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Molecular motors direct the fate of stem cells — ScienceDaily

Scientists at the University of Groningen and the University Medical Center Groningen used molecular motors to manipulate the protein matrix on which bone marrow-derived mesenchymal stem cells are grown. Rotating motors altered the protein structure, which resulted in a bias of the stem cells to differentiate into bone cells (osteoblasts). Without rotation, the stem cells tended to remain multipotent. These results, which could be used in tissue engineering, were published in Science Advances on 29 January.

‘Cells are sensitive to the structure of the surface that they attach to,’ explains Patrick van Rijn, associate professor in Materiobiology and Nanobiomaterials. ‘And movement is an important driver in biology, especially continuous movement.’ That is why Van Rijn and Feringa and their colleagues decided to use molecular motors to manipulate the protein matrix on which stem cells are grown. The light-driven motor molecules were designed by the 2016 Nobel Laureate in Chemistry Ben Feringa.

Structural changes

The scientists linked molecular motors to a glass surface. Subsequently, the surface was coated with protein and either exposed to UV irradiation to power the motors or not exposed to it at all. After about an hour, the motor movement was stopped and cells were seeded onto the protein layer and left to attach. Finally, differentiation factors were added. These experiments showed that cells grown on protein that was submitted to the rotary motion of the molecular motors tended to specialize into bone cells more often, while cells seeded on protein that was not disturbed were more inclined to maintain their stem-cell properties.

Observations of the protein layer using atomic force microscopy and simulations of the interaction between the motor molecules and proteins, performed by Prof. Marrink’s research group, showed that the movement induced subtle structural changes in the protein matrix. ‘The movement of motor molecules interferes with the alpha-helices in the proteins, which causes structural changes,’ explains Van Rijn. He compares it to the difference in texture between an unwhipped egg white and a whipped one.

Cell fate

The change in the surface structure of the adhered protein affects how the cells attach, for example how much they stretch out. This sets off a signaling cascade that eventually leads to altered behavior, such as the differentiation into bone cells. Thus, molecular movement leads to nanoscopic changes in surface structure, which in turn leads to differences in cell attachment, cell morphology and eventually, cell differentiation. ‘It’s like a domino effect, where smaller stones consecutively topple slightly larger ones so that a large effect can be achieved with a small trigger.’

‘Changing the properties of a surface to affect cell fate has been used before,’ says Van Rijn. However, this was done primarily with switches, so there was just a change from one state to another. ‘In our study, we had continuous movement, which is much more in line with the continuous motion found in biological transport and communication systems. The fact that the motors are driven by light is important,’ Van Rijn adds. ‘Light can be carefully controlled in space and time. This would allow us to create complex geometries in the growth matrix, which then result in different properties for the cells.’ Therefore, light-controlled molecular motors could be a useful tool in tissue engineering.

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Bone or cartilage? Presence of fatty acids determines skeletal stem cell development — ScienceDaily

In the event of a bone fracture, fatty acids in our blood signal to stem cells that they have to develop into bone-forming cells. If there are no blood vessels nearby, the stem cells end up forming cartilage. The finding that specific nutrients directly influence the development of stem cells opens new avenues for stem cell research. Biomedical scientists from KU Leuven and Harvard University published these results in Nature.

Bone fractures heal through the action of skeletal progenitor cells: stem cells that have evolved further but can still develop into different types of cells. Bone healing occurs in one of two ways: the progenitor cells evolve into bone-forming cells when the fracture is small, and into cartilage cells when the fracture is bigger. This cartilage is later replaced by bone. Until now, scientists did not know how progenitor cells decide whether to become bone or cartilage cells.

“Our hypothesis was that the presence of blood vessels plays a role,” explains first author Nick van Gastel. “Despite what many people think, our bones are full of blood vessels, while cartilage does not have any.” This new study on mice confirmed the team’s assumption: when blood vessels surrounding a fracture were blocked, cartilage was formed. When they were not, new bone was created immediately.

In a second phase of the study, the researchers tried to find out which signal the blood vessels actually send to the progenitor cells to make them evolve into either a bone or a cartilage cell. “Our previous research had already shown that nutrients play a role in the biology of progenitor cells,” explains Professor Geert Carmeliet from the Clinical and Experimental Endocrinology Unit at KU Leuven, who led the study. For the current study, the team tested how the presence of different nutrients influences progenitor cell fate. Their results show that the fatty acids present in blood cause progenitor cells to grow into bone-forming cells.

If there are no fatty acids nearby, progenitor cells activate the SOX9 gene, which plays an important role in skeletal development. This is the signal for the cell to become a cartilage cell. Cartilage cells do not need fatty acids to survive and form cartilage.

“This study is useful for researchers in regenerative medicine, since we still know little about cartilage formation,” says Professor Carmeliet. “Research into cartilage disorders such as osteoarthritis may also benefit from these findings. There are indications that cartilage cells receive more fatty acid signals and don’t produce enough of the SOX9 gene in patients with such disorders, which can have adverse effects on the joints. Finally, our study shows for the first time that specific nutrients can inform stem cells which type of cell they should become. That is an important step forward in stem cell research.” Eventually, the researchers hope to map out the effects of different nutrients on different types of progenitor cells.

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Materials provided by KU Leuven. Note: Content may be edited for style and length.

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Scientists boost gene-editing tools to new heights in human stem cells — ScienceDaily

During the past decade, the gene editing tool CRISPR has transformed biology and opened up hopeful avenues to correct deadly inherited diseases. Last fall, scientists began the first human clinical trials using CRISPR to combat diseases like cancer. They remove some of a person’s cells, CRISPR edit the DNA, and then inject the cells back in, where hopefully, they will cure the disease.

But along with this promise of regenerative, personalized medicine, CRISPR can also have significant safety limitations. CRISPR may not edit in the right place (so-called off-target gene effects) or not being terribly efficient (successful editing may only be achieved in about 10% of the time for every available cell target).

These limitations have frustrated scientists such as Arizona State University’s David Brafman, a cell bioengineer. Brafman initial hopes are to use gene editing to get at the heart of uncovering the causes of studies in his lab of neurodegenerative diseases like Alzheimer’s.

“We study neurodegenerative diseases like Alzheimer’s and use stem cells to study specific mutations or risk factors associated with Alzheimer’s disease,” said Brafman, a biomedical engineering faculty member in ASU’s Ira A. Fulton Schools of Engineering. “We are not necessarily a gene-editing tool development lab, but we were running into difficulty generating stem cell lines by using a traditional CRISPR-based editing approach. For reasons that are still unknown, stem cells are really resistant to that sort of genetic modification.”

Green light means go

Now, Brafman, using a new update to the CRISPR base editing technology originally developed in the lab of David Liu at Harvard, has vastly outperformed previous efforts by making highly accurate, single DNA base editing with an efficiency of up to 90% of human stem cells. The results were published in the journal Stem Cell Reports.

“Previously, with CRISPR, it’s just been a random guess,” said Brafman. “And so, if you are picking at random stem cells and the efficiency is low, you’ll likely get only 10% or 5% because you have no idea if the edits have been made — the cell isn’t telling you.”

Brafman’s lab has developed a new TREE method (an acronym short for transient reporter for editing enrichment, or TREE), which allows for bulk enrichment of DNA base-edited cell populations — -and for the first time, high efficiency in human stem cell lines.

“”Most of the studies are done in immortalized cell lines or cancer cell lines, which are relatively easy to edit,” said Brafman. “This is the first example of using base editors in pluripotent stem cells, which is a very valuable cell population to genetically modify. We envision this method will have important implications for the use of human stem cell lines in developmental biology, disease modeling, drug screening and tissue engineering applications,”

Last year, they had shown that their TREE approach can work in human cell lines, but wanted to further push the technology further to find a way to rapidly and efficiently edit human stem cell lines.

Unlike CRISPR, which cuts across both DNA stands, their TREE method only makes a single strand nick in DNA. For example, when a single DNA base is successfully edited from a C to a T, a protein gives off a signal, turning from blue to green.

“Now, if a cell is telling you, ‘if I’m glowing green I have a 90% chance of being edited you are going to have better luck identifying edited populations,” said Brafman. “Then, you can exclude all of the cells that are not edited. We isolate single cells that are glowing green, then grow those up into clonal populations that you are able to expand indefinitely.”

Targeting Alzheimer’s

Pluripotent stem cells are valued for regenerative medicine because they have the ability to become or differentiate into any cell type in the human body.

Brafman explains that there are two general sources, “embryonic stem cells, which are derived from the inner cell mass of a preimplantation blastocyst, and then there are induced pluripotent stem cells, which are derived from taking somatic cells like skin or blood from patients.”

Brafman’s lab uses the induced pluripotent stem cells for their research.

“For this study, we used pluripotent stem cells from both healthy patients and then patients with Alzheimer’s disease. Some of the genes that we were interested in modulating are related to Alzheimer’s disease. The majority of the patients suffering from Alzheimer’s disease suffer from late onset, or sporadic Alzheimer’s disease.”

To provide their proof-of-concept, they targeted the APOE gene, which can come in three flavors. One of the three gene variants, called APOE4, has been associated with a higher risk for late onset Alzheimer’s disease. For the study, they introduced single DNA based edits into the APOE gene.

“That’s why we are interested in having these cells,” said Brafman. “They are representative of the neurons and the various cell types in the central nervous system with patients with these various risk factors. Then, we can understand why an APOE variant can increase or decrease risk, and then we can start targeting those pathways that are affected.”

Not only could TREE make single DNA edits to the APOE4 gene, but unlike CRISPR, make highly accurate corrections to both copies of the APOE4 gene that humans possess.

“The traditional CRISPR approach is that you have to edit once to get a heterozygous edit , then isolate that clone, edit again to get another heterozygous edit,” said Brafman. “So, it’s very inefficient in that way. We are generating homozygous edits at an efficiency approaching 90%. I haven’t seen any other technologies that can do that in pluripotent stem cells.”

In addition, TREE could also be used to engineer critical gene knockout mutations into stem cell lines. “The most fundamental experiment you can do if a gene has important implications in disease, development or physiology is knock it out,” said Brafman. That opens up a whole bunch of questions that we can address. Using APOE as a case study, now we can knock out APOE in these cells if you don’t have APOE at all. Is it beneficial? Detrimental? Or no difference?”

Complex cases

While diseases like sickle-cell anemia or cystric fibrosis are caused by single mutations in DNA, for most diseases and leading causes of death, like heart disease or high blood pressure, are complex, and involve multiple genes. Brafman wanted to also address the complex, root causes of Alzheimer’s.

“Especially as it related to Alzheimer’s disease, there can be multiple risk factors that act in concert, so we wanted a way to introduce multiple edits simultaneously in pluripotent stem cells. Because otherwise, you would have to take this sequential iterative approach, where you introduce one edit, isolate a clonal population introduce another edit, and so on.

They successfully demonstrated that TREE could be used to make new stem cell lines that had been simultaneously edited at multiple gene locations. Their results showed that more than 80% of stem clones had been targeted at all three different gene sites, and with all clones editing both gene copies.

“We found that if you multiplex you still get the same efficiency of editing as you would if you just edited a single allele,” said Brafman. “Now, we can use these cells as in vitro models to study the disease and screen drugs.”

Brafman is hopeful that their new tools will generate excitement in the gene editing community, and spur others on to make new discoveries.

“We want to keep expanding on that toolbox,” said Brafman. “We’ve already gotten a high level of interest from other scientists who will be using this to generate their own cell lines. That’s a good thing.”

Funding for this work was provided by the National Institutes of Health (R01GM121698 to David Brafman, R21AG056706 to D.A.B, R01GM106081 to X.W.) and the Arizona Biomedical Research Commission (ADHS16-162401 to D.A.B).

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New hydrogels wither while stem cells flourish for tissue repair — ScienceDaily

Baby diapers, contact lenses and gelatin dessert. While seemingly unrelated, these items have one thing in common — they’re made of highly absorbent substances called hydrogels that have versatile applications. Recently, a type of biodegradable hydrogel, dubbed microporous annealed particle (MAP) hydrogel, has gained much attention for its potential to deliver stem cells for body tissue repair. But it is currently unclear how these jelly-like materials affect the growth of their precious cellular cargo, thereby limiting its use in regenerative medicine.

In a new study published in the November issue of Acta Biomaterialia, researchers at Texas A&M University have shown that MAP hydrogels, programmed to biodegrade at an optimum pace, create a fertile environment for bone stem cells to thrive and proliferate vigorously. They found the space created by the withering of MAP hydrogels creates room for the stem cells to grow, spread and form intricate cellular networks.

“Our research now shows that stem cells flourish on degrading MAP hydrogels; they also remodel their local environment to better suit their needs,” said Dr. Daniel Alge, assistant professor in the Department of Biomedical Engineering. “These results have important implications for developing MAP hydrogel-based delivery systems, particularly for regenerative medicine where we want to deliver cells that will replace damaged tissues with new and healthy ones.”

MAP hydrogels are a newer breed of injectable hydrogels. These soft materials are interconnected chains of extremely small beads made of polyethylene glycol, a synthetic polymer. Although the microbeads cannot themselves cling to cells, they can be engineered to present cell-binding proteins that can then attach to receptor molecules on the stem cells’ surface.

Once fastened onto the microbeads, the stem cells use the space between the spheres to grow and transform into specialized cells, like bone or skin cells. And so, when there is an injury, MAP hydrogels can be used to deliver these new cells to help tissues regenerate.

However, the health and behavior of stem cells within the MAP hydrogel environment has never been fully studied.

“MAP hydrogels have superior mechanical and biocompatible properties, so in principle, they are a great platform to grow and maintain stem cells,” said Alge. “But people in the field really don’t have a good understanding of how stem cells behave in these materials.”

To address this question, the researchers studied the growth, spread and function of bone stem cells in MAP hydrogels. Alge and his team used three samples of MAP hydrogels that differed only in the speed at which they degraded, that is, either slow, fast or not at all.

First, for the stem cells to attach onto the MAP hydrogels, the researchers decorated the MAP hydrogels with a type of cell-binding protein. They then tracked the stem cells as they grew using a high-resolution, fluorescent microscope. The researchers also repeated the same experiment using another cell-binding protein to investigate if cell-binding proteins also affected stem cell development within the hydrogels.

To their surprise, Alge’s team found that for both types of cell-binding proteins, the MAP hydrogels that degraded the fastest had the largest population of stem cells. Furthermore, the cells were changing the shape of the MAP hydrogel as they spread and claimed more territory.

“In the intact MAP hydrogel, we could still see the spherical microbeads and the material was quite undamaged,” said Alge. “By contrast, the cells were making ridges and grooves in the degrading MAP hydrogels, dynamically remodeling their environment.”

The researchers also found that as the stem cells grew, the quantity of bone proteins produced by the growing stem cells depended on which cell-binding protein was initially used in the MAP hydrogel.

Alge noted that the insight gained through their study will greatly inform further research and development in MAP hydrogels for stem-cell therapies.

“Although MAP hydrogel degradability profoundly affects the growth of the stem cells, we found that the interplay between the cell-binding proteins and the degradation is also important,” he said. “As we, as a field, make strides toward developing new MAP hydrogels for tissue engineering, we must look at the effects of both degradability and cell-binding proteins to best utilize these materials for regenerative medicine.”

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Materials provided by Texas A&M University. Original written by Vandana Suresh. Note: Content may be edited for style and length.

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Scientists solve long-debated puzzle of how the intestine heals itself — ScienceDaily

Deep within the lining of the human intestine lies the source of the organ’s ability to renew itself and recover from damage: intestinal stem cells (ISCs), lodged in pockets of tissue called crypts, generate the cells that continuously repopulate the intestinal lining. Even the stem cells themselves have a safety net: when they’re damaged, healthy replacements appear in less than a week.

For years, scientists have debated how the ISCs’ re-emergence occurs. Some have held that the intestine keeps a pool of ISCs on reserve — a kind of backup-backup supply — to replenish the cache of front-line ISCs that have been lost. Others have maintained that something more involuted is as work: The ISCs, like queen bees, give rise to more specialized, or “differentiated,” progeny — in this case, daughter cells that form the inner lining of the intestine. When the ISCs are damaged, this school of thought held, the daughter cells reverse course and “de-differentiate” — reverting into the ISCs from which they arose.

A new study by Dana-Farber Cancer Institute scientists comes down solidly on the latter option. Published online today by the journal Cell Stem Cell, the researchers found that ISCs and their daughter cells have a strikingly reciprocal relationship: under normal conditions, ISCs differentiate into daughter cells, and, if the ISCs are lost, the daughter cells simply reverse course and become ISCs.

“Our findings suggest that the restoration of intestinal stem cells occurs entirely by the process of de-differentiation,” says the study’s senior author, Ramesh Shivdasani, MD, PhD, of Dana-Farber, Brigham and Women’s Hospital (BWH), and the Harvard Stem Cell Institute. “We showed there’s no need for a reserve set of ISCs.”

Bolstering their findings, the researchers were also able to capture the de-differentiation process in real time. When cells begin to de-differentiate, they switch on a gene that that allows them to be isolated and collected with laboratory techniques, Shivdasani explains. Through this process, researchers were able to capture the cells along a continuum of de-differentiation. Shivdasani likens it to a baseball play in which a runner is tagged out between first and second base.

Heavy turnover

The intestine is one of just three tissues in the body, along with the skin and blood, in which cells are constantly turning over — dying and being replaced by freshly made cells. They share this quality because they are the tissues most intimately in contact with material from the environment, and therefore with potentially harmful substances. The constant turnover, it’s thought, is a way to prevent toxic substances from having lasting effects on cells and their offspring.

The crypts that hold ISCs are, in a sense, misnamed. Far from being enclosures where dead cells are entombed, they are the sites where ISCs daily generate the billions of daughter cells that take the place of defunct intestinal cells.

One of the chief characteristics of ISCs is that they are extremely radiosensitive, or vulnerable to radiation. People exposed to high levels of radioactivity, in the form of nuclear fallout, for example, can suffer severe intestinal damage because the loss of ISCs halts production of cells to regenerate the damaged tissue. But if ISCs succumb easily to radiation, they also make a rapid return. Patients with radiation-induced intestinal damage who can be kept alive for a week often recover as their ISC levels bounce back.

To determine whether this rebound is due to a reserve stockpile of ISCs or to de-differentiation of daughter cells, Shivdasani and his collaborators performed a kind of time-lapse experiment. They treated a collection of ISC cells with the drug tamoxifen, which caused the cells and their offspring to become fluorescent. They waited 48 hours for the label to take hold, then killed the ISC cells. If the daughter cells were indeed de-differentiating, any ISC cells produced after that point would be fluorescent. That’s exactly what researchers found.

While scientists have been able to convert many kinds of differentiated cells into stem cells using laboratory techniques, Shivdasani and his colleagues’ discovery demonstrates that de-differentiation ismore than a curious act of nature; it is the principal means to restore damaged stem cell in the intestine. It’s not known whether cells in other organs and tissues have this capability, but it remains an open avenue of investigation.

“It also isn’t clear how the crypt knows that stem cells have died and need to be replaced,” Shivdasani remarks, “or how the daughter cells receive the signal to de-differentiate. This is a subject we’re currently exploring.”

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Scientists have discovered a combination of two commonly available drugs that could help the body heal spinal fractures — ScienceDaily

The early-stage research in rats, by a group of scientists led by Imperial College London, revealed two existing medications can boost the body’s own repair machinery, by triggering the release of stem cells from the bone marrow.

The scientists published their research in the journal npj Regenerative Medicine.

The team say the two drugs (currently used for bone marrow transplants and bladder control) could be used for different types of bone fractures, including to the spine, hip and leg, to aid healing after surgery or fractures.

When a person has a disease or an injury, the bone marrow (the spongy tissue within bone) mobilises different types of stem cells to help repair and regenerate tissue.

The new research, involving scientists from Beaumont Health in the U.S, suggests it may be possible to boost the body’s ability to repair itself and speed repair, by using new drug combinations to put the bone marrow into a state of ‘red alert’ and send specific kinds of stem cells into action.

In the new study, funded by Wellcome, the researchers used drugs to trigger the bone marrow of healthy rats to release mesenchymal stem cells, a type of adult stem cell that can turn into bone, and help repair bone fractures.

Professor Sara Rankin, corresponding author of the study from the National Heart and Lung Institute at Imperial College London, said: “The body repairs itself all the time. We know that when bones break they will heal, and this requires the activation of stem cells in the bone. However, when the damage is severe, there are limits to what the body can do of its own accord. We hope that by using these existing medications to mobilise stem cells, as we were able to do in rats in our new study, we could potentially call up extra numbers of these stem cells, in order to boost our bodies own ability to mend itself and accelerate the repair process. Further down the line, our work could lead to new treatments to repair all types of bone fracture.”

The two treatments used in the research were a CXCR4 antagonist, used for bone marrow transplants, and a beta-3 adrenergic agonist, that is used for bladder control.

The rats were given a single treatment with the two drugs, which triggered enhanced binding of calcium to the site of bone injury, speeding bone formation and healing.

The researchers stress they did not analyse restoration of movement in the bone, or repair to additional tissue such as nerves.

One of the drugs used in the study was found to trigger fat cells in the bone marrow to release endocannabinoids, which suggests they may have a role in mobilising the stem cells and thereby promoting healing. However, the researchers add that phytocannabinoids (such as cannabis) would not have the same effect, as they act on the brain rather than the bone marrow.

The researchers say the drug combinations now need to be tested in humans.

Dr Tariq Fellous, first author of the research from Imperial’s National Heart and Lung Institute (NHLI) said: “We first need to see if these medications release the stem cells in healthy volunteers, before we can then test them in patients with fractures. We have the drugs and know they are safe to use in humans — we just need the funding for the human trials.”

Dr Andia Redpath, co-first author from the NHLI, added that repurposing existing medications that help the body heal itself — so called Regenerative Pharmacology — could have great potential as an efficient and cost-effective approach for a range of diseases. “Rather than devising new stem cell treatments from scratch that involve lengthy and expensive trials, our approach harnesses the power of the body’s own stem cells, using existing drugs. We already know the treatments in our study are safe, it’s now just a matter of exploring further if they help our bodies heal.”

This work was funded by Wellcome. Further support was provided by the National Heart and Lung Institute Foundation and the Lumbar Spine Research Society.

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Materials provided by Imperial College London. Original written by Kate Wighton. Note: Content may be edited for style and length.

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Curing genetic disease in human cells — ScienceDaily

Scientists show for the first time that a newer type of CRISPR, called base-editing, can safely cure cystic fibrosis in stem cells derived from patients.

While the genome editing tool CRISPR/Cas9, developed in 2012, cuts a mutation out of a gene and replaces it with a gene-piece, a newer type of CRISPR, called base-editing, can repair a mutation without cutting the DNA. Therefore, genome editing using base-editor is considered safer. Scientists from the research groups of Hans Clevers (Hubrecht Institute) and Jeffrey Beekman (UMC Utrecht) show for the first time that this base-editing can safely cure cystic fibrosis in stem cells derived from patients. The results of this study were published in Cell Stem Cell on the 20th of February.

In 2018 a new CRISPR-enzyme was developed that makes the CRISPR technique more precise and less error-prone, according to biologists Maarten Geurts (Hubrecht Institute) and Eyleen de Poel (UMC Utrecht). Maarten: “In traditional CRISPR/Cas9 genome editing a specific piece of the DNA is cut out resulting in DNA damage. This is done with the aim that the cell repairs this cut using a lab-made piece of ‘healthy’ DNA. However, in the new CRISPR-technique, called base editing, the Cas-part is altered in such a way that it no longer creates a cut, but still detects the mutation. So, instead of creating a cut and replacing the faulty DNA, the mutation is directly repaired on site, making this a more effective genome editing tool.” The current research shows that this new version of CRISPR/Cas9 can be safely and effectively applied in human stem cells.

Miniguts

The Hubrecht Organoid Technology foundation and the UMC Utrecht have generated a biobank consisting of intestinal organoids. These are tiny versions of the gut, that are established in the lab using the stem cells of cystic fibrosis (CF) patients. The miniguts are used for disease modeling and the development of new therapies. The biobank was set up together with many CF centers across Europe and the Dutch CF Foundation (NCFS). In this study, the miniguts were used to test whether the new base-editing technique can be applied in human stem cells. Maarten explains how this exactly works: “CF is caused by a mistake, a mutation, in the CFTR-gene leading to malfunctioning of the gene. As a consequence, the mucus in many organs, including the lungs, is less hydrated, resulting in mucus build-up and organ failure. With the new base-editing technique the mutation in the CFTR-gene can be detected and repaired without creating further damage in the genome.”

Even though this research shows that this novel CRISPR tool is effective in the lab, this does not mean that patients can already benefit from it. Eyleen: “This research represents a big step towards genetic repair of diseases in patients. However, a big question that remains is how to deliver the CRISPR-enzyme to the appropriate organs in the patient. Cystic fibrosis might also not be the most suitable disease to treat with CRISPR, as many organs are affected by the disease. Currently, the first medical applications with CRISPR gene editing are showing impressive clinical effects in diseases that affect a single organ or tissue such as sickle cell anemia. Further research is needed before the base-editor can be used for clinical application. However, in part due to this study, the first clinical applications may already happen in the coming five years.

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Abnormal bone formation after trauma explained and reversed in mice — ScienceDaily

Hip replacements, severe burns, spinal cord injuries, blast injuries, traumatic brain injuries — these seemingly disparate traumas can each lead to a painful complication during the healing process called heterotopic ossification. Heterotopic ossification is abnormal bone formation within muscle and soft tissues, an unfortunately common phenomenon that typically occurs weeks after an injury or surgery. Patients with heterotopic ossification experience decreased range of motion, swelling and pain.

Currently, “there’s no way to prevent it and once it’s formed, there’s no way to reverse it,” says Benjamin Levi, M.D., Director of the Burn/Wound/Regeneration Medicine Laboratory and Center for Basic and Translational Research in Michigan Medicine’s Department of Surgery. And while experts suspected that heterotopic ossification was somehow linked to inflammation, new U-M research explains how this happens on a cellular scale — and suggests a way it can be stopped.

To help explain how the healing process goes awry in heterotopic ossification, the research team, led by Levi, Michael Sorkin, M.D. and Amanda Huber, Ph.D., of the Department of Surgery’s section of plastic surgery, took a closer look at the inflammation process in mice. Using tissue from injury sites in mouse models of heterotopic ossification, they used single cell RNA sequencing to characterize the types of cells present. They confirmed that macrophages were among the first responders and might be behind aberrant healing.

Macrophages are white blood cells whose normal job is to find and destroy pathogens. Upon closer examination, the Michigan team found that macrophages are more complex than previously thought — and don’t always do what they are supposed to do.

“Macrophages are a heterogenous population, some that are helpful with healing and some that are not,” explains Levi. “People think of macrophages as binary (M1 vs. M2). Yet we’ve shown that there are many different macrophage phenotypes or states that are present during abnormal wound healing.”

Specifically, during heterotopic ossification formation, the increased presence of macrophages that express TGF-beta leads to an errant signal being sent to bone forming stem cells.

For now, the only way to treat heterotopic ossification is to wait for it to stop growing and cut it out which never completely restores joint function. This new research suggests that there may be a way to treat it at the cellular level. Working with the lab led by Stephen Kunkel, Ph.D. of the Department of Pathology, the team demonstrated that an activating peptide to CD47, p7N3 could alter TGF-beta expressing macrophages, reducing their ability to send signals to bone-forming stem cells that lead to heterotopic ossification.

“During abnormal wound healing, we think there is some signal that continues to be present at an injury site even after the injury should have resolved,” says Levi. Beyond heterotopic ossification, Levi says the study’s findings can likely be translated to other types of abnormal wound healing like muscle fibrosis.

The team hopes to eventually develop translational therapies that target this pathway and further characterize not just the inflammatory cells but the stem cells responsible for the abnormal bone formation.

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Scientists discover how rogue communications between cells lead to leukemia — ScienceDaily

New research has deciphered how rogue communications in blood stem cells can cause Leukemia.

The discovery could pave the way for new, targeted medical treatments that block this process.

Blood cancers like leukemia occur when mutations in stem cells cause them to produce too many blood cells.

An international team of scientists, including researchers at the University of York, have discovered how these mutations allow cells to deviate from their normal method of communicating with each other, prompting the development of blood cells to spiral out of control.

The scientists used super-resolution fluorescent microscopy to study the way blood stem cells talk to each other in real time.

They observed how cells receive instructions from ‘signalling proteins’, which bind to a receptor on the surface of another cell before transmitting a signal telling the cell how to behave.

Blood stem cells communicate via cytokines, which are one of the largest and most diverse families of signalling proteins and are critical for the development of blood cells and the immune system.

Understanding this process led researchers to the discovery that mutations associated with certain types of blood cancers can cause blood stem cells to ‘go rogue’ and communicate without cytokines.

The stem cells begin to transmit uncontrolled signals causing the normal system of blood cell development to become overrun, producing an imbalance of healthy white and red blood cells and platelets.

Professor Ian Hitchcock from the York Biomedical Research Institute and the Department of Biology at the University of York, said: “Our bodies produce billions of blood cells every day via a process of cells signalling between each other. Cytokines act like a factory supervisor, tightly regulating this process and controlling the development and proliferation of the different blood cell types.

“Our observations led us to a previously unknown mechanism for how individual mutations trigger blood stem cells to start signalling independently of cytokines, causing the normal system to become out of control and leading to diseases like leukemia.

“Understanding this mechanism may enable the identification of targets for the development of new drugs.”

This research team used a combination of molecular modelling, structural biology, biophysics, super-resolution microscopy and cell biology to demonstrate for the first time that these specific receptors on the surface of blood stem cells are linked by cytokines to form pairs.

Co-author of the study, Professor Jacob Piehler from Osnabrück University, said: “By directly visualising individual receptors at physiological conditions under the microscope, we were able to resolve a controversy that has preoccupied the field for more than 20 years.”

Professor Ilpo Vattulainen from the University of Helsinki, added: “Our biomolecular simulations unveiled surprising features concerning the orientation of active receptor pairs at the plasma membrane, explaining how mutations render activation possible without a ligand (such as a cytokine). These predictions were subsequently confirmed experimentally”

First author Dr. Stephan Wilmes, who started the project as a Postdoc at Osnabrück University before moving to the University of Dundee: “It was truly inspiring to tackle this highly relevant biomedical question by applying cutting-edge biophysical techniques. Here in Dundee, I had the chance to perform complementary activity assays, which corroborated our mechanistic model.”

The study is published in the journal Science and was carried out in collaboration between the universities of York, Dundee, Osnabruck (Germany), Helsinski (Finland), Stanford and New York (USA).

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Materials provided by University of York. Note: Content may be edited for style and length.

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Computer simulations visualize how DNA converts cells into stem cells — ScienceDaily

Researchers of the Hubrecht Institute (KNAW — The Netherlands) and the Max Planck Institute in Münster (Germany) have revealed how an essential protein helps to activate genomic DNA during the conversion of regular adult human cells into stem cells. Their findings are published in the Biophysical Journal.

A cell’s identity is driven by which DNA is “read” or “not read” at any point in time. Signalling in the cell to start or stop reading DNA happens through proteins called transcription factors. Identity changes happen naturally during development as cells transition from an undesignated cell to a specific cell type. As it turns out, these transitions can also be reversed. In 2012, Japanese researchers were awarded the Nobel prize for being the first to push a regular skin cell backwards to a stem cell.

A fuller understanding of molecular processes towards stem cell therapies

Until now, it is unknown how the conversion of a skin cell into a stem cell happens exactly, on a molecular scale. “Fully understanding the processes with atomic details is essential if we want to produce such cells for individual patients in the future in a reliable and efficient manner,” says research leader Vlad Cojocaru of the Hubrecht Institute. “It is believed that such engineered cell types may in the future be part of the solution to diseases like Alzheimer’s and Parkinson’s, but the production process would have to become more efficient and predictable.”

Pioneer transcription factor

One of the main proteins involved in the stem cell generation is a transcription factor called Oct4. It induces gene expression, or activity, of the proteins that ‘reset’ the adult cell into a stem cell. Those genes induced are inactive in the adult cells and reside in tightly packed, closed states of chromatin, the structure that stores the DNA in the cell nucleus. Oct4 contributes to the opening of chromatin to allow for the expression of the genes. For this, Oct4 is known as a pioneer transcription factor.

The data from Cojocaru and his PhD candidate — and first author of the publication — Jan Huertas show how Oct4 binds to DNA on the so-called nucleosomes, the repetitive nuclear structures in chromatin. Cojocaru: “We modelled Oct4 in different configurations. The molecule consists of two domains, only one of which is able to bind to a specific DNA sequence on the nucleosome in this phase of the process. With our simulations, we discovered which of those configurations are stable and how the dynamics of nucleosomes influence Oct4 binding. The models were validated by experiments performed by our colleagues Caitlin MacCarthy and Hans Schöler in Münster.”

One step closer to engineered factors

This is the first time computer simulations show how a pioneer transcription factor binds to nucleosomes to open chromatin and regulate gene expression. “Our computational approach for obtaining the Oct4 models can also be used to screen other transcription factors and to find out how they bind to nucleosomes,” Cojocaru says.

Moreover, Cojocaru wants to refine the current Oct4 models to propose a final structure for the Oct4-nucleosome complex. “For already almost 15 years now, we know that Oct4 together with three other pioneer factors transforms adult cells into stem cells. However, we still do not know how they go about. Experimental structure determination for such a system is very costly and time consuming. We aim to obtain one final model for the binding of Oct4 to the nucleosome by combining computer simulations with different lab experiments. Hopefully, our final model will give us the opportunity to engineer pioneer transcription factors for efficient and reliable production of stem cells and other cells needed in regenerative medicine.”

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Materials provided by Hubrecht Institute. Note: Content may be edited for style and length.

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