue Engineering and Regenerative Medicine at BWH and senior author of the study. “This study would not have been possible without the significant international collaboration between BWH and the RIKEN Center,” he added.
The inspiration for this research was an observation in plant cells – the ability of a plant callus, which is made by an injured plant, to grow into a new plant. These relatively dated observations led Vacanti and his collaborators to suggest that any mature adult cell, once differentiated into a specific cell type, could be reprogrammed and de-differentiated through a natural process that does not require inserting genetic material into the cells.
“Could simple injury cause mature, adult cells to turn into stem cells that could in turn develop into any cell type?” hypothesized the Vacanti brothers.
Vacanti and others used cultured, mature adult cells. After stressing the cells almost to the point of death by exposing them to various stressful environments including trauma, a low oxygen and acidic environments, researchers discovered that within a period of only a few days, the cells survived and recovered from the stressful stimulus by naturally reverting into a state that is equivalent to an embryonic stem cell. With the proper culture conditions, those embryonic-like stem cells were propagated and when exposed to external stimuli, they were then able to redifferentiate and mature into any type of cell and grow into any type of tissue.
To examine the growth potential of these STAP cells, Vacanti and his team used mature blood cells from mice that had been genetically engineered to glow green under a specific wavelength of light. They stressed these cells from the blood by exposing them to acid, and found that in the days following the stress, these cells reverted back to an embryonic stem cell-like state. These stem cells then began growing in spherical clusters (like plant callus tissue). The cell clusters were introduced into developing mouse embryos that came from mice that did not glow green. These embryos now contained a mixture of cells (a “chimera”). The implanted clusters were able to differentiate into green-glowing tissues that were distributed in all organs tested, confirming that the implanted cells are pluripotent.
Thus, external stress might activate unknown cellular functions that set mature adult cells free from their current commitment to a particular cell fate and permit them to revert to their naïve cell state.
“Our findings suggest that somehow, through part of a natural repair process, mature cells turn off some of the epigenetic controls that inhibit expression of certain nuclear genes that result in differentiation,” said Vacanti.
Of course, the next step is to explore this process in more sophisticated mammals, and, ultimately in humans.
“If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells. But for me the most interesting questions will be the ones that let us gain a deeper understanding of the basic principles at work in these phenomena,” said first author Haruko Obokata, PhD.
If human cells can be made into embryonic stem cells by a similar process, then someday, a simple skin biopsy or blood sample might provide the material to generate embryonic stem cells that are specific to each individual, without the need for genetic engineering or killing the smallest among us. This truly creates endless possibilities for therapeutic options.
Scientists might be able to offer people with less that optimal amounts of hair new hope when it comes to reversing baldness. Researchers from the University of Pennsylvania say they’ve moved closer to using stem cells to treat thinning hair — at least in mice.
This group said that the use of stem cells to regenerate missing or dying hair follicles is considered a potential way to reverse hair loss. However, the technology did not exist to generate adequate numbers of hair-follicle-generating stem cells.
But new findings indicate that this may now be achievable. “This is the first time anyone has made scalable amounts of epithelial stem cells that are capable of generating the epithelial component of hair follicles,” Dr. Xiaowei Xu, an associate professor of dermatology at Penn’s Perelman School of Medicine, said in a university news release.
According to Xu, those cells have many potential applications that extend to wound healing, cosmetics and hair regeneration.
In their new study, Xu’s team converted induced pluripotent stem cells (iPSCs) – reprogrammed adult stem cells with many of the characteristics of embryonic stem cells – into epithelial stem cells. This is the first time this has been done in either mice or people.
The epithelial stem cells were mixed with certain other cells and implanted into mice. They produced the outermost layers of the skin and hair follicles that are similar to human hair follicles. This study was published in the Jan. 28 edition of the journal Nature Communications.
This suggests that these cells might eventually help regenerate hair in people.
Xu said this achievement with iPSC-derived epithelial stem cells does not mean that a treatment for baldness is around the corner. Hair follicles contain both epithelial cells and a second type of adult cells called dermal papillae.
“When a person loses hair, they lose both types of cells,” Xu said. “We have solved one major problem — the epithelial component of the hair follicle. We need to figure out a way to also make new dermal papillae cells, and no one has figured that part out yet.”
Experts also note that studies conducted in animals often fail when tested in humans.
Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.
In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.
“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.
Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.
Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”
Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.
“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.
Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.
With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.
“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.
Japanese and American scientists have made a fantastic discovery. An incurable type of chromosomal abnormality, in which one end of the chromosome has fused with the other, is known as a “ring chromosome.” The presence of ring chromosomes often correlates with neurological abnormalities. The collaborative work by these two researcher groups has shown that if induced pluripotent stem cells are derived from abnormal adult cells that contain ring chromosomes will spontaneously repair themselves.
This research team, which included researchers from Kyoto University professor and iPS cell pioneer Shinya Yamanaka, also included Yohei Hayashi and other researchers from the U.S.-based Gladstone Institutes.
“I was very surprised at the results,” said Hayashi. “There still remains a risk, but the findings may lead to the development of breakthrough treatment for chromosomal abnormalities.”
Normal chromosomes pairs consist of two rod-shaped chromosomes, but in the case of a ring chromosome, the arms of one of the two chromosomes are fused to form a ring.
Ring chromosomes tend to be associated with mental disabilities and growth retardation, and there are no therapeutic strategies for ring chromosomes.
Hayashi and his colleagues developed iPS cells from skin cells of patients with ring chromosome disorder to study the effects of this chromosomal defect in stem cells. However, the ring chromosomes could not be observed in the engineered iPS cells made from this patient’s cells.
For reasons still unknown, only normal chromosomes survived, according to the researchers. Typically, a cell that contains ring chromosomes divides into two abnormal cells in the normal mitotic process.
These findings were published in the online edition of the British science journal Nature on Jan. 13, 2014.
Researchers from the Centre for Genomic Regulation in Barcelona, Spain, have discovered an even faster and more efficient way to reprogram adult cells to make induced pluripotent stem cells (iPSCs).
This new discovery decreases the time it takes to derived iPSCs from adult cells from a few weeks to a few days. It also elucidated new things about the reprogramming process for iPSCs and their potential for regenerative medical applications.
iPSCs behave similarly to embryonic stem cells, but they can be created from terminally differentiated adult cells. The problem with the earlier protocols for the derivation of iPSCs is that only a very small percentage of cells were successfully reprogrammed (0.1%-2%). Also this reprogramming process takes weeks and is a rather hit-and-miss process.
The Centre for Genomic Regulation (CRG) research team have been able to reprogram adult cells very efficiently and in a very short period of time.
“Our group was using a particular transcription factor (C/EBPalpha) to reprogram one type of blood cells into another (transdifferentiation). We have now discovered that this factor also acts as a catalyst when reprogramming adult cells into iPS,” said Thomas Graf, senior group leader at the CRG and ICREA research professor.
“The work that we’ve just published presents a detailed description of the mechanism for transforming a blood cell into an iPS. We now understand the mechanics used by the cell so we can reprogram it and make it become pluripotent again in a controlled way, successfully and in a short period of time,” said Graf.
Genetic information is compacted into the nucleus like a wadded up ball of yarn. In order to access genes for gene expression, that ball of yarn has to be unwound so that the cell can find the information it needs.
The C/EBPalpha (CCAAT/Enhancer Binding Protein alpha) protein temporarily unwinds that region of DNA that contains the genes necessary for the induction of pluripotency. Thus, when the reprogramming process begin, the right genes are activated and they enable the successful reprogramming all the cells.
“We already knew that C/EBPalpha was related to cell transdifferentiation processes. We now know its role and why it serves as a catalyst in the reprogramming,” said Bruno Di Stefano, a PhD student. “Following the process described by Yamanaka the reprogramming took weeks, had a very small success rate and, in addition, accumulated mutations and errors. If we incorporate C/EBPalpha, the same process takes only a few days, has a much higher success rate and less possibility of errors, said Di Stefano.
This discovery provides a remarkable insight into stem cell-forming molecular mechanisms, and is of great interest for those studies on the early stages of life, during embryonic development. At the same time, the work provides new clues for successfully reprogramming cells in humans and advances in regenerative medicine and its medical applications.
Research groups at the University of Manchester, and University College, London, UK, have developed a new technique for reprogramming adult cells into induced pluripotent stem cells that greatly reduces the risk of tumor formation.
Kostas Kostarelos, who is the principal investigator of the Nanomedicine Lab at the University of Manchester said that he and his colleagues have discovered a safe protocol for reprogramming adult cells into induced pluripotent stem cells (iPSCs). Because of their similarities to embryonic stem cells, many scientist hope that iPSCs are a viable to embryonic stem cells.
How did they do it? According to Kostarelos, “We have induced somatic cells within the liver of adult mice to transient behave as pluripotent stem cells,” said Kostarelos. “This was done by transfer for four specific gene, previously described by the Nobel-prize winning Shinya Yamanaka, without the use of viruses but simply plasmid DNA, a small circular, double-stranded piece of DNA used for manipulating gene expression in a cell.”
This technique does not use viruses, which was the technique of choice in Yamanaka’s research to get genes into cells. Viruses like the kind used by Yamanaka, can cause mutations in the cells. Kostarelos’ technique uses no viruses, and therefore, the mutagenic properties of viruses are not an issue.
Kostarelos continued, “One of the central dogmas of this emerging field is that in vivo implantation of (these stem) cells will lead to their uncontrolled differentiation and the formation of a tumor-like mass.”
However, Kostarelos and his team have determined that the technique they designed does not show this risk, unlike the virus-based methods.
“[This is the ] only experimental technique to report the in vivo reprogramming of adult somatic cells to plurpotentcy using nonviral, transient, rapid and safe methods,” said Kostarelos.
Since this approach uses circular plasmid DNA, the tumor risk is quite low, since plasmid DNA is rather short-lived under these conditions. Therefore, the risk of uncontrolled growth is rather low. While large volumes of plasmid DNA are required to reprogram these cells, the technique appears to be rather safe in laboratory animals.
Also, after a burst of expression of the reprogramming factors, the expression of these genes decreased after several days. Furthermore, the cells that were reprogrammed differentiated into the surrounding tissues (in this case, liver cells). There were no signs in any of the laboratory animals of tumors or liver dysfunction.
This is a remarkable proof-of-principle experiment that shows that reprogramming cells in a living body is fast and efficient and safe.
A great deal more work is necessary in order to show that such a technique can use useful for regenerative medicine, but it is certainly a glorious start.
Using induced pluripotent stem cells to have heart muscle cells is one of the goals of regenerative medicine. Successful cultivation of heart muscle cells from a patient’s own cells would provide material to replace dead heart muscle, and could potentially extend the life of a heart-sick patient.
Unfortunately, induced pluripotent stem cells, which are made by applying genetic engineering techniques to a patient’s own adult cells, like embryonic stem cells, will cause tumors when implanted into a living organism. To beat the problem of tumor formation, scientists must be able to efficiently isolate the cells that have properly differentiated from those cells that have not differentiated.
A new paper from a laboratory the Emory University School of Medicine in Atlanta, Georgia, have used “molecular beacons” to purify heart muscle cells from induced pluripotent stem cells, thus bringing us one step closer to a protocol that isolates pure heart muscle cells from induced pluripotent stem cells made from a patient’s own cells.
Molecular beacons are nanoscale probes that fluoresce when they bind to a cell-specific messenger RNA molecule. Because heart muscle cells express several genes that are only found in heart muscle cells, Kiwon Ban in the laboratory of Young-Sup Yoon designed heart muscle-specific molecular beacons and used them to purify heart muscle cells from cultured induced pluripotent stem cells from both mice and humans.
The molecular beacons made by this team successfully isolated heart muscle cells from an established heart muscle cell line called HL-1. Then Ban and co-workers applied these heart-specific molecular beacons to successfully isolate heart muscle cells that were made from human embryonic stem cells and human induced pluripotent stem cells. The purity of their isolated heart muscle cells topped 99% purity.
Finally, Ban and others implanted these heart muscle cells into the hearts of laboratory mice that had suffered heart attacks. When heart muscle cells that had not been purified were used, tumors resulted. However, when heart muscle cells that had been purified with their molecular beacons were transplanted, no tumors were observed and the heart function of the mice that received them steadily increased.
Because the molecular beacons are not toxic to the cells, they are an ideal way to isolate cells that have fully differentiated to the desired cell fate away from potentially tumor-causing undifferentiated cells. in the words of Ban and his colleagues, “This purification technique in combination with cardiomyocytes (heart muscle cells) generated from patient-specific hiPSCs will be of great value for drug screening and disease modeling, as well as cell therapy.”
The aorta is the largest blood vessel in our bodies and it emerges from the left ventricle of the heart, takes a U-turn, and swings down toward the legs (descending or dorsal aorta). There are several branches of the aorta as it sharply turns that extend towards the head and upper extremities.
Sometimes, as a result of inflammation of the aorta or other types of problems, the elastic matrix that surrounds and reinforces the aorta breaks down. This weakens the wall of the aorta and it bulges out. This bulge is called an aortic aneurysm and it is a dangerous condition because the aneurysm can burst, which will cause the patient to bleed to death.
If an aneurysm is discovered through medical imaging techniques, drugs are given to lower blood pressure and take some of the pressure off the aorta. Also, drugs that prevent further degradation of the elastic matrix are also used. Ultimately, for large or fast-growing aneurysms, surgical repair of the aorta is necessary. For aneurysms of the abdominal aorta, a surgical procedure called abdominal aortic aneurysm open repair is the “industry standard.” For this surgery, the abdomen is cut open, and the aneurysm is repaired by the use of a long cylinder-like tube called a graft. Such grafts are made of different materials that include Dacron (textile polyester synthetic graft) or polytetrafluoroethylene (PTFE, a nontextile synthetic graft). The surgeon sutures the graft to the aorta, and connects one end of the aorta at the site of the aneurysm to the other end.
A “kinder, gentler” way to fix an aneurysm is to use a procedure called endovascular aneurysm repair (EVAR). EVAR uses these devices called “stents” to support the wall of the aorta. A small insertion is made in the groin and the collapsed stent is inserted through the large artery in the leg. Then the stent, which is long cylinder-like tube made of a thin metal framework and covered with various materials such as Dacron or polytetrafluoroethylene (PTFE), is inserted into the aneurysm. Once in place, the stent-graft will be expanded in a spring-like fashion to attach to the wall of the aorta and support it. The aneurysm will eventually shrink down onto the stent-graft.
In some cases, the patient is too weak for surgery, and is not a candidate for EVAR. A much better option would be to non-surgically repair the elastic support framework that surrounds the aorta, and stem cells are candidates for such repair.
To repair the elastic mesh work that surrounds the wall of the aorta, smooth muscle cells that secrete the protein “elastin” must be introduced into the wall of the aorta. Also, using the patient’s own stem cells offers a better strategy at this point, since this circumvents such issues as immune rejection of implanted tissues and so on. The sources of stem cells for smooth muscle cells include bone marrow stem cells, fat-based stem cells, and stem cells from peripheral blood. All three of these stem cell sources have problems with finding enough cells in the body and expanding them to high enough numbers in order to properly treat the aneurysm.
Fortunately, the use of induced pluripotent stem cells, which are made from a patient’s mature cells and have many, though not all of the characteristics of embryonic stem cells, can provide large quantities of elastin-secreting smooth muscle cells. Also, one laboratory in particular has reported differentiating human induced pluripotent stem cells into smooth muscle cells (Lee TH, Song SH, Kim KL, et al. Circ Res 106:120–128). While there are challenges to making functional elastin, there are possibilities that many of these can be overcome.
In addition to induced pluripotent stem cells, other laboratories have examined umbilical cord mesenchymal stem cells and their ability to decrease the inflammation within the aorta that leads to aneurysms. The researchers discovered that all the indicators of inflammation decreased, but the synthesis of new elastin was not examined. However, a Japanese laboratory used mouse mesenchymal stem cells from bone marrow and found that not only did these cells shut down enzymes that tend to degrade elastin, but also initiated new elastin synthesis in culture. The same study also showed that MSCs implanted into the vessel walls of an aorta that was experiencing an aneurysm stabilized the aneurysm by inhibiting the elastin-degrading enzymes, and increasing the elastin content of the vessel wall. This had the net effect of stabilizing the aneurysms and preventing them from growing further (see Hashizume R, Yamawaki-Ogata A, Ueda Y, et al. J Vasc Surg 54:1743–1752).
These experiments show that stem cell treatments for abdominal aneurysms are feasible and would definitely be a much-needed nonsurgical treatment option for the high-risk elderly demographic, which is rapidly growing in the developed world.
For more information on this interesting topic, see Chris A. Bashura, Raj R. Raob and Anand Ramamurthia. Perspectives on Stem Cell-Based Elastic Matrix Regenerative Therapies for Abdominal Aortic Aneurysms. Stem Cells Trans Med June 2013 vol. 2 no. 6 401-408.
Stem cell treatments for muscular dystrophy and other degenerative diseases of muscle might be a realistic possibility, since scientists have discovered protocols to make muscle cells from human pluripotent stem cells.
Tiziano Barberi, Ph.D., chief investigator in the Australian Regenerative Medicine Institute (ARMI) at Monash University in Clayton, Victoria, and Bianca Borchin, a graduate student in the Barberi laboratory, have developed techniques to generate skeletal muscle cells. Barberi and Borchin isolated muscle precursor cells from human pluripotent stem cells (hPSCs), after which they applied a purification technique that allows these cells to differentiate further into muscle cells.
Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), have the ability to become any cell in the human body, including skeletal muscles, which control movement. Once the stem cells begin to differentiate, controlling that process is very challenging, but essential in order to produce only the desired cells. Barberi and Borchin used a technique known as fluorescence activated cell sorting (FACS) to identify those cells that contained the precise combination of protein markers that are expressed in muscle precursor cells. FACS also enabled them to successfully isolate those muscle precursor cells.
“There is an urgent need to find a source of muscle cells that could be used to replace the defective muscle fibers in degenerative disease. Pluripotent stem cells could be the source of these muscle cells,” Dr. Barberi said. “Beyond obtaining muscle from hPSCs, we also found a way to isolate the muscle precursor cells we generated, which is a prerequisite for their use in regenerative medicine.”
Borchin said there were existing clinical trials based on the use of specialized cells derived from hPSCs in the treatment of some degenerative diseases, but deriving muscle cells from pluripotent stem cells proved to be challenging. “These results are extremely promising because they mark a significant step towards the use of hPSCs for muscle repair,” she said.
“The production of a large number of pure muscle precursor cells does not only have potential therapeutic applications, but also provides a platform for large-scale screening of new drugs against muscle disease,” Dr. Barberi added.
This study was published early online Nov. 27 in Stem Cell Reports. This study does not address the immune response against dystrophin that has plagued gene therapy and stem cell-based muscular dystrophy clinical trials that has been noted in previous posts. The use of embryonic stem cells, in particular, would create muscles that are not tissue matched to the patient and would generate robust inflammation against the implanted muscles. Thus embryonic stem cells would generate a “cure” that would be much worse than the disease itself. Nevertheless, adapting the Barberi-Borchin protocol to induced pluripotent stem cells would produce skeletal muscle cells that are tissue matched to the patient.
Scientists who work in the pharmaceutical industry have seen this time and time again: A candidate drug that works brilliantly in laboratory animals fails to work in human trials. So what’s up with this?
Now a research consortium from the University of Bonn and the biomedical company Life & Brain GmbH has shown that animal models of Alzheimer’s disease fail to recapitulate the results observed with cultured human nerve cells made from stem cells. Thus, they conclude that candidate Alzheimer’s disease drugs should be tested in human nerve cells rather than laboratory animals.
In the brains of patients with Alzheimer’s disease beta-amyloid protein deposits form that are deleterious to nerve cells. Scientists who work for drug companies are trying to find compounds that prevent the formation of these deposits. In laboratory mice that have a form of Alzheimer’s disease, over-the-counter drugs called NSAIDs (non-steroidal anti-inflammatory drugs), which include such population agents as aspirin, Tylenol, Advil, Nuprin and so on prevent the formation of beta-amyloid deposits. However in clinical trials, the NSAIDs royally flopped (see Jaturapatporn D, Isaac MG, McCleery J, Tabet N. Cochrane Database Syst Rev. 2012 Feb 15;2:CD006378).
Professor Oliver Brüstle, the director of the Institute for Reconstructive Neurobiology at the University of Bonn and Chief Executive Officer of Life and Brain GmbH, said, “The reasons for these negative results have remained unclear for a long time.”
Jerome Mertens, a former member of Professor Brüstle’s research, and the lead author on this work, said, “Remarkably, these compounds were never tested directly on the actual target cells – the human neuron.”
The reason for this disparity is not difficult to understand because purified human neurons were very difficult to acquire. However, advances in stem cell biology have largely solved this problem, since patient-specific induced pluripotent stem cells can be grow in large numbers and differentiated into neurons in large numbers.
Using this technology, Brüstle and his collaborators from the University of Leuven in Belgium have made nerve cells from human patients. These cells were then used to test the ability of NSAIDs to prevent the formation of beta-amyloid deposits.
According to Philipp Koch, who led this study, “To predict the efficacy of Alzheimer drugs, such tests have to be performed directly on the affected human nerve cells.”
Nerve cells made from human induced pluripotent stem cells were completely resistant to NSAIDs. These drugs showed no ability to alter the biochemical mechanisms in these cells that eventually lead to the production of beta-amyloid.
Why then did they work in laboratory animals? Koch and his colleagues think that biochemical differences between laboratory mice and human cells allow the drugs to work in one but not in the other. In Koch’s words, “The results are simply not transferable.”
In the future, scientists hope to screen potential Alzheimer’s disease drugs with human cells made from the patient’s own cells.
“The development of a single drug takes an average of ten years,” said Brüstle. “By using patient-specific nerve cells as a test system, investments by pharmaceutical companies and the tedious search for urgently needed Alzheimer’s medications could be greatly streamlined.”
Scientists from the Columbia University Medical Center have succeeded in transforming human stem cells into functional lung and airway cells. This finding has significant potential for modeling lung disease, screening lung-specific drugs, and, hopefully, generating lung tissue for transplantation.
Study leader, Hans-Willem Snoeck, professor of medicine and affiliated with the Columbia Center for Translational Immunology and the Columbia Stem Cell Initiative, said, “Researchers have had relative success in turning human stem cells into heart cells, pancreatic beta cells, intestinal cells, liver cells, and nerve cells, raising all sorts of possibilities for regenerative medicine. Now, we are finally able to make lung and airway cells. This is important because lung transplants have a particularly poor prognosis. Although any clinical application is still many years away, we can begin thinking about making autologous lung transplants – that is, transplants that use a patient’s own skin cells to generate functional lung tissue.”
The research builds on Snoeck’s earlier discoveries in 2011 that a set of chemical factors could induce the differentiation of embryonic or induced pluripotent stem cells into “anterior foregut endoderm,” which is the embryo in the tissue from which the lungs form (Green MD, et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011 Mar;29(3):267-72).
In his new study, Snoeck and his colleagues found new factors that can transform anterior foregut endoderm cells into lung and airway cells. In particular, Snoeck and his co-workers were able to establish the presence of “type 2 alveolar epithelial cells,” which secrete the lung surfactant that maintains the lung alveoli (those tiny sacs in the lung where all the oxygen exchange takes place).
With these techniques, lung researchers hope to study diseases like idiopathic pulmonary fibrosis (IPF), in which type 2 epithelial cells seem to divide and produce scarring in the lungs.
“No one knows what causes the disease, and there’s no way to treat it,” said Snoeck. “Using this technology, researchers will finally be able to create laboratory models of IPF, study the disease at the molecular level, and screen drugs for possible treatments or cures. In the longer term, we hope to use this technology to make an autologous lung graft. This would entail taking a lung from a donor, removing all the lung cells, leaving only the lung scaffold; and seeding the scaffold with new lung cells derived from the patient. In this way, rejection problems could be avoided.”
Snoeck is investigating this approach in collaboration with researchers in the Columbia University Department of Biomedical Engineering.
Italian researchers have derived stem cells from skin cells that can reduce the damage to the nervous system cause by a mouse version of multiple sclerosis. This experiment provides further evidence that stem cells from patients might be a feasible source of material to treat their own maladies.
The principal investigators in this work, Cecilia Laterza and Gianvito Martino, are from the San Raffaele Scientific Institute, Milan and the University of Milan, respectively.
Because multiple sclerosis results from the immune system attacking the myelin sheath that surrounds nerves, most treatments for this disease consist of agents that suppress the immune response against the patient’s own nerves. Unfortunately, these treatments have pronounced side effects, and are not effective in the progressive phases of the disease when damage to the myelin sheath might be widespread.
The symptoms of loss of the myelin sheath might one or more of the following: problems with touch or other such things, muscle cramping and muscle spasms, bladder, bowel, and sexual dysfunction, difficulty saying words because of problems with the muscles that help you talk (dysarthria), lack of voluntary coordination of muscle movements (ataxia), and shaking (tremors), facial weakness or irregular twitching of the facial muscles, double vision, heat intolerance, fatigue and dizziness; exertional exhaustion due to disability, pain, or poor attention span, concentration, memory, and judgment.
Clinically, multiple sclerosis is divided into the following categories on the basis of the frequency of clinical relapses, time to disease progression, and size of the lesions observed on MRI. These classifications are:
A) Relapsing-remitting MS (RRMS): Approximately 85% of cases and there are two types – Clinically isolated syndrome (CIS): A single episode of neurologic symptoms, and Benign MS or MS with almost complete remission between relapses and little if any accumulation of physical disability over time.
B) Secondary progressive MS (SPMS)
C) Primary progressive MS (PPMS)
D) Progressive-relapsing MS (PRMS)
The treatment of MS has 2 aspects: immunomodulatory therapy (IMT) for the underlying immune disorder and therapies to relieve or modify symptoms.
To treat acute relapses:
A) Methylprednisolone (Solu-Medrol) can hasten recovery from an acute exacerbation of MS.
B) Plasma exchange (plasmapheresis) for severe attacks if steroids are contraindicated or ineffective (short-term only).
C) Dexamethasone is commonly used for acute transverse myelitis and acute disseminated encephalitis.
For relapsing forms of MS, the US Food and Drug Administration (FDA) include the following:
A) Interferon beta-1a (Avonex, Rebif)
B) Interferon beta-1b (Betaseron, Extavia)
C) Glatiramer acetate (Copaxone)
D) Natalizumab (Tysabri)
F) Fingolimod (Gilenya)
G) Teriflunomide (Aubagio)
H) Dimethyl fumarate (Tecfidera)
For aggressive MS:
A) High-dose cyclophosphamide (Cytoxan).
In order to treat multiple sclerosis, restoring the damaged myelin sheath is essential for returning patients to their former wholeness.
In this study, this research team reprogrammed mouse skin cells into induced pluripotent skin cells (iPSCs), and then differentiated them into neural stem cells. Neural stem cells can differentiate into any cell type in the central nervous system.
Next, Laterza and her colleagues administered these neural stem/progenitor cells “intrathecally,” which simply means that they were injected into the spinal cord underneath the meninges that cover the brain and spinal cord to mice that had a rodent version of multiples sclerosis called EAE or experimental autoimmune encephalomyelitis.
EAE mice are made by injecting them with an extract of myelin sheath. The mouse immune system mounts and immune response against this injected material and attacks the myelin sheath that surrounds the nerves. EAE does not exactly mirror multiple sclerosis in humans, but it comes pretty close. While multiple sclerosis does not usually kill its patients, EAE either kills animals or leaves them with permanent disabilities. Animals with EAE also suffer severe nerve inflammation, which is distinct from multiple sclerosis in humans in which some nerves suffer inflammation and others do not. Finally, the time course of EAE is entirely different from multiple sclerosis. However, both conditions are caused by an immune response against the myelin sheath that strips the myelin sheath from the nerves.
The transplanted neural stem cells reduced the inflammation within the central nervous system. Also, they promoted healing and the production of new myelin. However, most of the new myelin was not made by the injected stem cells. Instead the injected stem cells secreted a compound called “leukemia inhibitory factor” that promotes the survival, differentiation and the remyelination capacity of both internal oligodendrocyte precursors and mature oligodendrocytes (these are the cells that make the myelin sheath). The early preservation of tissue integrity in the spinal cord limited the damage to the blood–brain barrier damage. Damage to the blood-brain barrier allows immune cells to infiltrate the central nervous system and destroy nerves. By preserving the integrity of the blood-brain barrier, the injected neural stem cells prevented infiltration of blood-borne of the white blood cells that are ultimately responsible for demyelination and axonal damage.
“Our discovery opens new therapeutic possibilities for multiple sclerosis patients because it might target the damage to myelin and nerves itself,” said Martino.
Timothy Coetzee, chief research officer of the National Multiple Sclerosis Society, said of this work: “This is an important step for stem cell therapeutics. The hope is that skin or other cells from individuals with MS could one day be used as a source for reparative stem cells, which could then be transplanted back into the patient without the complications of graft rejection.”
Obviously, more work is needed, but this type of research demonstrates the safety and feasibility of regenerative treatments that might help restore lost function.
Martino added, “There is still a long way to go before reaching clinical applications but we are getting there. We hope that our work will contribute to widen the therapeutic opportunities stem cells can offer to patients with multiple sclerosis.”
See Cecilia Laterza, et al. iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF. NATURE COMMUNICATIONS 4, 2597: doi:10.1038/ncomms3597.
Using stem cells to model neurodegenerative diseases shows terrific promise, but because the stem cells tend to produce young cells, they often fail to accurately model disorders that show late-onset. To solve this problem, a research group has published a paper in the December 5th edition of the journal Cell Stem Cell that describes an ingenious new method that converts induced pluripotent stem cells (iPSCs) into nerve cells that recapitulate features associated with aging as well as Parkinson’s disease. This simple approach, which involves exposing iPSC-derived cells to a protein associated with premature aging called “progerin,” could provide a way for scientists to use stem cells to model a range of late-onset disorders. This technique could potentially open new avenues for preventing and treating these devastating diseases.
“With current techniques, we would typically have to grow pluripotent stem cell-derived cells for 60 or more years in order to model a late-onset disease,” says senior study author Lorenz Studer of the Sloan-Kettering Institute for Cancer Research. “Now, with progerin-induced aging, we can accelerate this process down to a period of a few days or weeks. This should greatly simplify the study of many late-onset diseases that are of such great burden to our aging society.”
Induced pluripotent stem cells allow scientists to model a specific patient’s disease in a culture dish. By extracting a small sample of skin cells and genetically engineering them with pluripotency factors, the cells are reprogrammed into embryonic-like stem cells that have the ability to differentiate into other disease-relevant cell types like neurons or blood cells. However, iPSC-derived cells are immature and they can take months to become functional. Consequently, their slow maturation process causes iPSC-derived cells to be too young to effectively model diseases that emerge later in life.
To overcome this hurdle, Studer’s team exposed iPSC-derived skin cells and neurons that originated from both young and old donors, to a protein called “progerin.” Progerin is a mutant form of the nuclear lamin proteins that provide structure to the nuclear membrane. Mutations in these proteins cause premature aging and an early death from old age. Short-term exposure of these iPSC-derived cells to progerin caused them to manifest age-associated markers that are normally present in older cells.
Then Studer and others used iPSC technology to reprogram skin cells taken from patients with Parkinson’s disease and differentiated them into dopaminergic neurons; the type of neuron that is defective in these patients. After exposure to progerin, these cultured neurons recapitulated disease-related features, including neuronal degeneration and cell death as well as mitochondrial defects.
“We could observe novel disease-related phenotypes that could not be modeled in previous efforts of studying Parkinson’s disease in a dish,” says first author Justine Miller of the Sloan-Kettering Institute for Cancer Research. “We hope that the strategy will enable mechanistic studies that could explain why a disease is late-onset. We also think that it could enable a more relevant screening platform to develop new drugs that treat late-onset diseases and prevent degeneration.”
Skeletal muscle – that type of voluntary muscle that allows movement – has proven difficult to grow in the laboratory. While particular cells can be differentiated into skeletal muscle cells, forming a coherent, structurally sound skeletal muscle is a tough nut to crack from a research perspective.
Another problem dogging muscle research is the difficulty growing new muscle in patients with muscle diseases such as muscular dystrophy or other types of disorders that weaken and degrade skeletal muscle.
Now research groups at the Boston Children’s Hospital Stem Cell Program have reported that they can boost the muscle mass and even reverse the disease of mice that suffer from a type of murine muscular dystrophy. To do this, this group use a combination of three different compounds that were identified in a rapid culture system.
This ingenious rapid culture system uses the cells of zebrafish (Danio rerio) embryos to screen for these muscle-inducing compounds. These single cells are placed into the well of a 96-well plate, and then treated with various compounds to determine if those chemical induce the muscle formation. To facilitate this process, the zebrafish embryo cells express a very special marker that consists of the myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.” When cells become muscle, they express the myosin light polypeptide 2 gene at high levels. Therefore, any embryo cell that differentiates into muscle should glow a red color.
Once a cocktail of muscle-inducing chemicals were identified in this assay, those same chemicals were used to treat induced pluripotent stem cells made from cells taken from patients with muscular dystrophy. Those iPSCs were treated with the combination of chemicals identified in the zebrafish embryo screen as muscle inducing agents.
The results were outstanding. Leonard Zon from the Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and his colleagues showed that a combination of basic Fibroblast Growth Factor, an adenylyl cyclase activator called forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs). Furthermore, these muscle cells produced engraftable myogenic progenitors that contributed to muscle repair when implanted into mice with a rodent form of muscular dystrophy.
Zon hopes that clinical trials can being soon in order to translate these remarkable results into patients with muscle loss within the next several years. Zon and his co-workers are also screening compounds to address other types of disorders beyond muscular dystrophy.
This paper represents the application of shear and utter genius. However, there is one caveat. The mice into which the muscles were injected were immunodeficient mice whose immune systems are unable to reject transplanted tissues. In human patients with muscular dystrophy, an immune response against dystrophin, the defective protein, has been an enduring problem (for a review of this, see T. Okada and S. Takeda, Pharmaceuticals (Basel). 2013 Jun 27;6(7):813-836). While there have been some technological developments that might circumvent this problem, transplanting large quantities of muscle cells might be beyond the pale. Muscular dystrophy results from disruption of an important junction between the muscle and substratum to which the muscle is secured. This connection is mediated by the “dystrophin-glycoprotein complex.” Structural disruptions of this complex (shown below) lead to unanchored muscle that cannot contract properly, and eventually atrophies and degrades.
This is a remarkable advance, but until the host immune response issue is satisfactorily addressed, it will remain a problem.
Wolfram syndrome is a rare form of diabetes characterized by high blood sugar levels that result from insufficient levels of the hormone insulin. The chronically high blood sugar levels cause degeneration of the optic nerve, leading to progressive vision loss (optic atrophy). Wolfram syndrome patients often also have abnormal pituitary glands that release abnormally low levels of the hormone vasopressin (also known as antidiuretic hormone or ADH), which causes hearing loss caused by changes in the inner ear (sensorineural deafness), urinary tract problems, reduced amounts of the sex hormone testosterone in males (hypogonadism), or neurological or psychiatric disorders.
Diabetes mellitus is typically the first symptom of Wolfram syndrome, usually diagnosed around age 6. Nearly everyone with Wolfram syndrome who develops diabetes mellitus requires insulin replacement therapy. Optic atrophy is often the next symptom to appear, usually around age 11. The first signs of optic atrophy are loss of color vision and peripheral (side) vision. Over time, the vision problems get worse, and people with optic atrophy are usually blind within approximately 8 years after signs of optic atrophy first begin.
Mutations in the WFS1 gene cause more than 90 percent of the cases of Wolfram syndrome type 1. The WFS1 gene encodes a protein called wolframin that regulates the amount of calcium in cells. A proper calcium balance is important for a whole host of cellular processes, including cell-to-cell communication, the tensing (contraction) of muscles, and protein processing. Wolframin protein is found in many different tissues, such as the pancreas, brain, heart, bones, muscles, lung, liver, and kidneys. Inside cells, wolframin is in the membrane of a cell structure called the endoplasmic reticulum that is involved in protein production, processing, and transport. Wolframin is particularly important in the pancreas, where it helps process proinsulin into mature hormone insulin, the hormone that helps control blood sugar levels.
WFS1 gene mutations lead to the production of a sub-functional versions of wolframin. As a result, calcium levels within cells are not properly regulated and the endoplasmic reticulum does not work correctly. When the endoplasmic reticulum does not have enough functional wolframin, the cell triggers its own cell death (apoptosis). In the pancreas, the cells that make insulin (beta cells) die off, which causes diabetes mellitus. The gradual loss of cells along the optic nerve eventually leads to blindness, and the death of cells in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1.
A certain mutation in the CISD2 gene also causes Wolfram syndrome type 2. The CISD2 gene provides instructions for making a protein that is in the outer membrane of cell structures called mitochondria,the energy-producing centers of cells. Even though the function of the CISD2 protein is unknown, CISD2 mutations produce nonfunctional CISD2 protein that causes mitochondria to eventually break down. This accelerates the onset of cell death. Cells with high energy demands such as nerve cells in the brain, eye, or gastrointestinal tract are most susceptible to cell death due to reduced energy, and people with mutations in the CISD2 gene have ulcers and bleeding problems in addition to the usual Wolfram syndrome features.
Some people with Wolfram syndrome do not have an identified mutation in either the WFS1 or CISD2 gene. The cause of the condition in these individuals is unknown.
Now that you have a proper introduction to Wolfram syndrome, scientists from the New York Stem Cell Foundation and Columbia University Medical Center have produce induced pluripotent stem cells (iPSCs) from skin samples provided by Wolfram syndrome patients. All of the patients who volunteered for this study were recruited from the Naomi Berrie Diabetes Center and had childhood onset diabetes and required treatment with injected insulin, and all had vision loss. Control cell lines that did not have mutations in WFS1 were obtained from Coriell Research for Medical Research.
These skin samples contained cells known as fibroblasts and these were reprogrammed into induced pluripotent stem cells. In order to show that these cells were truly iPSCs, this group implanted them underneath the kidney capsule of immuno-compromised mice, and they formed the teratoma tumors so characteristic of these cells.
When these iPSCs were differentiated into insulin-secreting pancreatic beta cells, Linshan Shang and her colleagues discovered that the beta cells made from cells that did not come from Wolfram syndrome patients secreted normal levels of insulin. However, those beta cells made from iPSCs derived from Wolfram patients failed to secrete normal quantities of insulin either in culture or when transplanted into the bodies of laboratory animals. Further investigations of these cells showed these beta cells showed elevated levels of stress in the endoplasmic reticulum as a result of an accumulation of unfolded proteins.
What on earth is endoplasmic reticulum protein-folding stress? First some cell biology. When the cell needs to make a protein that will be secreted, embedded in a membrane or vesicle. that protein begins its life on ribosomes (protein synthesis factories of the cell) in the cytoplasm, but later those ribosomes are dragged to a cellular structure called the endoplasmic reticulum. While on the surface of the endoplasmic reticulum, the ribosome completes the synthesis of the protein and extrudes the protein into the interior of the endoplasmic reticulum or embeds the protein into the endoplasmic reticulum membrane. From there, the protein is trafficked in a vesicle to another subcellular structure called the Golgi apparatus, were it undergoes further modification, and from the Golgi apparatus, the protein goes to the membrane, secretory vesicle or other places.
If the proteins in the endoplasmic reticulum cannot fold properly, they clump and build up inside the endoplasmic reticulum, and this induces the ERAD or Endoplasmic Reticulum-Associated Protein Degradation response. The players in the ERAD response are shown below. As you can see, this response is rather complicated, but if it fails to properly clear the morass of unfolded proteins in the endoplasmic reticulum, then the cell will undergo programmed cell death.
However, this research team did not stop there. When they treated the cultured beta cells made from cells taken from Wolfram syndrome patients with a chemical called 4-phenyl butyric acid, the stress on the cells was relieved and the cells survived. This experiment shows that relieving this unfolded protein stress is a potential target for clinical intervention.
“These cells represent an important mechanism that causes beta-cell failure in diabetes. This human iPS cell model represents a significant step forward in enabling the study of this debilitating disease and the development of new treatments,” said Dieter Egli, the principal investigator of the study, and senior research fellow at the New York Stem Cell Foundation.
Because all forms of diabetes mellitus ultimately result from an inability of the pancreatic beta cells to provide sufficient quantities of insulin in response to a rise in blood sugar concentrations, this Wolfram patient stem cell model enables an analysis of a more specific pathway that leads to beta-cell failure in more prevalent forms of diabetes. Furthermore, this strategy enables the testing of strategies to restore beta-cell function that may be applicable to all types of diabetes.
Susan L. Solomon of the New York Stem Cell Foundation, said, “Using stem cell technology, we were able to study a devastating condition to better understand what causes the diabetes syndromes as well as discover possible new drug targets.”
Rudolph L. Leibel, a professor of diabetes research and co-author of this study, said, “This report highlights again the utility of close examination of rare disorders as a path to elucidating more common ones. Our ability to create functional insulin-producing cells using stem cell techniques on skin cells from patients with Wolfram’s syndrome has helped to uncover the role of ER stress in the pathogenesis of diabetes. The use of drugs that reduce such stress may prove useful in the prevention and treatment of diabetes.”
The ERAD response seems to play a role in the survival of insulin-producing beta cells in both type 1 and type 2 diabetes. The ERAD response opposes the stress of the immune assault in type 1 diabetes and the metabolic stress of high blood glucose levels in both types of diabetes. When the ERAD response fails, cell death ensues and this reduces the number of insulin-producing cells.
Lawrence Goldstein, director of the UC San Diego Stem Cell Program and a member of the Departments of Cellular and Molecular Medicine and Neurosciences, has an abiding interest in Alzheimer’s disease (AD). To that end, he and his colleagues have used genetically engineered human induced pluripotent stem cells to determine the role a particular protein plays in the causation of familial AD. Apparently, a simple loss-of-function model does not contribute to the inherited form of this disorder. Goldstein hopes that his findings might be able to better explain the mechanisms behind AD and help drug makers design better drugs to treat this disease.
Familial AD is a subset of the larger group of conditions known as early-onset AD. The vast majority of cases of AD are “sporadic” and do not have a precise known cause, even though age is a primary risk factor (an estimated 5.2 million Americans have AD). Familial AD is causes by mutations in particular genes. One of these genes, PS1, encodes a protein called “presenilin 1,” which acts as a protease (an enzyme that clips other proteins in half). Presenilin 1 is the catalytic component of a protein complex called “gamma-secretase.” Presenilin 1 forms a complex with three other proteins (Nicastrin, Aph1, Pen2) to form gamma-secretase, and this enzyme attacks specific proteins that are embedded in the cell membrane and clips them into smaller pieces.
By clipping these cell membrane proteins into smaller pieces, gamma-secretase helps the cell transport cellular material from one side of the cell membrane to the other side or form the outside of the cell to the inside.
One of the substrates of gamma-secretase is a protein called amyloid precursor protein (APP). While the function of APP remains unknown, APP cleavage by the gamma-secretase produces small protein fragments known as amyloid beta.
A consensus among AD researchers is that the accumulation of specific forms of amyloid beta causes the formation of the amyloid plaques that kills off neurons and leads to the onset of AD. The most abundant product of gamma-secretase cleavage of APP is a protein called “Aβ40.” This protein is forty amino acids long and does not cause any brain damage. However, a minority product of APP cleave by the gamma-secretase is “Aβ42,” which is 42 amino acids long and forms the amyloid plaques and neurofibillar tangles that are so characteristic of AD (see Scheuner, D., et al., Nat. Med. 2, 864–870).
According to Goldstein, most of the time, gamma-secretase clips APP without causing any problems, but some 20% of the time, the protein clips APP incorrectly and this results in the plaque-forming forms of amyloid beta. Goldstein explained: “Our research demonstrates very precisely that mutations in PS1 double the frequency of bad cuts.”
To demonstrate this, Goldstein and his co-workers purchased human induced pluripotent stem cells and differentiated them into neurons. These neurons contained different alleles (forms) of the PS1 gene, and some of these mutant forms of PS1 contained the types of mutations that cause familial AD. Once PS1 allele in particular called PS1 ΔE9 increased the ratio of Aβ42 to Aβ40 dose-dependent manner. Since the PS1 ΔE9 causes familial AD, this research elucidates precisely why it does so.
“We were able to investigate exactly how specific mutations and their frequency change the behavior of neurons. We took finely engineered cells that we knew and understood and then looked how a single mutation causes changed in the molecular scissors and what happened next.”
Goldstein further notes, “In some ways, this is a powerful technical demonstration of the promise of stem cells and genomics research in better understanding and ultimately treating AD. We were able to identify and assign precise limits on how a mutations works in familial AD. That’s an important step in advancing the science, in finding drugs and treatments that can slow, maybe reverse, the disease’s devastating effects.”
Bioengineers from the laboratory of Song Li at UC Berkeley have used physical cues to help push mature cells to de-differentiate into embryonic-like cells known as induced pluripotent stem cells.
Essentially, Li and his coworkers grew skin fibroblasts from human skin and mouse ears on surfaces with parallel grooves 10 micrometers apart and 3 micrometers high, in a special culture medium. This procedure increased the efficiency of reprogramming of these mature cells four-fold when compared to cells grown on a flat surface. Growing cells in scaffolds of nanofilbers aligned in parallel had similar effects.
Li’s study could significantly advance the protocols for making induced pluripotent stem cells (iPSCs). Normally iPSCs are made by genetically engineering adult cells so that they overexpress four different genes: Oct-4, Sox-2, Klf-4, and c-Myc. To put these genes into the cells, genetically modified viruses are used, or plasmids (small circles of DNA). Initially, Shinya Yamanaka, the scientist who invented iPSCs, and his co-workers used retroviruses that contained these four genes. When fibroblasts were infected with these souped-up retroviruses, the viruses inserted their viral DNA into the genomes of the host cells and expressed these genes.
Shinya Yamanaka won the Nobel Prize for this work in Physiology or Medicine in 2012 for this work. Unfortunately, retroviruses and can cause insertional mutations when they integrate into the genome (Zheng W., et al., Gene. 2013 Apr 25;519(1):142-9), and for this reason they are not the preferred way of making iPSCs. There are other viral vectors that do not integrate into the genome of the host cell (e.g., Sendai virus; see Chen IP, et al., Cell Reprogram. 2013 Dec;15(6):503-13). There are also techniques that use plasmids, which encode the four genes but do not integrate into the genome of the host cell. Finally, synthetic messenger RNAs that encode these four genes have also been used to make iPSCs (Tavernier G,, et al., Biomaterials. 2012 Jan;33(2):412-7).
The use of physical cues to make iPSCs may replace the need for gene overexpression, just as the use of particular chemicals can replace the need for particular genes (Zhu, S. et al. Cell Stem Cell 7, 651–655 (2010); Li, Y. et al. Cell Res. 21, 196–204 (2011)). If physical cues can replace the need for the overexpression of particular genes, then this discovery could revolutionize iPSC derivation; especially since the overexpression of particular genes in mature cells tends to cause genome instability in cells (Doris Steinemann, Gudrun Göhring, and Brigitte Schlegelberger. Am J Stem Cells. 2013; 2(1): 39–51).
“Our study demonstrates for the first time that the physical features of biomaterials can replace some of these biochemical factors and regulate the memory of a cell’s identity,” said study principal investigator Song Li, UC Berkeley, Professor of bioengineering. “We show that biophysical signals can be converted into intracellular chemical signals that coax cells to change.”
To boost the efficiency of mature cell reprogramming, scientists also use a chemical called valproic acid, which dramatically affects global DNA structure and expression.
“The concern with current methods is the low efficiency at which cells actually reprogram and the unpredictable long-term effects of certain imposed genetic or chemical manipulations,” said the lead author of this study Timothy Downing. “For instance, valproic acid is a potent chemical that drastically alters the cell’s epigenetic state and can cause unintended changes inside the cell. Given this, many people have been looking at different ways to improve various aspects of the reprogramming process.”
This new study confirms and extends previous studies that showed that mechanical and physical cues can influence cell fate. Li’s group showed that physical and mechanical cues can not only affect cell fate, but also the epigenetic state and cell reprogramming.
“Cells elongate, or example, as they migrate throughout the body,” said Downing, who is a research associate in Li’s lab. “In the case of topography, where we control the elongation of a cell by controlling the physical microenvironment, we are able to more closely mimic what a cell would experience in its native physiological environment. In this regard, these physical cues are less invasive and artificial to the cell and therefore less likely to cause unintended side effects.”
Li and his colleagues are studying whether growing cells on grooved surfaces eventually replace valproic acid and even replace other chemical compounds in the reprogramming process.
“We are also studying whether biophysical factors could help reprogram cells into specific cell types, such as neurons,” said Jennifer Soto, a UC Berkeley graduate student in bioengineering who was also a co-author on this paper.
Timothy Downing, et al., Nature Materials 12, 1154–1162 (2013).
Induced pluripotent stem cells are made from the adult cells of an individual by means of genetic engineering techniques. After introducing four different genes into adult cells, some of the cells de-differentiate to form cells that grow indefinitely in culture and have most of the characteristics of embryonic stem cells. However, if iPSCs are made from a patient who suffers from a genetic disease, then those stem cells will have the same mutation as the patient, and any derivatives of those iPSCs will show the same behaviors and pathologies of the tissues from the patient. This strategy is called the “disease in a dish” model and it is being increasingly used to make seminal discoveries about diseases and treatment strategies.
Scientists from Cedars-Sinai Regenerative Medicine Institute have used iPSC technology to study Lou Gehrig’s disease, and their research has provided a new approach to treat this horrific, debilitating disease.
Because I have previously written about Lou Gehrig’s disease or Amyotrophic Lateral Sclerosis (ALS), I will not describe it further.
Cedar Sinai scientists isolated skin scrapings from each patient and used the skin fibroblasts from each sample to make iPSCs. According to Dhruv Sareen, the director of the iPSC facility and faculty research scientist with the Department of Biomedical Sciences and the first author on this article, skins cells of patients who have ALS were converted into motor neurons that retained the genetic defects of the disease, thanks to iPSC technology. Then they focused on gene called C9ORF72, which was found to be the most common cause of familial ALS and frontotemporal lobar disease, and is even responsible for some cases of Alzheimer’s and Parkinson’s disease.
Mutations in a gene that has the very non-descriptive name “chromosome 9 open reading frame 72” or C9ORF72 for short seems to play a central role in the onset of Lou Gehrig’s disease. Mutations in C9orf72 have been linked with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). FTD is a brain disorder that typically leads to dementia and sometimes occurs in tandem with ALS.
Mutations in C9ORF72 result from the expansion of a hexanucleotide repeat GGGGCC. When the C9ORF72 gene is replicated, the enzyme that replicates DNA (DNA polymerase) has a tendency to slip when comes to this stretch of nucleotides and this polymerase slip causes the hexanucleotide GGGGCC sequence to wax and wane (expand and shrink). Normally, there are up to 30 repeats of this GGGCC sequence, but in people with mutations in C9ORF72, this GGGGCC repeat can occur many hundreds of times. Massive expansions of the GGGGCC repeat interferes with normal expression of the protein made by C9ORF72. The presence of messenger RNAs (mRNAs) with multiple copies of GGGGCC in the nucleus and cytoplasm is toxic to the cell, since it gums up protein synthesis, RNA processing and other RNA-dependent functions. Also the lack of half of the C9ORF72 protein contributes to the symptoms of this conditions.
Robert Baloh, director of Cedars-Sinai’s Neuromuscular Division and the lead researcher of this research project, said, “We think this buildup of thousands of copies of the repeated sequence GGGGCC in the nucleus of patient’s cells may become toxic by altering the normal behavior of other genes in the motor neurons. Because our studies supported the toxic RNA mechanism theory, we used to small segments of genetic material called antisense oligonucleotides – ASOs – to block the buildup and degrade the toxic RNA. One ASO knocked down overall C9ORF72 levels. The other knocked down the toxic RNA coming from the gene without suppressing overall gene expression levels. The absence of potentially toxic RNA, and no evidence of detrimental effect on the motor neurons, provides a strong basis for using this strategy to treat patients suffering from these diseases.”
Baloh continued: “In a sense, this represents the full spectrum of what we are trying to accomplish with patient-based stem cell modeling. It gives researchers the opportunity to conduct extensive studies of a disease’s genetic and molecular makeup and develop potential treatments in the laboratory before translating them into patient trials.”
Researchers from another institution recently began a phase one clinical trial that used a similar ASO strategy to treat ALS caused by a different mutation. No safety issues were reported in this clinical trial.
Clive Svendsen, director of the Regenerative Medicine Institute and one of the authors, has investigated ALS for more than a decade, said, “ALS may be the cruelest, most severe neurological disease, but I believe the stem cell approach used in this collaborative effort holds the key to unlocking the mysteries of the and other devastating disorders. Within the Regenerative Medicine Institute, we are exploring several other stem cell-based strategies in search of treatments and cures.”
ALS affects 30,000-50,000 people in the US alone, but unlike other neurodegenerative diseases, it is almost always fatal within three to five years.
A source of stem cells from the digestive tract can repair a type of inflammatory bowel disease when transplanted into mice has been identified by British and Danish scientists.
This work resulted from a collaboration between stem cell scientists at the Wellcome Trust-Medical Research Council/Cambridge Stem Cell Institute at Cambridge University, and the Biotech Research and Innovation Centre (BRIC) at the University of Copenhagen, Denmark. This research paves the way for patient-specific regenerative therapies for inflammatory bowel diseases such as ulcerative colitis.
All tissues in out body probably contain a stem cell population of some sort, and these tissue-specific stem cells are responsible for the lifelong maintenance of these tissues, and, ultimately, organs. Organ-specific stem cells tend to be restricted in their differentiation abilities to the cell types within that organ. Therefore, stem cells from the digestive tract will tend to differentiate into cell types typically found in the digestive tract, and skin-based stem cells will usually form cell types found in the skin.
When this research team examined developing intestinal tissue in mouse fetuses, they discovered a stem cell population that differed from the adult stem cells that have already been described in the gastrointestinal tract. These new-identified cells actively divided and could be grown in the laboratory over a long period of time without terminally differentiating into adult cell types. When exposed to the right conditions, however, these cells could differentiate into mature intestinal tissue.
Could these cells be used to repair a damaged bowel? To address this question, this team transplanted these cells into mice that suffered from a type of inflammatory bowel disease, and within three hours the stem cells has attached to the damaged areas of the mouse intestine. integrated into the intestine, and contributed to the repair of the damaged tissue.
“We found that the cells formed a living plaster (British English for a bandage) over the damaged gut,” said Jim Jensen, a Wellcome Trust researcher and Lundbeck Foundation fellow, who led the study. “They seemed to response to the environment they had been placed in and matured accordingly to repair the damage. One of the risks of stem cell transplants like this is that the cells will continue to expand and form a tumor, but we didn’t see any evidence of that with this immature stem cell population from the gut.”
Because these cells were derived from fetal intestines, Jensen and his team sought to establish a new source of intestinal progenitor cells. Therefore, Jensen and others isolated cells with similar characteristics from both mice and humans, and made similar cells similar cells by reprogramming adult human cells in to induced pluripotent stem cells (iPSCs) and growing them in the appropriate conditions. Because these cells grew into small spheres that consisted of intestinal tissue, they called these cells Fetal Enterospheres (FEnS).
Established cultures of FEnS expressed lower levels of Lgr5 than mature progenitors and grew in the presence of the Wnt antagonist Dkk1 (Dickkopf). New cultures can be induced to form mature intestinal organoids by exposure to the signaling molecule Wnt3a. Following transplantation in a model for colon injury, FEnS contributed to regeneration of the epithelial lining of the colon by forming epithelial crypt-like structures that expressed region-specific differentiation markers.
“We’ve identified a source of gut stem cells that can be easily expanded in the laboratory, which could have huge implications for treating human inflammatory bowel diseases. The next step will be to see whether the human cells behave in the same way in the mouse transplant system and then we can consider investigating their use in patients,” Jensen said.