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The Immune System and HD

            The human immune system consists of various cells circulating in blood and lymph vessels that can localize to sites of damage, injury, or infection and help in repairing damaged cells and destroying foreign or unhealthy substances. The immune system is complex, involving innate mechanisms such as inflammation and fever as well as adaptive mechanisms like cells that recognize specific antigens and respond to them faster upon second exposure. In any case, the body’s immune system is largely immaterial when it comes to neurodegenerative diseases like Huntington’s Disease, as there is a blood-brain barrier that prevents immune cells from crossing from the body’s circulation into the brain’s bloodstream. There has been some research that suggests in HD, there is either some crossing over the blood-brain barrier or the brain and body’s immune systems are activated in synchronization (see Dr. Paul Muchowski’s research presentation at the UCSF research symposium here), when studying HD scientists have traditionally regarded the blood-brain barrier as intact, and focused on the components of the immune system specific to the brain and how they affect and are effected in HD. The potential of focusing therapies on immune mechanisms in the brain is an interesting new area of research in the search for HD treatments.

The Blood Brain Barrier^

While the blood-brain barrier seems an abstract concept, it has a tangible counterpart in reality: tight junctions in the capillaries separating the brain’s circulation from the rest of the body do not allow cells like immune system macrophages through but allowing for the passage of oxygen and other small molecules such as hormones.  This makes it necessary for the brain to have its own immune system against foreign pathogens. It has been observed that in HD and other neurodegenerative diseases, cytokines, small signaling proteins involved in immune function, are found in increased levels in both the central nervous system of the body and in the brain.  Although it is possible that the cytokines are flowing through the blood-brain barrier, scientists think it is more likely that the cytokines are being released from glial cells in the brain and immune cells in the body in concurrence, causing this correlation. The blood brain barrier’s seeming impermeability has implications for any potential drugs administered orally or intravenously for HD: these chemicals must be able to cross the blood-brain barrier if they are to be of any direct use in slowing disease progression.

Inflammation, a key immune response that can be harmful if prolonged chronically, plays a large role in the immune response to HD and recent research has shown that inflammation may lead to increased permeability of the blood-brain barrier, with potential harmful results for patients whose immune systems would be thus compromised. In inflammation, increased activity of microglia, the major immune cell of the brain, increases the permeability of the blood-brain barrier and allows in macrophages from the body’s immune system. While this may be helpful in combating the disease in the short-run, in the long run the loss of this protection of the brain from harmful foreign cells and substances in the body is threatening to the patient.  The permeability of the blood-brain barrier also increases with age, furthering the risk of compromising the immune system of the brain. It has also been proposed that this increased permeability does not result from increased activity of the brain immune system but actually increased activity of the macrophages of the body’s immune system. As both macrophages and microglia promote inflammation when activated, either could be the potential cause of this increased permeability. Inflammation in the brain and heightened immune activity is more risky than in the body as the brain is more susceptible to disorganization and damage when inflamed.

Innate immune system and inflammation^

The innate immune system is the first line of defense against foreign pathogens or cancerous cells, and while rapid it is neither complete nor specific in the way the adaptive (also called acquired) immune system is. HD affects cells of the innate immune system; for instance it was seen that monocytes expressing mutant huntingin were hyperactive, which can cause an auto-immune response, where the immune system turns on and destroys healthy cells of the body, mistaking them as foreign or infected.   The rise in cytokine levels in HD also suggested overactivation of the innate immune response, as the cytokines increased earliest are part of the innate response. This overactivation of the innate immune system in both the body and the brain (microglia are overactive in HD patients) corresponds to HD progression and so is a potential target for HD therapies.

            One aspect of the innate immune system that plays a particularly prominent role in HD is inflammation, as mentioned above.  In HD, molecules promoting inflammation are released in increased and sustained levels, causing irregularly high inflammation. As inflammation activates the adaptive immune system, including microglia, which release inflammatory factors, this cycle is only perpetuated as microglia activity is increased, in turn further increasing inflammation. Many neurodegenerative diseases are associated with chronic neuroinflammation, which contributes directly to neuronal death. For instance, normally neuroinflammation prevents the accumulation of amyloid plaques. However, in Alzheimer’s Disease, the accumulation of amyloid plaques may be a result of chronic increased neuroinflammation.  In HD, inflammation is chronic in both the peripheral and central immune systems. Mutant huntingtin promotes inflammatory factors, such the cytokine IL-6, which promotes inflammation in glial cells.

While neuroinflammation in response to pathogens in usually beneficial, minimizing injury and expediating tissue repair, chronic neuroinflammation’s self-perpetuating nature causes it to persist long after the initial injury had rendered it  beneficial. Whether neuroinflammation is beneficial or harmful in the brain clearly depends on its duration, with prolonged inflammation tipping the scale to harm. Inflammation also causes increased oxidative stress, another factor that leads to neurodegeneration. It is also a factor contributing to the compromise of the blood-brain barrier, which allows macrophages to infiltrate the brain, furthering inflammation even more. An important part of an inflammatory response is the activation of anti-inflammatory regulation that is not present in HD and other neurodegenerative diseases. The neuronal death in HD also further feeds inflammation, perpetuating the cycle.

 

Oxidative stress^

            One the major ways chronic inflammation leads to neurodegeneration is through oxidative stress and the damage this causes to neuronal cells. In diseased brains, peroxidases, enzymes that oxidize cells, are present in elevated level. Oxidizing agents such as hypochlorous acid (a precursor to hydrogen peroxide—a very harmful oxidizing agent) are produced by active immune cells, and in this way immune responses promotes oxidation that leads to neurodegeneration and cell death, to a harmful degree if the response is chronic. It is also important to remember that neuronal death leads to a less functional nervous system, less able to combat these oxidative stresses. While neuronal death is the direct cause of neurodegeneration, the pathways to neuronal death in HD and other diseases is still not completely clear. Oxidative stress is recognized as at least one of these pathways. Oxidative stress occurs when the cellular defenses against reactive oxygen species (oxidants such as hydrogen peroxide or peroxidases) are compromised. As the brain has the highest metabolic rate of any organ, it is the most sensitive to oxidative stress. Brain cells also have a high content of unsaturated fatty acids in their membranes as well as high levels of iron, both of which promote and are particularly susceptible to oxidative stress (for an article on omega-3 fatty acids and neuronal membranes, click here).  The brain also has much fewer antioxidants than other organs.

In immune cells such as phagocytes, NADPH oxidase, an enzyme involved in electron transport chains in the membrane is activated in immune response, creating oxygen radicals that are precursors to hydrogen peroxide. Immune cells also produce nitric oxide, which activates hydrogen peroxide activity. In Alzheimer’s Disease, accumulation of amyloid plaques promotes NADPH oxidase in immune cells such as microglia and macrophages, stimulating hydrogen peroxide production. Again, the inflammatory response causes an increase in microglia and macrophage response in the brain, furthering oxidative stress.  In Parkinson’s Disease, microglia also activate NADPH oxidation in dopaminergic neurons, the cells harmed and killed in PD. The extracellular matrix in brains suffering from neurodegenerative diseases is at risk from inflammation and oxidative stress, as are unsaturated lipids common to neuronal membranes (such as membranes containing omega-3 fatty acids).

The heat shock protein response to oxidative stress prevents misfolding and aggregation of damaged proteins.  These proteins also reduce oxidative damage to lipids and DNA. In neurodegenerative diseases, this response is compromised and cannot keep up with increased oxidative stress. As oxidative stress is such an important cause of cellular death in neurodegenerative diseases, it and its causes, inflammation and chronic immune system activation, are promising targets for disease therapies.

Microglia^

Microglia have been mentioned above, and as the brain’s counterpart to macrophages, they are the primary cells of the central nervous system’s innate immune response. Unlike other cells in the brain, microglia are not derived from neuronal precursors but rather myeloid precursors, and have their own independent replication cycle. It has been shown that microglia are irregularly activated in HD, even before symptoms appear, and their activation is correlated with disease severity. Mutant huntingtin is expressed in microglia, which may be the reason for their increased activation in HD. The hyperactivation of microglia leads to the increased production of cytokines by the cells, which further increases immune activation. Microglia also produce oxidizing agents such as hydrogen peroxide.

In AD, it has been seen the microglia-mediated inflammation contributes to neurodegeneration, and anti-inflammatory drugs that decrease this inflammatory response has had positive results in Alzheimer patients. In Parkinson’s Disease as well it has been shown that microglial activation leads directly to the death of dopaminergic neurons.   As mentioned above, the destruction of the blood-brain barrier means microglia overactivation is augmented by infiltrating macrophages from the body’s immune system.

As the principal cause of inflammation in diseased brains, microglia react to many external stimuli to cause neuronal dysfunction. In healthy brains, microglia have mechanisms that monitor their environment, and when activated, begin protection and repair of damaged tissue. In this controlled activation, microglia take up and destroy neurotoxins, remove dying neurons and debris, and secrete neurotrophic factors to promote neuronal survival. Pathogens and neurotoxins activate microglia either by damaging cells, which are recognized by the microglia or being recognized themselves as foreign agents. While not all microglia activation contributes to neurodegeneration, when overactivated, the microglia become neurotoxic and contribute to disease progression, accelerating and exacerbating disease mechanisms. Inflammation shifts microglia activity from plaque removal to plaque deposition in AD, and a similar switch occurs in other neurodegenerative diseases, showing that the brain’s environment and circumstances influence whether microglia are helpful or harmful.

Interestingly, patients with PD have elevated microglia activity in many brain regions regardless of how long the disease has progressed. Similarly, in HD, microglia are activated before symptoms are manifest. Once symptoms are observed, microglial activation corresponds to disease severity. Microglia regulate both inflammation levels and adaptive immune response, and are responsible for scavenging cellular debris. Microglia cells are also the reason for graft rejection in the central nervous system, and this immune rejection would have to be taken into account with any potentials for tissue regeneration treatments for HD. The importance of microglia in the brain’s immune system cannot be overstated, and their chronic activation in HD contributes directly to disease progression.  

The microglia’s counterpart in the body’s immune system are phagocytic cells, specifically monocytes. Though HD pathology mainly affects the brain and its functions, it has been shown that monocytes in HD patients also express the mutant huntingtin allele. As mentioned above, even before HD symptoms show up monocytes in HD positive patients can behave abnormally, increasing production of the cytokine IL-6. This overactivity of monocytes is harmful to the body’s immune system and makes the cells more able to cross the blood brain barrier and turn into microglia, further overactivating the brain’s immune system.

Cytokines^

            The first indicators that immune activity was upregulated in HD patients came from the observation of cytokine levels. Cytokines, as protein signalers of the immune system, play many roles in activating various components of the immune response. It was seen that in HD patients the cytokines IL-6 and IL-8 increased enormously, and IL-4 and IL-10 increased substantially as well. The increase in IL-6 was shown to happen approximately sixteen years before motor symptoms of HD appeared, which indicates IL-6 may be a potential early indicator of disease progression. IL-8 levels corresponded directly with disease progression, and all the cytokines were upregulated not only in the brain, but all around the body. While in healthy subjects there is low levels of cytokine production that activate the immune system when necessary, the neuroinflammation in HD causes the release of cytokines to spiral out of control, worsening the disease. Activated microglia secrete cytokines that perpetuate inflammation and activation of microglia.  HD is not the only neurodegenerative disease connected to cytokine activity; in Alzheimer’s Disease plaque aggregation increases the secretion of IL-6 and IL-8 as well, which in turn lead to increased aggregation, and in Parkinson’s patients the cerebrospinal fluid has been seen to have high levels of IL-6. In HD patients, the increases of cytokines were most marked in the striatum, where the disease pathology is the worst. Cytokines are an indispensible part of the chronic inflammation and immune activation indicative of neurodegenerative diseases, and their heightened levels before symptoms occur in HD have potential to be used in HD therapies.

Early indicators^

Many of the immune system changes in HD occur many years before motor symptoms, such as the increase in cytokine levels and inflammation. Because of this occurrence, the immune activation in HD must be better characterized so there are markers of disease progression (state biomarkers) that connect immune activation to their stage in HD.  The changes in the body’s immune system are easy to monitor and most likely mirror the neurodegeneration in the brain that is much less easy to track. Biomarkers in AD in the peripheral immune system have already been characterized, with the goal of finding therapies that can treat the disease in its earlier stages, before symptoms even occur.  Immune activation in neurodegenerative diseases occurs before significant neuronal death, and the prospect of arresting disease progression in these early stages holds much promise for these diseases.  Anti-inflammatory treatments given early on to HD patients may alter the disease pathology and slow its progression. In this way, the immune system and inflammatory agents are both useful in the detection of HD and as potential targets for its preemptive treatment.

 

The complement system^

The complement system is yet another component of the innate immune system that overactivates in neurodegenerative disease. The complement system recognizes and kills foreign pathogens or infected cells, as part of the immune system’s distinction between self and non-self. If it is activated in inappropriate regions or overactivated, it can attack host tissue and become harmful. The tissues of the brain create a complete complement system in parallel to the body’s system, and also produce inhibitors to the complement system to prevent overactivation and lysis by the complement system. Complement activation induces a cell to lyse, especially neurons, and also influences inflammation, alternatively in pro and anti-inflammatory activities. Research has shown that complement activation may also allow tissue remodeling to repair cells after injury, especially in the brain and the central nervous system, clearing toxic and damaging deposits. A major role of the complement system is recognizing and clearing away cell debris and lysed cells by microglia that have receptors recognizing components of the complement system. This prevents further inflammation that is caused by an excess of toxic debris. It can additionally form a membrane attack complex (MAC) on a target cell,  that destroys the cell membrane by creating pores into the cell and lysing the cell. Inflammation by the complement system causes neuronal apoptotic activity as well.  

Because the complement system initiates inflammation and lysis, if unregulated, it can lead to severe tissue damage. While complement activity is helpful in repairing tissue after injury, its roles are potentially threatening, especially if upregulated. For this reason, the system is normally tightly regulated by soluble and membrane proteins produced by the liver and immune and endothelial cells. In AD, amyloid plaques further activate the complement system and in HD microglia increase production of complement proteins and receptors.  In healthy brains complement activation is minimal, as it is inhibited by the specific inhibitory proteins produced by the brain, to avoid the self-destructive activity of an overactivated system.  If a healthy brain becomes infected, its cells can synthesize complement proteins to kill the pathogen while preserving healthy, uninfected cells. Glial cells and neurons in are capable of producing a full complement system, and increase complement expression after brain infection. Neurons are extremely susceptible to attack by the complement system, especially by membrane attack complexes, as neurons, unlike all other brain cells, do not express high levels of complement inhibitor proteins in their membranes. Increased complement synthesis and activation can in this way lead to neuronal loss in HD and other neurodegenerative disease, though it is under debate to what extent complement activation is a cause of neurodegeneration or an effect of it.

In neurodegenerative diseases, the complement system is particularly increased in the areas of the brain most affected by the disease. Complement inhibitors do not increase their levels in these diseases, leaving the brain vulnerable to the increased complement activity. Some research has suggested that the complement system plays a role in the apoptosis of neurons caused by mutant huntingtin and also causes damage to surrounding cells by lysing them as well. The complement system is further activated by the intracellular components that are released in lysed cells, such as nucleic acids and mitochondrial membranes. While normally microglia with complement cells aid in the removal of pathogens and toxic debris, overactive complement system can be signaled by inflammatory activity. Activation of the membrane attack complex has been shown to increase complement regulators and inhibitors and protect against overactivation of the complement system. Once this mechanism is further elucidated, it may hold potential towards treating the overactivation of the complement system in neurodegenerative diseases. Inhibitors of the complement system would have to be specifically delivered or expressed in the brain, as the complement system continues to perform a beneficial role in the body during neurodegenerative diseases.

Cannabinoid receptors^

A different immune system overactivation in neurodegenerative diseases is overactivation of endocannabinoids.  Normally, endocannabinoids act protectively in the brain, reducing neurodegenerative and inflammatory damage. In HD, the peripheral endocannabinoid system (ECS) mirrors the problem occurring in the ECS of the central nervous system. Because of these parallels, blood ECS levels may serve as an early, non-invasive diagnosis for HD and other neurological diseases, like cytokines with which the ECS is intimately linked. Also like cytokines, the ECS may be a potentially promising target for HD therapies. Endocannabinoid receptors, which come in two primary types, CB1R and CB2R, are activated by the neurotransmitter endocannabinoid anandamide (AEA). In neurodegenerative diseases, AEA production is increased and flows through the blood brain barrier into the brain. AEA activity depends greatly on its life span which begins when it is taken up by a cell and ends when it is degraded in the cell.  It is not known whether there is a transport protein that ferries AEA across the blood-brain barrier. CB1R is the cannabinoid receptor primarily found in the central nervous system, and CB2R is predominantly expressed by immune cells in the body. When the ECS is overactivated, it increases neuroinflammation and displays autoimmune behavior, harming healthy neurons. ECS activation has been studied in Parkinson’s Disease, where it becomes overactive in dopaminergic neurons, elevating AEA levels, which is also true in multiple sclerosis (MS). ECS activation is connected to the neuroinflammation that precedes neuronal loss and onset of symtoms of many neurological diseases. CB1 receptors are densely expressed in the striatum, the main region of the brain affected by HD.  A severe loss of CB1 receptors has been seen in HD patients. This loss correlates directly to the length of the CAG repeats, which determines the onset and severity of disease pathology. In mouse models, preventing this loss of CB1 receptors slowed progression of HD, which may point to potential therapies for HD involving the CB receptors.

Immune privilege^

Although it has been seen that the blood-brain barrier is not immutable, the brain’s immune system still poses a formidable barrier to HD therapies, as even a compromised brain can recognize drugs as foreign and may destroy cell and tissue therapies that are intended to treat the disease. It was formerly thought that the brain was an “immunologically privileged site”, that does not reject implants of foreign cells and tissue as it does not recognize them as foreign. This has been shown to be false, for although the blood-brain barrier does allow a degree of “ignorance” (unawareness that foreign material is present), and “tolerance” (incomplete ability of the immune system to reject tissue), it does not display absolute immunological privilege as activated lymphocytes can cross the blood brain barrier. There are multiple factors that contribute to the brain’s ability to reject grafts of tissue, such as the type of tissue transplanted, the degree of immunological difference between the host and introduced tissue, and the way in which the tissue is transplanted.  Interestingly, transplantation to a site where microglia are activated to produce inflammation results in more successful grafts, most likely due to the microglia’s production of growth factors. As this characterizes HD brains, tissue transplantation therapies may not face as many obstacles in HD and other neurodegenerative diseases. But there have been no successful neural transplantations for HD yet; twelve patients have been grafted with embryonic neural pig tissue, with no benefit and in fact harm done to the patients’ brains.

Cell therapies hold promise to combat the mass injury done in neurodegenerative brains, and the need for successful transplantation is vital. Using multiple donors has shown to be beneficial to transplantations.  Transplantation surgery will inevitably damage the blood-brain barrier. T cells are the main cellular causes of immune rejection of transplants, as are microglial cells. Microglia in a healthy brain are typically immature and ineffective, but when activated they mature into active macrophages.  Healthy brains also have a much lower level of antibodies, complement proteins, and other immune proteins than is found in the body’s bloodstream, but in response to a transplant these levels increase dramatically. For drugs taken orally or through the bloodstream, the blood-brain barrier poses a obstacle towards these therapies reaching their targets in the brain. The blood-brain barrier is a capillary barrier of tight junctions restricting cell passage, as well as enzymes that degrade substances before they can pass through.  Active transport proteins across the blood-brain barrier are highly specific and do not easily carry drugs.

Peripheral immune activation^

HD symptoms do not occur exclusively in the brain, and the body’s innate immune system is abnormally activated in HD. The earliest blood abnormality associated with HD is an increase in IL-6 levels sixteen years before symptoms occur.  The parallel central nervous system and peripheral pathways of immune activation in HD occurs because the mutant protein is expressed all over the body. It is unknown what immune effects (such as cytokine levels rising) is due directly to the mutant protein and what is due to inflammation that is itself caused by the mutant proteinComplement proteins are also up-regulated peripherally by mutant huntingtin or inflammation. Because monocytes in the body display immune dysfunction in premanifest HD patients, they offer a non-invasive way to track disease progression.

 

Therapies^

Anti-inflammatory drugs have reduced onset of Alzheimer’s Disease, with a five-year use of ibuprofen protecting against disease development.  These drugs inhibit excessive microglia activation and the inflammation this causes. Unfortunately, clinical trials of anti-inflammatory drugs have yielded inconclusive results, as the key inflammatory proteins of AD have yet to be indentified and as such cannot be directly targeted. Drugs must also be delivered more effectively to the brain across the blood-brain barrier, and the beneficial effects of neuroinflammation cannot be shut down with the administration of anti-inflammatory drugs. Therapies must cross the blood-brain barrier and target destructive inflammatory mediators without compromising beneficial survival-promoting effects and overall immune function.

While this is a tall order for any drug, the importance of the immune system’s activation in HD points to its promise as a target for potential treatments. As the role of the immune system in HD, constantly fluctuating between beneficial and harmful, is better understood, its manipulation to track disease progression and slow the disease by arresting harmful immune responses may become a subject of HD research that leads to the next stage of knowledge about HD and how it affects the brain and body’s function.

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Induced Pluripotent Stem Cells: The Future of Tissue Generation

As viable human brain tissue is not available for use in studying disease development and creating therapies for neurological disorders like Huntington’s disease (HD), researchers desperately needed an alternative cell source for this purpose.  Embryonic stem cells fit this role but have many disadvantages, especially for treatments, including immune rejection by the recipient. Some of these drawbacks have been overcome by a recent discovery that revolutionized the face of stem cell biology.  In 2006, Shinya Yamanaka’s research group at Kyoto University made a groundbreaking announcement: they had discovered that adult cells could be genetically engineered to revert back to apluripotent, stem cell-like state.  As iPSC (induced pluripotent stem cell) production rapidly improved, the cells were soon able to compete with traditional fetal, embryonic, and adult stem cells. The primary advantages of iPSCs compared to other stem cells are: a) iPSCs can be created from the tissue of the same patient that will receive the transplantation, thus avoiding immune rejection, and b) the lack of ethical implications because cells are harvested from a willing adult without harming them.  These patient-specific cells can be used to study diseases in vitro, to test drugs on a human model without endangering anyone, and to hopefully act as tissue replacement for diseased and damaged cells.

Like other stem cells, iPSCs have the ability to proliferate indefinitely in vitro, creating a theoretically unlimited source of cells. Like embryonic stem cells,  iPSCs can also differentiate into any cell of the body, regardless of the original tissue from which they are created.  Scientists have found how to direct the differentiation of pluripotent stem cells into many types of target tissue, including neural tissue.  iPSCs demonstrate that by the introduction of just four genes into somatic cells that normally cannot differentiate at all, cells can be created that can differentiate into every cell type in the body.  The early results of iPSC differentiation studies look promising. For example, human fibroblasts have been successfully turned into iPSCs that then are differentiated into insulin-producing cells, a result that holds much potential for the treatment of diabetes.  Mouse iPSCs have been differentiated into cardiovascular (heart muscle) cells, that actually show the contractile beating expected of heart tissue.

Although there are many problems that still must be addressed for iPS technology, such as the tendency for tumors to evolve after iPSC transplantation and the low efficiency of the technology, iPSCs could completely change how diseases are approached in biomedical research.  For HD and other neurological disorders, iPSCs could create perfect models for the cells of the central nervous system that are harmed in the diseases.

Stem cell biology is a very hot topic in modern medicine, yet much is still unknown about the mechanisms underlying pluripotency and differentiation. In order for safe, controllable, and efficient cellular reprogramming to be achieved, there must be more knowledge on the regulation of stem cell states and transitions. iPSCs show that specialized cells and tissue can be transformed into other types of cells, proving cells are much more flexible than previously thought.  As the study of HD will greatly benefit from this new, unlimited source of neural cells for research and cell therapy, iPSCs may be able to provide new and innovative treatments for HD.

The Discovery of iPSCs^

The creation of pluripotent cells has been widely studied for decades. In 1976, the first method of fusion of an adult somatic cell with embryonic cells to create pluripotent stem cells was reported. However, fusion with embryonic cells created unstable cells that were rejected by immune systems after transplantation. If the genes that induced pluripotency could be isolated from their parent embryos and injected into somatic cells, these problems could be avoided.

Yamanaka’s research team studied twenty-four genes expressed by embryonic stem cells in an effort to track down these essential genes that induce pluripotency. To detect pluripotency, they looked for cells expressing genes that were traditionally expressed only in embryos.  They discovered that the addition of four genes induced a cell into a pluripotent state capable of then becoming many different cell types.

The Four Factors^

Subsequent studies showed that other gene combinations were also successful in reengineering cells into iPSCs, but none were as efficient as the first four. Adding other genes that are expressed in early development was shown to increase reprogramming efficiency, and the specific genes needed varied depending on the cell type that was being forced back to its pluripotent state.  As the four factors and their alternatives were largely discovered by trial and error, it is not known how the genes induce pluripotency. Discovering how genes work may point to ways of improving the efficiency of the process and assessing the quality of iPSCs.

The specific genes that induce iPSCs tell scientists a lot about the characteristics of the cells themselves. Pluripotent stem cells are very closely related to tumor cells. Both can survive and proliferate indefinitely, and a test of pluripotency is whether a cell can create a tumor.  It is therefore no surprise that two tumor-related genes, c-Myc and Klf4, are needed to create iPSCs.  Another requirement of pluripotent stem cells is open and active chromatin structures (for more information on chromosomes, click here and DNA transcription click here). The c-Myc gene codes for proteins that loosen the chromatic structure, stimulating differentiation.  Klf4 impedes proliferation.  c-Myc and Klf4 in this way  regulate the balance between proliferation and differentiation.  If only c-Myc and Klf4 are used in the engineering of iPSCs, tumor cells will arise—instead of pluripotent stem cells. Oct3/4 and Sox2 are required to direct cell fate towards a more embryonic stem cells (ESC)-like phenotype.  Oct3/4 directs specific differentiation, such as neural and cardiac differentiation, while Sox2 maintains pluripotency.  Oct3/4 and Sox2 together ensure that iPSCs are indeed pluripotent stem cells and not tumor cells.

Amounts and Timing of Reprogramming Factors^

The programming of iPSCs depends both on the original cell type being transformed and the levels of each reprogramming factor that is expressed.  Expressing Oct3/4 more than the other genes increases efficiency.  Increasing the expression of any of the other three genes decreases the efficiency.  There is clearly a correlation between gene expression ratio and reprogramming efficiency, but the optimal ratio is likely to vary depending on the cell type being reprogrammed. For instance, when neural progrenitor cells are reprogrammed, they do not require Sox2 as they express this gene sufficiently already.  The level of expression of other important genes for maintaining pluripotency also can affect the reprogramming process and the quality of the resulting cells.

The effect gene expression ratio has on reprogramming may explain why efficiency is typically so low (less than 1% of cells are reprogrammed successfully).  Reprogramming is a slow process, and so the timing of various events may also exert a great influence over thecell’s success. The minimum time for the full reprogramming of a mouse somatic cell into an iPSC is between eight and twelve days. The timing of the mechanism for cellular reprogramming may also be a reason for low efficiency, as the cells can only proceed if the right molecular events happen in the correct order.

Creation of a Germline Competent Model^

In the first studies of iPSCs, the cells were shown to be similar to ESCs in morphology and proliferation.  But the cells were not germline-competent, in other words they were unable to differentiate into cells that expressed genes of the parent cells, and so they could not give rise to adult chimeras when transplanted into blastocysts.  As chimeras play key roles in biomedical research, scientists identified iPSCs through a stricter gene marker that only identified iPSCS that were germline competent. It was found that cells that expressed Nanog, a gene closely tied to pluripotency, were germline competent. These cells also were virtually indistinguishable from ESCs in gene expression, and were more stable.  The transgenes were better silenced in the Nanog identified cells although 20% of the iPSCs still developed tumors due to the reactivation of c-Myc.  Unfortunately this stricter criterion also decreased efficiency to only 0.001-0.03%. While subsequent studies improved this efficiency by varying methods, the fact remains that iPSCs are generated with incredibly low efficiency.

Characteristics of iPSCs^

iPSCs exhibit many characteristics that are related to their pluripotency. They lose proteins that are common to somatic cells and gain proteins common to embryonic cells. They also lose the G1 checkpoint in their cell cycle control mechanism, which embryonic stem cells lack as well. During the reprogramming of somatic cells in the iPS mechanism, the cell cycle structure of stem cells must be reestablished. Another distinguishing characteristic of pluripotent stem cells is their open chromatin structure, as this is needed to maintain pluripotency and to access genes rapidly for differentiation. iPSCs have the open chromatin structure associated with ESCs and other pluripotent cells.  Finally, female iPSCs show reactivation of the somatically silenced X chromosome.  A very early step of stem cell differentiation is the inactivation of one of the two X chromosomes in female mammals, a random process. By the reactivation of this X chromosome, iPSCs show that they are truly pluripotent and identical to ESCs.

Non-retroviral Methods of iPSC Production^

A huge barrier to the eventual use of iPSC-derived treatments is the use of retroviruses to force the expression of the four key genes, discussed above, and activating their transcription factors. Retroviruses can carry target DNA that is inserted into a host cell’s genome upon injection, making them ideal for incorporating the four genes into target cells. However, this DNA and the rest of the viruses’ genomes remain in the host genome, which can lead to transcription of unwanted genes and greatly increases the risk of tumors. The expression of the four transgenes must be silenced after reprogramming to avoid harmful gene expression.  c-Myc, a tumor-promoting gene, especially must be silenced after cellular reprogramming or the risk of tumor development becomes too great for clinical use. These retroviral methods in which the transgenes are still present in the pluripotent cells pose a danger to safety, and also are less closely related to ESCs in gene expression than their non-retroviral alternatives.  Methods of reprogramming iPSCs without transgene expression in the reprogrammed cell is therefore essential not only for potential therapies and clinical applications, but also for reliable and accurate in vitro models of diseases.  Yet, the low efficiency of alternatives remains a worry. Whether these methods will be viable for human clinical use remains to be seen.

Excisions^

The excision strategy (transient transfection) of iPSC generation allows the transgenes to briefly integrate into the genome but then removes them once reprogramming is achieved. An example of this site/enzyme combination is the loxP site and the Cre enzyme.  In a study of Parkinson’s disease (PD), specific iPSCs, this loxP/Cre combination was used to generate the iPSCs. Neural differentiation was then induced on the iPSCs to test whether they could differentiate into dopaminergic neurons, the cells harmed in PD.  The differentiation was successful, indicating the transgenes had been excised. However, a loxP site remains in iPSC genome as does some residual viral DNA, so there is still a small potential for insertional mutagenesis. The piggyBac site/enzyme system on the other hand is capable of excising itself completely, not leaving any remnants of external DNA in the iPSC genome.  The piggyBac system also was much more efficient than other non-retroviral methods, with comparable efficiency to retroviral methods, but with the added benefits of safety and ease of application.

Adenoviral Methods^

Adenoviral methods do not pose the same threats as retroviral methods of generating iPSCs. Adenoviruses work like all viruses by hijacking their hosts’ cellular machinery to replicate their own genome and reproduce, but unlike retroviruses they do not incorporate their genome into the host DNA. Because the transgenes are never even incorporated into the host’s genome they do not have to be excised. Instead, the genes are expressed directly from the virusgenome. iPSCs created by adenoviral methods demonstrated pluripotency, but have extremely low reprogramming efficiency.   Viralgenomic material could not be detected in any of the iPSCs, and no tumor formation was reported. This suggests that the use of non-integrating adenoviral methods substantially lowers the threat of tumorgenesis. The successful creation of iPSCs from adenoviral methods proves definitively that safer, non-retroviral methods can also successfully reengineer cells.

Non-DNA methods of iPSC generation^

Recent studies have implied that perhaps genetic material is not required for iPS cellular reprogramming.  The substitution of transgenes with small molecules that promote iPSC generation would be a safe, clinically appropriate way of creating iPSCs, though it remains to be seen if small molecules will be able to completelyreplace genetic methods of iPSC generation or are just useful as supplementary aids to the process.  Protein transduction is a different method shown to entirely replace gene delivery. In this method fusion proteins are created, which fuse each of the transgenes to a cell-penetrating peptide sequence that allows it to cross the cellular membrane. Reprogramming without DNA intermediates should eliminate the risk of tumorgenesis and distorted gene expression due to the reactivation of the transgenes.

Issues Facing the Use of iPSCs^

With iPSC research being a hotspot for several years now, many of the problems the technology first faced have been studied and resolved.  iPSCs are now germline competent,  can be generated from many different types of human and animal somatic tissue, and can be generated in a  variety of retrovirus-free methods. This lack of retroviruses ended worries about transgene reactivation and subsequent tumorgenesis. The nature of the transgenes in question made the risk of tumor development particularly prevalent, as two of the genes, c-Myc and Klf4, directly inducing tumorgenesis.  Retroviral delivery posed a threat to safety in its increased risk of tumorgenesis and in its tendency to alter gene expression. When other methods were established that did not require retroviruses, these concerns were put to rest, yet these new methods’ efficiencies must be improved and some issues still remain concerning the safety of iPSCs and their abilities to act on par with any other pluripotent cell.

Tumorgenesis^

Even without the use of retrovirsues, tumorgenesis is still a large concern for iPSCs, especially if they are ever to be used as cell replacement therapies. Using retroviral methods, twenty percent of iPSCs developed tumors in one study, and though this number has significantly lowered, it must become negligible for iPSCs to be considered for clinical use.  It is telling that the assay for pluripotency in stem cells is the ability to form teratomas, or tumors. This test of “stemness” illustrates the precariously close link between stem cells and tumor cells.  There are several proposals on how to prevent this tumor formation.  The idea to sort cells before transplantation and after differentiation, so that only well-differentiated neural progenitors will be transplanted, is one such proposal.  Another proposal is to genetically modify iPSCs so that they will have a suicide gene to self-destruct when tumors are created. Finally, some antioxidants, such as Resveratrol, have been shown to have tumor-suppressing qualities, and could potentially aid in any treatment proposed to prevent tumors (for an article about the potential of Resveratrol for the treatment of HD, click here).

Differentiation^

Directed differentiation has been a perennial problem in stem cell biology, and iPSCs bring their own unique characteristics to the dilemma.  As with ESCs, iPSCs sometimes have the tendency to not fully differentiate. Also, as with all stem cell research with neurodegenerative diseases, a more efficient and comprehensive method to differentiate cells into neural progenitors and specific neuronal tissue must be discovered, as current methods are imperfect and slow.

Quality Assessment and Variability^

In iPSC research there is a need to establish methods to evaluate the reprogramming process and the final quality of the cells.  To create human iPSCs suitable for cell replacement therapies, there must be tests to ensure that all pluripotent cells have differentiated, and that the cells have not been genetically altered during reprogramming or during differentiation.  With cells derived from diseased individuals for an autologous treatment, there is naturally the concern that the underlying genetic cause of the disease remains in the iPSCs and will manifest itself in the same way. Some studies have indicated that iPSC lines differ drastically, which makes the reproducibility of any particular phenotype difficult.  Analyzing this variability may help discover which somatic tissue is best for generating iPSCs.

Efficiency^

A problem that has not been significantly improved upon since the beginnings of iPSC research is the technology’s low efficiency. Some hypothesize that the addition of other factors would greatly aid the reprogramming process, and that reprogramming success depends on specific amounts and ratios of the four factors, which are only achieved by chance in a small percentage of the cells. Modifying the culture conditions is another area of study for increasing efficiency and rate of iPSC production. For cellular transplantation therapies, other questions must also be considered, such as the optimal cell dose and source tissue, and the best way to deliver the cells. There are potential solutions to this problem, though. Induction efficiencies have been improved up to a hundred times initial values by use of different somatic starting cells and the aid of small molecules. Although there are barriers to iPSC production, research in this field is still in its infancy and has made impressive gains for the short time it has been going on. As more studies are conducted on iPSCs, these issues may be resolved and iPSCs may enter a state capable of clinical use.

Origin Cells Used^

Another potential way to improve iPSC generation efficiency is to establish the best somatic cells type to reprogram for the cleanest, easiest reprogramming. Many different tissue types have been reprogrammed, including fibroblasts, neural progenitor cell, and stomach epithelial (stomach lining) cells. Certain cell types are much more efficient and rapid than others. There is also the probability that subtly varying iPSCs are generated from different types of starting tissue, some of which may prove to be useful for research or replacement purposes.

An interesting “type” of somatic cell was used in studies of secondary iPSCs.  iPSCS were initially generated and then implanted into blastocysts to create chimeric animals. Somatic cells from these chimeras were then removed and iPSCs were generated from these cells, creating secondary iPSCs.  These secondary iPSCs were generated more efficiently. The differentiation status of thecells to be reprogrammed also affects efficiency, as adult progenitor cells are reprogrammed at three hundred times the efficiency of completely differentiated somatic cells.

Transdifferentiation^

An interesting possibility for the reprogramming methods of iPSCs is the potential for transdifferentiation. It may not always be necessary to reprogram cells all the way back to their most primitive pluripotent stem cell state, and instead reprogram one type of adult somatic tissue directly into a different type, bypassing the lengthy processes of complete reprogramming and subsequent differentiation.  For example, in theory fibroblasts that can be easily and safely obtained from a patient’s skin could be converted into neurons or heart muscle cells without ever passing through a pluripotent stage. This would have advantages not only in the conservation of time and resources but also for safety, as transdifferentiation does not pose the risk of tumorgenesis as the cells never are pluripotent. Unfortunately, the technology for such processes is very difficult. To reprogram cells directly into a different cell type, the qualities and characteristics of the desired cell type must be comprehensively understood. For iPSCs the desired cell type was embryonic stem cells, which were very well researched and characterized, but for many types of cell of interest, including cells of the central nervous system, there are still many unanswered questions about the target cell population. Excitingly, the Wernig lab at Stanford has recently created induced neurons (iN) directly from mouse fibroblasts.

Paramedic functions^

A potential use of iPSCs for cellular therapy that can be applied much more quickly than actual replacement of damaged tissue is the transplant of pluripotent cells as support cells rather than replacement neurons. These cells offer neuroprotection by preventing inflammation and producing neurotrophic factors (for the therapeutic use of neurotrophic factors in HD, click here).  In various studies, the transplantation of iPSCs has significantly improved host neuronal survival and function.  This bystander mechanism of therapy is of huge immediate potential in iPSCs, and Dr. Nolta’s lab recently submitted a request for a clinical study of the same mechanism using mesenchymal stem cells to the FDA. For a detailed study of the use of iPSCs for this purpose click here.

Conclusions^

Stem cell biology has been an area of great interest and intense debate since its inception, and iPSC technology has furthered this research and created hope for potential therapeutic applications. While there are still many barriers to the clinical use of stem cells, iPSCs may help elucidate the nature of both pluripotent stem cells and of many disease pathologies to reach an eventual concrete connection between the two. With their potential for autologous cell replacement and disease modeling in vitro iPSCs are the future of stem cell research, and as such they are key players in the battle against HD.

For Further Reading^

Abeliovich, Asa and Claudia A. Doege. “Reprogramming Therapeutics: iPS Cell Prospects for Neurodegenerative Disease.” Neuron. 12 Feb, 2009, 61 (3): 337-39.

Short, approachable article reviewing two studies deriving iPSCs from patients with neurological disorders.

Cox, Jesse L. and Angie Rizzino. “Induced pluripotent stem cells: what lies beyond the paradigm shift.” Experimental Biology and Medicine. Feb 2010, 235 (2): 148-58.

Very detailed, mostly accessible review of the state of iPS research and the discoveries to date, as well as what iPS cells mean for stem cell biology and modern medical approaches. Perfect thorough introduction to iPS technology.

Crook, Jeremy Micah, and Nao Rei Kobayashi. “Human stem cells for modeling neurological disorders: Accelerating the drug discovery pipeline.” Journal of Cellular Biochemistry. 105 (6): 1361-66.

Accessible, interesting article that argues the greatest potential for iPSCs is to test potential drugs for neurological diseases in vitro and find problems early on in the drug development, saving time and resources.

Gunaseeli, I., et al. “Induced Pluripotent Stem Cells as a Model for Accelerated Patient- and Disease-specific Drug Discovery.” Current Medicinal Chemistry. 2010, 17: 759-766.

Readable review on the future of iPS cells, comparing them with other stem cells and elucidating their pontential drawbacks. Good summary of the landmark discoveries in iPS technology to date.

Haruhisa, Inoue. “Neurodegenerative disease-specific induced pluripotent stem cell research.” Experimental Cell Research. 2010.

General overview of use of iPS cells specific to neurological diseases for modeling diseases in vitro and eventually using as a cellular replacement therapy. Good, non-technical overview of the various potential pathways of iPS technology.

Hung, Chia-Wei, et al.  “Stem Cell-Based Neuroprotective and Neurorestorative Strategies.” International Journal of Molecular Science. 2010, 11(5): 2039–2055.

Overview of various neurological diseases and the potential of stem cell therapeutics, either using adult neural stem cells or iPS stem cells. Experiment descriptions are fairly technical, but the review’s reflections and discussion are accessible and interesting.

Laowtammathron, Chuti, et al. “Monkey hybrid stem cells develop cellular features of Huntington’s disease.” BioMed Center Cell Biology. 2010, 11 (12).

Detailed article on the establishment of pluripotent HD monkey model cell line and its use in the study of Huntington’s.

Marchetto, Maria C.N., et al. “Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases.” Human Molecular Genetics. 2010, 19 (1).

Fairly technical review describing the use of iPSCs for modeling neurological disorders.

Niclis, J.C., et al. “Human embryonic stem cell models of Huntington’s Disease.” Reproductive Biomedicine Online. July 2009, 19 (1): 106-13.

Detailed, technical article on the use of human embryonic stem cell lines for HD.

O’Malley, James. “New strategies to generate induced pluripotent stem cells”. Current Opinions in Biotechnology. Oct. 2009: 20 (5): 516-21.

Longer technical article on the various strategies to generate iPS cells without using potentially dangerous viral vectors.

Okita, Keisuke, et al. “Generation of germline-competent induced pluripotent stem cells.” Nature. 19 Jul, 2007, 448(7151):313-17.

Fairly technical article about an early study in iPS research, where cells were selected for Nanog expression rather than the less pertinent gene Fbx15. This higher caliber of selected cells were germline-competent.

Okita, Keisuke, et al. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors.” Science. 7 Nov, 2008, 322 (5903): 949-53.

Technical article about the advancements in finding non-viral, clinically applicable methods of creating iPS cells.

Orlacchio, A., et al. “Stem Cells: An Overview of the Current Status of Therapies for Central and Peripheral Nervous System Diseases.” Current Medicinal Chemistry. 2010, 17: 595-608.

Technical review on the various types of stem cells used in the studies of neurological diseases and the progress made to date with these cells.

Park, In-Hyun, et al. “Disease-Specific Induced Pluripotent Stem Cells.” Cell. 2008, 134 (5): 877-86.

Fairly accessible article on the creation of iPS cells with genetic defects, as tools for studying the symptoms and experimenting with treatments of various diseases.

Robbins, Reisha D., et al. “Inducible pluripotent stem cells: not quite ready for prime time?” Current Opinion in Organ Transplantation. 15 (1): 61-57.

Clear review of the barriers facing clinical use of iPSCs, accessible and realistic.

Soldner, Frank, et al.Parkinson’s Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors.” Cell. 6 Mar, 2009, 136 (5): 964-77.

Technical article about first successful derivation of iPS cells from a patient with a neurodegenerative disease without using viral vectors. Relevant to HD research as a protocol that will likely be followed for subsequent creation of neurodegenerative iPSC lines for in vitro study.

Stradtfeld, Matthias, et al. “Induced Pluripotent Stem Cells Generated Without Viral Integration.” Science. 7 Nov, 2008, 322 (5903): 945-49.

Technical article outlining a method for creating iPS cells using excisable adenoviruses, rather. than retroviruses that have the potential to harm the cells.

Takahashi, Kazutoshi, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors” Cell. 30 Nov, 2007, 131(5): 861-72.

Landmark article in the discovery of induced pluripotent stem cells and the factors that create them. Short, but fairly technical.

Yamanaka, Shinya. “Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors.” Cell Proliferation. Feb, 2008, 41 (Suppl. 1):51-6

Short review, less technical summary of first iPS discovery by Yamanaka. Perfect for quick overview of the basics of iPS cell generation.

Yamanaka, Shinya. “Strategies and New Developments in the Generation of Patient-Specific Pluripotent Stem Cells.” Cell: Stem Cell. 7 June 2007, 1(1): 39-49.

Comprehensive review of various methods for creating pluripotent stem cells with a detailed introduction to iPSC methods. Fairly accessible, and very thorough.

A. Lanctot 2011

More

Adipose Derived Stem Cells and HD: Real Applications of iPSCs

While stem cells have always been heralded as the future of cellular replacement therapies, recent stem cell research has explored the potential “bystander” or “paramedic” effects of stem cells, which use stem cells to repair damaged cells rather than replacing them. Bystander therapies do not require the stem cells to become the type of cell that is damaged (to differentiate into this cell type),  but rather can help damaged neurons by changing the host environment. The phenotype of the undifferentiated, stem cell that are still pluripotent, able to differentiate into many different cell types, may provide therapeutic benefits in Huntington’s Disease (HD) by releasing neurotrophic factors that promote neuron growth and survival and arrest the mutant huntingtin protein’s negative influence on key cellular survival and energetic pathways. This paramedic function of stem cells might be harnessed to prevent mechanisms of HD that cause harmful symptoms rather than replacing damaged cells, as an alternative approach to traditional drug therapy for the treatment of neurodegenerative diseases.

Researchers in South Korea have recently found that adipose-derived stem cells (ASCs) can serve the same “paramedic” function in HD as is observed in the mesenchymal stem cells that Dr. Nolta is currently researching at the University of California, Davis. Similar to mesenchymal stem cells, adipose-derived stem cells do not create the ethical debates that embryonic stem cells do, as they are removed from adults during elective surgery, not from embryos in vitro. But in contrast to mesenchymal stem cells, ASCs are not found naturally in the body, but are rather multipotent stem cells created by iPS, or induced pluripotent stem cells (for more information about the new technology of iPS, click here). Derived from fat tissue taken from consenting patients, ASCs have the double advantage of easy access and minimal ethical implications. Like all stem cells, ASCs have the ability to differentiate into different somatic cells, though the mechanisms of differentiation are still unclear and scientists do not know how to direct differentiation of ASCs into certain types of tissue.  This study was not concerned with the differentiation of ASCs, but rather their neuroprotective abilities, such as the release of growth factors that are essential in combating many of the symptoms of HD.

How the Bystander Mechanism Works^

Current research using HD mouse models indicates that the use of fetal striatal tissue to replace the damaged striatal tissue in HD mouse models is not possible at this time, as the replacement tissue does not alter the toxicity of the mutant huntingtin protein.  This is like replacing a wall that has been eaten away by termites without doing anything to remove the termites: the new wall will soon also be harmed by the pests. Even if this barrier was overcome, the use of stem cells to replace damaged tissue is hampered by many other problems, such as a lack of donor tissue and rejection by the immune system, identical problems to organ replacement.  Rather than replacing damaged tissue, a different approach could use stem cells to preemptively prevent HD from harming neuronal tissue.  Scientists have noted that with other diseases, stem cells often act through a bystander mechanism, preventing the symptoms of the disease from manifesting, rather than directly replacing damaged cells. This novel approach to stem cell use does not require the extensive technology of differentiation, transplantation, and incorporation with host tissue that cell replacement needs. Instead, the bystander mechanism approach of stem cells takes advantage of the cells’ ability to release factors in their pluripotent state that combat the symptoms of HD. The researchers in this study wished to test this mechanism using ASCs, knowing that the cells could not only differentiate into cells such as neurons and glial cells that could be useful in combating HD, but could also release growth factors that may slow the symptoms of HD (to learn about growth factors and their role in fighting HD, click here.

HD symptoms may be caused by the alterations the mutant huntingtin protein makes to the transcription of DNA by interfering with transcription factors (for more information about the HD’s affect on transcription, click here) Transcriptional interference and mitochondrial dysfunction are key aspects of HD pathology. Mutant huntingtin protein aggregates impede important transcription factors such as the CREB-binding protein which is essential for transcription of pathways key to cell survival.  In HD, one way neural cell death is induced is by inhibiting this transcription factor.  ASCs could slow neurodegeneration and neural death by releasing neurotrophic factors that help prevent premature cell death. Neurotrophic factors encourage the maintenance, growth, and survival of neurons and so serve as a counterforce to mutant huntingtin’s stimulation of cell death. Another transcription factor, PGC-1, controls the creation of mitochondria, and when it is repressed by mutant huntingtin, reduced numbers of mitochondria causes the cell to receive insufficient energy. This makes the cell susceptible to glutamate, an excitatory neurotransmitter that can harm neurons and may be involved in many of the symptoms of HD. ASCs increase PGC-1 expression by preventing the huntingtin protein from impeding its transcriptional regulation, and so prevent glutamate levels from becoming harmfully high in the neuron.

Effects of ASCs in Three Different Models^

The effect of ASC transplantation was tested in three models, a knockout rat model, a transgenic mouse model and an in vitro cell model (for more information about different types of experimental models, click here). All models showed the positive effects of ASCs. The rat model showed that the ASCs were neuroprotective, reducing neuronal death. More specifically, this was observed by comparing the size of the rats’ ventricles of the brain, which are enlarged by the loss of neural tissue in HD. The ventricles of the ASC-treated rats were smaller than those of the control HD model rats, indicating less tissue loss. Ventricle volume is closely correlated with HD progression, as larger ventricles indicate greater loss of neural tissue. By showing a decreased ventricle volume, ASC rats seem to exhibit less cell death, resulting in less loss of neural tissue. In addition, compared to the HD control mice, the mouse model with ASC transplantation had a delayed decline in motor function, had less mutant huntingtin aggregates, and  lived longer.

Potential Problems with ASCs for Clinical Use^

The promising results of the three models hint at the potential of ASCs to delay the onset of HD symptoms, but there is much that still needs to be researched.  It was found that the transplanted ASCs were not evenly distributed across the striatum as expected, but rather most ASCs remained at the initial site of transplantation. This may have impeded the effectiveness of the ASCs and may even be harmful in long-term models. The proliferation of the ASCs was slow once transplanted, so the documented ability of stem cells to proliferate in actual organisms does not seem to apply to ASC transplantation in HD models.  This study did not test whether the same results could be achieved in model organisms that are longer lived. Whether ASCs would be effective in humans, for extended periods of time, has not yet been determined.  Furthermore, the ASCs must be prevented from differentiating into cells that would be harmful in the brain. Currently, the research for directed differentiation of pluripotent cells is rudimentary, and there is particular risk associated with the spontaneous differentiation of ASCs in vivo. The potential risk of the cells to differentiate into other tissue like heart or skin cells should be tested. Another problem of stem cells is that they may be rejected by the patient’s immune system, though this problem is greatly reduced with ASCs. In this study, human ASCs were successfully injected into rat and mouse models without the aid of immunosuppressants, which is encouraging. To further prevent the risk of rejection, the patient’s own adipose cells could be used to create the ASCs. But ASCs derived from patients with the mutant huntingtin protein have yet to be tested and it is possible that these cells may be damaged or not fully effective.

Conclusions^

Researchers have shown that ASCs may have the potential to protect mechanisms of transcription and rescue degenerating neurons by combating the detrimental actions of huntingtin aggregates through the release of growth factors. This bystander mechanism is a novel approach to using stem cells, as they have been traditionally thought of as replacement cells for damaged tissue. The value of stem cells to replace damaged tissue with healthy, fully-differentiated replacement cells cannot be dismissed, especially with new iPS technology and the ability to engineer replacement cells from the patient who is to receive them, reducing the risk of immune rejection. Unfortunately, much more research must be done before stem cells will be used in clinical therapies for cellular replacement, but a more immediate potential for stem cells is in a paramedic capacity, where differentiation and incorporation with the host tissue is not required. By influencing key events in the pathogenesis of HD, ASCs may delay the onset of harmful symptoms. Current research has shown that ASC transplantation may allow for the expression of transcription pathways that HD suppresses, reduce the number of toxic huntingtin aggregates, and decrease the extent of neuron death in mouse models. Its therapeutic use still requires much more research and exploration, and then must make the leap from animal models to human trials, but ASCs have the potential to rescue degenerating neurons and prevent HD symptoms. This ability of stem cells to not only replace damaged tissue, but also prevent tissue damage, holds promise for the treatment of HD.

For Further Reading

Lee, et al. “Slowed Progression in Models of Huntington Disease by Adipose Stem Cell Transplantation.Annals of Neurology. 2009, 66(5): 671-681.

Technical but well-explained article on a specific study of the use iPS cells derived from adipose cells in three HD models: induced rat, transgenic mouse, and in vitro.

A. Lanctot 2011


While stem cells have always been heralded as the future of cellular replacement therapies, recent stem cell research has explored the potential “bystander” or “paramedic” effects of stem cells, which use stem cells to repair damaged cells rather than replacing them. Bystander therapies do not require the stem cells to become the type of cell that is damaged (to differentiate into this cell type), but rather can help damaged neurons by changing the host environment. The phenotype of the undifferentiated, stem cell that are still pluripotent, able to differentiate into many different cell types, may provide therapeutic benefits in Huntington’s Disease (HD) by releasing neurotrophic factors that promote neuron growth and survival and arrest the mutant huntingtin protein’s negative influence on key cellular survival and energetic pathways. This paramedic function of stem cells might be harnessed to prevent mechanisms of HD that cause harmful symptoms rather than replacing damaged cells, as an alternative approach to traditional drug therapy for the treatment of neurodegenerative diseases.

Researchers in South Korea have recently found that adipose-derived stem cells (ASCs) can serve the same “paramedic” function in HD as is observed in the mesenchymal stem cells that Dr. Nolta is currently researching at the University of California, Davis. Similar to mesenchymal stem cells, adipose-derived stem cells do not create the ethical debates that embryonic stem cells do, as they are removed from adults during elective surgery, not from embryos in vitro. But in contrast to mesenchymal stem cells, ASCs are not found naturally in the body, but are rather multipotent stem cells created by iPS, or induced pluripotent stem cells (for more information about the new technology of iPS, click http://hopes.stanford.edu/n3821/research-and-hd/induced-pluripotent-stem-cells). Derived from fat tissue taken from consenting patients, ASCs have the double advantage of easy access and minimal ethical implications. Like all stem cells, ASCs have the ability to differentiate into different somatic cells, though the mechanisms of differentiation are still unclear and scientists do not know how to direct differentiation of ASCs into certain types of tissue. This study was not concerned with the differentiation of ASCs, but rather their neuroprotective abilities, such as the release of growth factors that are essential in combating many of the symptoms of HD.

How the Bystander Mechanism Works

Current research using HD mouse models indicates that the use of fetal striatal tissue to replace the damaged striatal tissue in HD mouse models is not possible at this time, as the replacement tissue does not alter the toxicity of the mutant huntingtin protein. This is like replacing a wall that has been eaten away by termites without doing anything to remove the termites: the new wall will soon also be harmed by the pests. Even if this barrier was overcome, the use of stem cells to replace damaged tissue is hampered by many other problems, such as a lack of donor tissue and rejection by the immune system, identical problems to organ replacement. Rather than replacing damaged tissue, a different approach could use stem cells to preemptively prevent HD from harming neuronal tissue. Scientists have noted that with other diseases, stem cells often act through a bystander mechanism, preventing the symptoms of the disease from manifesting, rather than directly replacing damaged cells. This novel approach to stem cell use does not require the extensive technology of differentiation, transplantation, and incorporation with host tissue that cell replacement needs. Instead, the bystander mechanism approach of stem cells takes advantage of the cells’ ability to release factors in their pluripotent state that combat the symptoms of HD. The researchers in this study wished to test this mechanism using ASCs, knowing that the cells could not only differentiate into cells such as neurons and glial cells that could be useful in combating HD, but could also release growth factors that may slow the symptoms of HD (to learn about growth factors and their role in fighting HD, click (http://hopes.stanford.edu/treatmts/neurotrophicfactors/nf0.html|here).

HD symptoms may be caused by the alterations the mutant huntingtin protein makes to the transcription of DNA by interfering with transcription factors (for more information about the HD’s affect on transcription, click http://hopes.stanford.edu/causes/huntprot/p5.html |here) Transcriptional interference and mitochondrial dysfunction are key aspects of HD pathology. Mutant huntingtin protein aggregates impede important transcription factors such as the CREB-binding protein which is essential for transcription of pathways key to cell survival. In HD, one way neural cell death is induced is by inhibiting this transcription factor. ASCs could slow neurodegeneration and neural death by releasing neurotrophic factors that help prevent premature cell death. Neurotrophic factors encourage the maintenance, growth, and survival of neurons and so serve as a counterforce to mutant huntingtin’s stimulation of cell death. Another transcription factor, PGC-1http://www3.interscience.wiley.com/giflibrary/12/alpha.gif, controls the creation of mitochondria, and when it is repressed by mutant huntingtin, reduced numbers of mitochondria causes the cell to receive insufficient energy. This makes the cell susceptible to glutamate, an excitatory neurotransmitter that can harm neurons and may be involved in many of the symptoms of HD. ASCs increase PGC-1http://www3.interscience.wiley.com/giflibrary/12/alpha.gif expression by preventing the huntingtin protein from impeding its transcriptional regulation, and so prevent glutamate levels from becoming harmfully high in the neuron.

Effects of ASCs in Three Different Models

The effect of ASC transplantation was tested in three models, a knockout rat model, a transgenic mouse model and an in vitro cell model (for more information about different types of experimental models, click http://hopes.stanford.edu/rltdsci/studyhd/am00.html|here). All models showed the positive effects of ASCs. The rat model showed that the ASCs were neuroprotective, reducing neuronal death. More specifically, this was observed by comparing the size of the rats’ ventricles of the brain, which are enlarged by the loss of neural tissue in HD. The ventricles of the ASC-treated rats were smaller than those of the control HD model rats, indicating less tissue loss. Ventricle volume is closely correlated with HD progression, as larger ventricles indicate greater loss of neural tissue. By showing a decreased ventricle volume, ASC rats seem to exhibit less cell death, resulting in less loss of neural tissue. In addition, compared to the HD control mice, the mouse model with ASC transplantation had a delayed decline in motor function, had less mutant huntingtin aggregates, and lived longer.

Potential Problems with ASCs for Clinical Use

The promising results of the three models hint at the potential of ASCs to delay the onset of HD symptoms, but there is much that still needs to be researched. It was found that the transplanted ASCs were not evenly distributed across the striatum as expected, but rather most ASCs remained at the initial site of transplantation. This may have impeded the effectiveness of the ASCs and may even be harmful in long-term models. The proliferation of the ASCs was slow once transplanted, so the documented ability of stem cells to proliferate in actual organisms does not seem to apply to ASC transplantation in HD models. This study did not test whether the same results could be achieved in model organisms that are longer lived. Whether ASCs would be effective in humans, for extended periods of time, has not yet been determined. Furthermore, the ASCs must be prevented from differentiating into cells that would be harmful in the brain. Currently, the research for directed differentiation of pluripotent cells is rudimentary, and there is particular risk associated with the spontaneous differentiation of ASCs in vivo. The potential risk of the cells to differentiate into other tissue like heart or skin cells should be tested. Another problem of stem cells is that they may be rejected by the patient’s immune system, though this problem is greatly reduced with ASCs. In this study, human ASCs were successfully injected into rat and mouse models without the aid of immunosuppressants, which is encouraging. To further prevent the risk of rejection, the patient’s own adipose cells could be used to create the ASCs. But ASCs derived from patients with the mutant huntingtin protein have yet to be tested and it is possible that these cells may be damaged or not fully effective.

Conclusions

Researchers have shown that ASCs may have the potential to protect mechanisms of transcription and rescue degenerating neurons by combating the detrimental actions of huntingtin aggregates through the release of growth factors. This bystander mechanism is a novel approach to using stem cells, as they have been traditionally thought of as replacement cells for damaged tissue. The value of stem cells to replace damaged tissue with healthy, fully-differentiated replacement cells cannot be dismissed, especially with new iPS technology and the ability to engineer replacement cells from the patient who is to receive them, reducing the risk of immune rejection. Unfortunately, much more research must be done before stem cells will be used in clinical therapies for cellular replacement, but a more immediate potential for stem cells is in a paramedic capacity, where differentiation and incorporation with the host tissue is not required. By influencing key events in the pathogenesis of HD, ASCs may delay the onset of harmful symptoms. Current research has shown that ASC transplantation may allow for the expression of transcription pathways that HD suppresses, reduce the number of toxic huntingtin aggregates, and decrease the extent of neuron death in mouse models. Its therapeutic use still requires much more research and exploration, and then must make the leap from animal models to human trials, but ASCs have the potential to rescue degenerating neurons and prevent HD symptoms. This ability of stem cells to not only replace damaged tissue, but also prevent tissue damage, holds promise for the treatment of HD.

More

Induced Neurons

Despite predictions of transdifferentiation being a technology of the future, Dr. Marius Wernig’s lab at Stanford has recently discovered a method of reengineering neurons directly from fibroblasts by the the forced expression of transgenes. This is the same method by which induced pluriptent stem cells (iPSCs) are produced, and transdifferentiation, the engineering of cells so they change their type without preceding through an intermediate stage, has been suggested as the logical progression from iPSCs. Its success suggests that, along with iPSC technology, cellular differentiation and cell fate are far more flexible than previously thought.  The new discovery that somatic cells can be turned into completely different cell types by a cocktail of a few genes revolutionizes previous thought about the unchangeable nature of fully differentiated cells, and adds new theories as to how cell fate is determined. The research also importantly shows that reengineering cells with transcription factors can create the complex structures and functions of somatic cells, not simply undifferentiated stem cells.  A combination of three transcription factors that are specifically found in the brain were shown to be able to convert both embryonic and postnatal fibroblasts directly into neurons, in much the same way iPSCs are created, but without preceding through any pluripotent stem cell intermediate. The lab tested nineteen potential genes to see what combination could efficiently convert mouse fibroblasts into neurons in vitro. The gene Asc11 alone could generate immature neurons with undeveloped properties. Another eighteen genes were then tested in combination with Ascl1, and five genes (Brn2, Brn4, Myt1l, Zic1, and Olig2) substantially improved neuron development. While none of these five genes generated induced neuronal (iN) cells when tested individually, it was found that the three factor combination of Ascl1, Brn2, and Myt1l produced the highest quantity of neurons with the most mature action potentials. As a result this combination was concluded to be the ideal grouping of factors for inducing neuron phenotypes.

Induced Neurons: Are they just like normal neurons?^

A difficulty in evaluating whether transdifferentation is successful is that unlike the relatively simple phenotypes of undifferentiated cells, somatic cells have far too many characteristics that must be tested to ensure that complete reengineering occurred. Induced neuronal cells (iNs) express neuron-specific proteins, generate action potentials and form functional synapses but it is still not known how, if at all, the iNs differ from normal neurons. The cells expressed three neuron marker proteins, MAP2, NeuN, and synapsin, and produced spontaneous action potentials, which are the distribution of charged ions across a neuron membrane that causes a signal to be propelled down the neuron’s axon and to the next neuron. Action potentials promote neural communication as they allow a message to cross a cell before neurotransmitters become involved in its movement between two cells. These action potentials were blocked when a sodium ion inhibitor, tetradotoxin, was introduced, as would occur in normal brain tissue. In normal brains, the concentration of sodium across the membrane of the neuron is essential for the cells to generate action potential and relay electrochemical messages through the neuron and onto the connecting cells. The cells’ expression of functional membrane channel proteins that allow sodium ions to flow in and out of the neuron supports the claim that iN cells and normal neurons exhibit identical membrane properties.

Problems in iN Generation^

The iN cells had various phenotypes, but did not exhibit all types of nervous tissue. Most iNs were excitatory neurons, while almost none contained periperin, a protein characteristic of neurons in the peripheral nervous system. The majority of iN cells were excitatory cells that expressed markers indicative of cortical identity, i.e. specific to cells typically found in the cortex. Further research may focus on the generation of iN cells of other specific neuronal subtypes, not to mention the generation of iNs from human cells (currently only mouse iNs have been successfully engineered).  Additional neural transcription factors may aid in creating neurons of more specific phenotypes.

Like induced pluripotent stem cells, iNs go through a gradual process of reengineering. While immature neuron-like cells can been seen as early as three days after infection, it takes five days for branching neuronal cells to form. Physical maturation continues over several weeks.  The efficiency of converting cells into iNs ranged from 1.8–7.7%, which is substantially better than efficiency of iPSC production. However, iNs cannot proliferate like iPSCs, so creating a larger quantity of cells is crucial.

An important question concerning iNs involves their ability to function as neurons by forming functional communication with other cells. This is a crucial requirement if they are to be used as tissue replacement in the future. The researchers tested whether iN cells have the capacity to form functional synapses with other iN cells and whether iN cells were capable of integrating into preexisting neural networks. When iN cells were grown with actual neurons, spontaneous and rhythmic neural activity was observed. The cells could receive synaptic inputs from the normal neurons, demonstrating their ability to integrate into preexisting tissue. It was also shown that iN cells are capable of forming functional synapses with each other.

Transgenes that Engineer Neurons^

Like iPSCs, the gene combination for creating iN cells allows some leniency and variation, but a certain “recipe” seems the most effective. It remains to be seen if different genes or different ratios of the genes that Dr. Wernig’s lab identified will even further improve efficiency of iN production. While Ascl1 alone is sufficient to induce some neuronal traits, such as expression of proteins that generate action potentials, the addition of Brn2 and Myt1l creates more mature cells with increased efficiency up to 19.5%. The highly efficient production of iNs makes it unlikely that they are merely formed from rare stem or precursor cells in the starting cell population, as great care was taken to exclude neural tissue in the isolation of the initial cell population, and no neurons or neural progenitor cells were detected in the culture. Future studies are nonetheless needed to unequivocally demonstrate that cells that have their own unique morphologies can be directly converted into neurons, and that the iNs are not mere derivatives of stem cells.

Why certain neuronal subpopulations (such as the cortical neurons) are more favored than others is another aspect of iN technology that remains to be researched. It may be that high expression of neural cell-fate determining factors directs certain cell types to form, so they are reengineered more often. Different cell types are produced during development by sets of transcription factors that cause cell type specific proteins to be produced. Each cell can be thought of as a person who walks by a lot of doors, but only has one key (specific transcription factors) that allows him to open one door, i.e. the cell can only become the type specified by its transcription factors.

iN cells are a possible alternative to iPSCs for generating patient-specific neurons. The generation of iN cells is fast, efficient, and has the major advantage over iPSCs that it does not go through a stage of pluripotent stem cells that are susceptible to tumor production.  The iN cells could also provide new methods for studying cellular identity and neural development. They have potential uses in neurological disease-modeling, drug discovery, and regenerative medicine. Formerly, transdifferentiation was never thought of as anything besides a futuristic version of cell engineering that would take many years to accomplish. The Wernig lab has shown that cells with more complex morphologies can indeed be generated directly from other cell types using much the same method as iPSCs, and although much remains to be tested, this new technology may revolutionize cell therapies as more cell types are derived.

For Further Reading^

Vierbuchen, et al. “Direct conversion of fibroblasts to functional neurons by defined factors.” Nature. 25 Feb. 2010, 463 (7284):1035-1041.

Well written, fairly accessible article. Some parts a bit technical but very nice section on the next steps for iN.

A. Lanctot 2011.


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