Regenerating Dead Cells In the Brain with Stem Cells

Neuroscientists at the Université Libre De Bruxelles (ULB) in Belgium have taken a very important step in cell therapy for diseases of the brain. This team generated cortical neurons from embryonic stem cells, which they then used to treat adult with brain problems. This research was recently published in the journal Neuron.

The ULB team was led by Pierre Vanderhaeghen, Kimmo Michelsen and Sandra Acosta (ULB Neuroscience Institute, in collaboration with the laboratory of Afsaneh Gaillard (INSERM/U. Poitiers, France). These results open new perspectives for the repair of damaged cells in the brain and replacing damage neurons.

The cerebral cortex is definitely the most complex and essential structure of our brain. The nerve cells or neurons that compose the cerebral cortex are the basic building blocks that help it do every job that it does. The loss of loss of cortical neurons is the cause of many neurological diseases as a result of stroke, Alzheimer disease, or physical trauma to the brain can seriously compromise brain function.

Previously, these same ULB researchers discovered how to generate cortical neurons in the laboratory cortical neurons from embryonic stem cells. Despite the triumph of these findings, it was completely unclear whether these findings could be translated into a living creature.

Now, the ULB team has successfully tested the use of their laboratory-generated cortical neurons in a living animal. In this study, Vanderhaeghen and others transplanted cortical pyramidal neurons made from embryonic stem cells into the brains of adult mice who had undergone chemically induced brain damage. This experiment cause rather massive neuronal losses in the visual cortex.

Remarkably, the implanted neurons integrated effectively into the brain after injury, but most importantly they could connect with the host brain, and some of them even responded to visual stimuli, like the visual cortex.

Integration only occurred, if the types of implanted neurons were matched to the lesioned area. In other words, since visual cortex neurons were lost, only the implantation of other cortical neurons allowed the cells to properly engraft into the visual cortex. However the grafted neurons displayed long-range patterns of connectivity with the host neurons.

This remains an experimental approach that has, to date, only been successfully performed with laboratory mice. A good deal more much research is required before any clinical application in humans will come to the clinic. Regardless, the success of these experiments combining cell engineering to generate nerve cells in a controlled and unlimited fashion, together with transplantation in to damaged brain, opens new avenues to repair the brain following damage or degeneration, such as following stroke or brain trauma.

Stem Cell Structure and Obesity

New research conducted at Queen Mary University of London (QMUL) has discovered that the regulation of the length of primary cilia, which are small hair-like projections on the surfaces of most cells, can prevent the production of fat cells taken from adult human bone marrow. Such a discovery might be used to develop a way of preventing obesity.

What are primary cilia?  For many years, almost all attention was focused on cilia that moved because their function was readily observable.  However, Alexander Kowalevsky first reported in 1867 the presence of single (nonmotile) cilia in a variety of vertebrate cells.  These solitary and nonmotile cilia are far more widespread than the motile type.  In humans, only a few cell types have motile cilia, namely epithelial cells in the bronchi and oviducts, and ependymal cells that line brain vesicles.  However, virtually all other cells have a primary cilium.

What makes primary cilia different from the motile form? First, they lack the central pair of microtubules, which would explain the lack of motility.  Primary cilia also seem to lack dynein, one of the molecular motors needed for motility.  In addition, some primary cilia do not project beyond the cell surface, and most, but not all, are very short.  What do these organelles do if they are not sticking out of the cell, or motile?

Further work has shown that primary cilia are important in intracellular transport and also in sensory function for cells.  Now it seems that primary cilia are also important in the process of adipogenesis.

Primary cilia

Adipogenesis refers to the differentiation of stem cells into fat cells. The QMUL research team showed that during adipogenesis, the length of primary cilia increases, which increases the movement of specific proteins associated with the cilia. When the QMUL team genetically restricted primary cilia elongation by genetic means, they were able to stop the formation of new fat cells.

One of the lead authors or this study, Melis Dalbay, said that it was the first time that subtle changes in primary cilia structure can influence the differentiation of stem cells into fat.

Since the length of primary cilia can be influenced by various factors including pharmaceuticals, inflammation and even mechanical forces, this study provides new insight into the regulation of fat cell formation and obesity.

This research points toward a new type of treatment known as “cilia-therapy” where manipulation of primary cilia may be used in the future to treat a growing range of conditions including obesity, cancer, inflammation and arthritis.

Testing Stem Cell Quality

A new paper published in the journal EMBO Molecular Medicine by a team from the Lausanne University Hospital describes a protocol that can ensure the safety of adult epidermal stem cells before they are used as treatments for patients. The approach devised by this team takes cultivated, genetically modified stem cells and isolates single cells that are then used to make clonal cell cultures. These cloned cells are then rigorously tested to ensure that they meet the highest possible safety criteria. This protocol was inspired by approaches designed in the biotechnology industry and honed by regulatory authorities for medicinal proteins produced from genetically engineered mammalian cells.

“Until now there has not been a systematic way to ensure that adult epidermal stem cells meet all the necessary requirements for safety before use as treatments for disease,” says EMBO Member Yann Barrandon, Professor at Lausanne University Hospital, the Swiss Federal Institute of Technology in Lausanne and the lead author of the study. “We have devised a single cell strategy that is sufficiently scalable to assess the viability and safety of adult epidermal stem cells using an array of cell and molecular assays before the cells are used directly for the treatment of patients. We have used this strategy in a proof-of-concept study that involves treatment of a patient suffering from recessive dystrophic epidermolysis bullosa, a hereditary condition defined by the absence of type VII collagen which leads to severe blistering of the skin.”

Barrandon and co-workers have cultivated epidermal cells from patients who suffer from epidermolysis bullosa. These cells were then genetically engineered in order to insert a normal copy of the type VIII collagen gene. Then the genetically fixed cells were grown in culture so that they can be used to regenerate skin. Barrandon and others subjected these cells to an array of tests in order to determine which of the genetically engineered cells meet the requirements for safety and “stemness,” which refers to the stem cell characteristics that distinguish it from regular cells; its developmental immaturity and its ability to grow and self-renew. Clonal analysis revealed that the cultured, genetically engineered stem cells varied in their ability to produce functional type VII collagen. When the most viable, modified stem cells were selected and transplanted into the skin of immunodeficient mice, the cells regenerated skin and produced skin that did not blister in the mouse model system for recessive dystrophic epidermolysis bullosa. Furthermore, the cells produced functional type VII collagen. The safety of the cells was assessed by mapping the sites of integration of the viral vector. Because such viruses and produce gene rearrangements other mutations, the chosen cell lines were subjected to whole genome sequencing. Only the cells with insertions in benign locations were considered for use in their mouse model.

Barrandon concluded: “Our work shows that at least for adult epidermal stem cells it is possible to use a clonal strategy to deliver a level of safety that cannot be obtained by other gene therapy approaches. A clonal strategy should make it possible to integrate some of the more recent technologies for targeted genome editing that offer more precise ways to change genes in ways that may further benefit the treatment of disease. Further work is in progress in this direction.”

This work is certainly fascinating, but I think that using integrating viral vectors is asking for trouble. Certainly it should be possible to fix or replace the abnormal type VII collagen gene. Viruses that randomly insert genes into the genome can cause genetic problems, and even sequencing the genome may not properly address the safety concerns of the use of such viral vectors.

Safer Culture Conditions for Stem Cells

Jeanne Loring from the Scripps Institute is the senior author of a very important study that examined the culture conditions for pluripotent stem cells.

Several scientists have discovered that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) can accumulate cancer-causing mutations when grown in culture for extended periods of time (for example, see Uri Weissbein, Nissim Benvenisty, and Uri Ben-David, J Cell Biol. 2014 Jan 20; 204(2): 153–163). However, some laboratories have managed to keep ESCs in culture for extended periods without observing instabilities.

To try to tease apart why this might be the case, Loring and her group examined various culture methods and determined that some stem cell culture methods are associated with increased incidence of mutations in the DNA of stem cells.

“This is about quality control; we’re making sure these cells are safe and effective,” said Loring, who is a professor of developmental neurobiology at Scripps Research Institute (SRI) in San Diego, CA.

All cells run the risk of accumulating mutations when they divide, but previous research from Loring and her colleagues showed that particular culture conditions could potentially select for faster growth and mutations that accelerate growth. Such growth-enhancing mutations are sometimes associated with tumors.

“Most changes will not compromise the safety of the cells for therapy, but we need to monitor the cultures so that we know what sorts of changes take place,” said Ibon Garitaonandia, who is a postdoctoral research fellow in Loring’s laboratory at SRI.

New research from Loring’s group has shown how particular culture conditions can reduce the likelihood of mutations. Loring and her colleagues tested several different types of surfaces upon which the cells were grown. They also used different ways of propagating or “passaging” the cultures. When cells are grown in culture, the culture dishes must be scraped to get the cells off them and then the cells must be transferred to a fresh culture dish. How you do this matters: do you use enzymes to detach the cells, or do you mechanically scrape them off? Other culture techniques use layers of “feeder cells” that do not divide, but are still able to secrete growth factors that improve the health of the growing stem cells.

Loring and her crew tested various combinations of surfaces, passaging methods and feeder cell populations and grew the cells for three years with over 100 passages. Over the course of this experiment, the cells were sampled and analyzed for the presence of new mutations in their genomes.

It turns out that stem cells grown on feeder cells that are passaged by hand (manually) show the fewest growth-enhancing mutations after being cultured for three years.

Loring’s study also demonstrated the importance of monitoring cell lines over time. In particular, deletion of the TP53 gene, a tumor suppressor gene, in whose absence cancer develops, should be closely watched.

“If you want to preserve the integrity of the genome, then grow your cells under those conditions with feeder cells and manual passaging,” said Loring. “Also, analyze your cells. It’s really easy, she added.

When Thomson made the first human ESC lines, he used feeder cells derived from mouse skin cells.  However, the use of animal materials to make ESCs might pollute them with animal viruses and specific sugars from the surfaces of the animal cells might also contaminate the surfaces of the ESCs, making them unsuitable for regenerative medicine (see Stem Cells 2006; 24:221-229).  To address this problem, several laboratories have made “Xeno-free” ESC lines that were made without touching any animal products.  Some of these Xeno-free lines were made without feeder cells (see C. Ellerström, et al., Stem Cells. 2006 Oct;24(10):2170-6)., but others were made with human feeder cell lines (see K Rajala, et al., Hum Reprod. 2007 May;22(5):1231-8). Therefore, it appears, that the use of human feeder cell lines are preferable to feeder-free systems, given Loring’s findings.  However, it is also possible that such culture systems are also preferable for iPSCs, which do not have the problem of immunological rejection for patients, and do not require the killing of the youngest members of humanity.  Therefore, Loring’s work could very well benefit iPSC cultures as well.

Monkey’s Own Cells Are Used to Treat Parkinson’s Disease

Neurologist Ole Isacson and his Harvard Medical School team successfully implanted neurons made from a monkey’s own cells to treat Parkinson’s disease in those animals. The implanted neurons were watched for two years, and they proved to be both safe and effect in the treatment of Parkinson’s disease.

Induced pluripotent stem cells or iPSCs are made from mature, adult cells by means of a combination of genetic engineering and cell culture techniques. The cells resemble embryonic stem cells in many of their growth characteristics and gene expression patterns, but they are have several differences as well. One of the biggest differences between iPSCs and embryonic stem cells is that the reprogramming process that makes iPSCs places cells under stresses that increase the mutation rate and makes iPSCs, on average, more likely to cause tumors than embryonic stem cells. However, it is also clear that not all iPSC lines are the same and careful screen protocols that determine safe lines from less safe lines.

A distinct advantage of iPSCs over embryonic stem cells is that they have the same set of cell surface proteins as the patient from whom they were made, which makes them less likely to be rejected by the patient’s immune system. Even though some experiments had shown that cells derived from iPSCs can be rejected by the patient’s immune system, these experiments used poor-quality iPSC lines. High-quality iPSCs lines are much less likely to be rejected by the immune system. Therefore, using a patient’s own stem cells has distinct advantages as opposed to embryonic stem cells.

Isacson and his colleagues made patient-specific iPSCs from cynomolgus monkeys and used them to produce midbrain dopamine-making neurons – the kind that die off in patients with Parkinson’s disease – and used them to treat those same monkeys that suffered from Parkinson’s disease.

Such an experiment is potentially risky because even though differentiation of pluripotent stem cells into midbrain dopamine-making neurons is feasible, getting pure cultures of these cells that do not have any non-differentiated cells that can cause tumors is not all that easy to do. Fortunately, some advances in these techniques in the past few years have increased the ability of laboratories to not only produce large quantities of midbrain dopamine-making neurons, but screen them properly before transplantation.

In this experiment, Isacson and his team analyzed their implants for up to 2 years. The implanted animals were subjected to routine observations and tests, and in one animal, with the most successful protocol, they observed that lateral engraftment of CM-iPSCs on one side of the animal’s brain produced a gradual onset of functional motor improvement on the side opposite to the that of dopamine neuron transplantation, and increased motor activity. These implantation also did not require any immunosuppression and the implants caused to evidence of graft rejection. Postmortem analyses of these implanted animals revealed robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the tissue into which the cells were transplanted; the putamen, which is one of the “basal ganglia” that help control voluntary movements.

This remarkable proof-of-concept experiment supports further development of iPSC-derived cell transplantation for treatment of Parkinson’s Disease.

Preconditioning Your Way to Better Stem Cells

When stem cells are implanted into injured tissues, they often face a hostile environment that is inimical to their survival. A stroke, for example, can produce brain tissue without ample blood flow, low oxygen levels, and lots of cell debris and inflammation. The same can be said for the heart after a heart attack. If stem cells are going to help anyone we have to find a way for them to survive.

The first hints came in the form of genetically-engineered stem cells that expressed a host of genes that can help cells survive in low oxygen, high stress environments. However, the FDA is unlikely to approve genetically engineered cells for therapeutic purposes. Therefore, a more “user-friendly” way to precondition cells was sought, and found. Instead of loading cells up with extra genes, all you had to do was grow the cells under low oxygen, high stress conditions, and they would adapt and survive when implanted into damaged tissue. This, however, has a drawback: if you want to treat a patient, you do not always have the time it takes for extract and isolate their cells, grow them in culture over a week or two, and then implant them. Is there a better way?

The answer turns out to be yes. Treating cells with particular compounds or growth factors can induce resistance to low-oxygen, high-stress conditions, and two papers show us how it’s done.

The first paper is from the laboratory of Ling Wei at Emory University School of Medicine in Atlanta who has shown in the past that low-oxygen adaptation of mesenchymal stem cells from bone marrow made them better able to treat acute heart attacks in laboratory animals. In this paper, Wei and her colleagues exploited a biochemical pathway known to induce resistance of low-oxygen conditions known as the HIF-1 pathway. The HIF-1 pathway consists of two proteins that work as a pair; HIF1alpha and HIF1beta. HIF1beta is made all the time and HIF1alpha is oxygen sensitive. In the presence of oxygen, enzymes called prolyl hydroxylases modify HIF1alpha, marking it for destruction. In the absence of oxygen, the prolyl hydroxylases do not have enough oxygen to modify HIF1alpha and the HIF1alpha/beta complex activates the expression of a host of genes necessary for increased tolerance to low oxygen levels. Therefore, to make cells more tolerant to low oxygen levels, we need to turn on the HIF1 pathway and to do that we need to inhibit the prolyl hydroxylases.

This turns out to be pretty straight forward. A small molecule called dimethyloxalyglycine or DMOG can effectively inhibit prolyl hydroxylase and induce survival in low-oxygen, high-stress environments. Therefore Wei and her group used DMOG to treat cells and test them out.

In culture, the DMOG-treated cells made proteins known to be important for the establishment of new blood vessels and for survival. When they were compared to cultured stem cells that had not been treated with DMOG, the DMOG-treated cells expressed significantly more of VEGF, Glut-1 and HIF1alpha, all of which are important for surviving in low-oxygen environments. In a Matrigel assay, the DMOG-treated cells also made more blood vessels that were longer than their non-DMOG-treated counterparts.

When used in laboratory animals that had suffered heart attacks, the DMOG-treated cells distinguished themselves once again. They survived better than the control cells and hearts that had received the DMOG-treated cells had much smaller heart scars after heart attacks. Functional assays of heart function illustrated that the DMOG-treated cells helped their heart perform above and beyond what was shown observed in the animals implanted with stem cells that had not bee treated with DMOG.

Thus it is possible to precondition cells without long culture periods or genetic engineering. One compound can accomplish it and the cells only needed to be exposed to DMOG for 24 hours.

In a similar vein, Genshan Ma and others from Zhongda Hospital in Nanjing, China used a small peptide called bradykinin to precondition human umbilical cord endothelial progenitor cells (EPCs); the cells that form blood vessels. In this paper, Ma and colleagues used bradykinin-treated EPCs to treat heart attacks in mice. One nice aspect of this paper is the large number of controls they ran with their experimental runs.

The bradykinin-treated cells outperformed their untreated counterparts when it came to the size of the heart scar, the number of dead cells in the heart, and heart performance parameters. Cell culture experiments established that the bradykinin-treated cells expressed the Akt kinase at high levels, and expressed higher levels of VEGF, the blood vessel-inducing growth factor. Bradykinn-treated cells also were more resistant to being starved for oxygen, and survived better under unusual culture conditions. All of these benefits could be abrogated by inhibiting the activity of the Akt kinase by treating cells with LY294002, a compound that specifically inhibits the activator of Akt.

In this case, cells were treated with bradykinin for 10 minutes to 12 hours.

Two papers, two success stories. Stem cell preconditioning certainly works in laboratory animals. Since stem cell trials have been completed in human patients, it might be time to try preconditioned stem cells in human patients.