Toward a stem cell model of human nervous system development Human cells could one day show us more about why neural tube birth defects occur and how to prevent them.

Human embryonic stem cells can be guided to become the precursor tissue of the central nervous system, research led by the University of Michigan has demonstrated. The new study also reveals the important role of mechanical signals in the development of the human nervous system.

While studying embryonic development using animal embryos can provide useful insights about what happens during human development, human embryos grow differently even at this early stage.

“There is a critical need to establish embryonic developmental models using human cells. Not only could they advance our fundamental understanding of human development, they are also essential for regenerative medicine and for testing the safety of drugs and chemicals that pregnant women may need or encounter,” said Jianping Fu, an associate professor of mechanical engineering, who has been supervising this research.

“For the first time, we are able to use human embryonic stem cells to develop a synthetic model of neuroectoderm patterning, the embryonic event that begins the formation of the brain and spinal cord in the human embryo.”

There is a critical need to establish embryonic developmental models using human cells.Jianping Fu, associate professor of mechanical engineering.

In humans, the cells that will later differentiate into the central nervous system (including the brain and spinal cord) are known as the neural plate, while those that stand between the neural plate and future skin cells are called the neural plate border. The neural plate folds in on itself about 28 days after conception, becoming the neural tube, and the border on either side of it fuses together along its length. When the neural tube fails to close properly, it typically results in paralysis or death.

“The exact causes of neural tube defects are not clear, and there is currently no cure for them. Environmental factors, such as certain drugs pregnant women take, may play roles in causing neural tube defects,” said Fu.

In the new study, Fu’s research team arranged human embryonic stem cells into circular cell colonies with defined shapes and sizes. The cells were then exposed to chemicals known to coax them to differentiate into neural cells. During the differentiation process, cells in circular colonies organized themselves with neural plate cells in the middle and neural plate border cells in a ring around the outside.

“Since all of the cells in a micropatterned colony are in the same chemical environment, it’s amazing to see the cells autonomously differentiate into different cells and organize themselves into a multicellular pattern that mimics human development,” said Xufeng Xue, a PhD student in mechanical engineering working in Fu’s research group.  Xue is a co-first author of the paper.

Disc-shaped colonies shown with phase contrast (top) and fluorescence (bottom) microscopy. Between day 3 and day 9, cells in the center of the colony grow faster and become much more densely packed. Confined space drives the cells in the center of the colony to become neural plate cells, whereas those cells at the colony border (experiencing less confinement) differentiate into neural plate border cells. Image: Xufeng Xue, Integrated Biosystems and Biomechanics Laboratory, University of Michigan.

 

Fu’s team observed that cells in the circular colony became more densely packed in the middle of the colony, where they became neural plate cells, versus the colony border, where they became neural plate border cells. Suspecting mechanical signals might affect their differentiation, they placed single human embryonic stem cells onto adhesive spots of different sizes.

In the same chemical environment, single human embryonic stem cells grown on larger spots began signaling events within the cells that drove them toward becoming neural plate border cells. These signaling events were inhibited in stem cells confined on smaller spots. The team also developed a system to stretch cells in the middle of a colony. Responding to this mechanical signal, the cells in the middle of a colony differentiated into neural plate border cells, rather than the neural plate cells at the center of an ordinary colony.

“While many current models attribute patterning of embryonic tissues to chemical gradients or cell migration, our results show that these factors may not be the only drivers,” said Yubing Sun (ME PhD ’15), a former doctoral student in Fu’s lab and now an assistant professor of mechanical and industrial engineering at the University of Massachusetts, Amherst. Sun is a co-first author of the paper.

The study, titled, “Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells,” is published in Nature Materials.

This work was supported by the National Science Foundation (grant numbers CMMI 1129611, CBET 1149401and CMMI 1662835), the American Heart Association (grant number 12SDG12180025) and the U-M Department of Mechanical Engineering.

Fu is also an associate professor of biomedical engineering, cell and developmental biology, and is an associate director of the Michigan Center for Integrative Research in Critical Care.

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Findings in mice show pill for breast cancer diagnosis may outperform mammograms A new kind of imaging could distinguish aggressive tumors from benign, preventing unnecessary breast cancer treatments.

As many as one in three women treated for breast cancer undergo unnecessary procedures, but a new method for diagnosing it could do a better job distinguishing between benign and aggressive tumors. Researchers at the University of Michigan are developing a pill that makes tumors light up when exposed to infrared light, and they have demonstrated that the concept works in mice.

Mammography is an imprecise tool. About a third of breast cancer patients treated with surgery or chemotherapy have tumors that are benign or so slow-growing that they would never have become life-threatening, according to a study out of Denmark last year. In other women, dense breast tissue hides the presence of lumps and results in deaths from treatable cancers. All that, and mammograms are notoriously uncomfortable.

“We overspend $4 billion per year on the diagnosis and treatment of cancers that women would never die from,” said Greg Thurber, an assistant professor of chemical engineering and biomedical engineering, who led the team. “If we go to molecular imaging, we can see which tumors need to be treated.”

The move could also catch cancers that would have gone undetected. Thurber’s team uses a dye that responds to infrared light to tag a molecule commonly found on tumor cells, in the blood vessels that feed tumors and in inflamed tissue. By providing specific information on the types of molecules on the surface of the tumor cells, physicians can better distinguish a malignant cancer from a benign tumor.

Compared to visible light, infrared light penetrates the body easily—it can get to all depths of the breast without an X-ray’s tiny risk of disrupting DNA and seeding a new tumor. Using a dye delivered orally rather than directly into a vein also improves the safety of screening, as a few patients in 10,000 can have severe reactions to intravenous dyes. These small risks turn out to be significant when tens of millions of women are screened every year in the US alone.

But it’s not easy to design a pill that can carry the dye to the tumor.

“To get a molecule absorbed into the bloodstream, it needs to be small and greasy. But an imaging agent needs to be larger and water-soluble. So you need exact opposite properties,” said Thurber.

Fortunately, they weren’t the only people looking for a molecule that could get from the digestive system to a tumor. The pharmaceutical company Merck was working on a new treatment for cancer and related diseases. They got as far as phase II clinical trials demonstrating its safety, but unfortunately, it wasn’t effective.

“It’s actually based on a failed drug,” said Thurber. “It binds to the target, but it doesn’t do anything, which makes it perfect for imaging.”

The targeting molecule has already been shown to make it through the stomach unscathed, and the liver also gives it a pass, so it can travel through the bloodstream. The team attached a molecule that fluoresces when it is struck with infrared light to this drug. Then, they gave the drug to mice that had breast cancer, and they saw the tumors light up.

“It’s actually based on a failed drug. It binds to the target, but it doesn’t do anything, which makes it perfect for imaging.”Greg Thurber

The research is described in a paper in the journal Molecular Pharmaceutics, titled, “Oral administration and detection of a near-infrared molecular imaging agent in an orthotopic mouse model for breast cancer screening.”

This work was done in collaboration with David Smith, the John G. Wagner Collegiate Professor of Pharmaceutical Sciences in the College of Pharmacy, and a member of the Comprehensive Cancer Center.

The study was supported by the Foundation for Studying and Combating Cancer and the National Institutes of Health.

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‘Nightmare bacteria:’ Michigan Engineers discuss how to combat antibiotic resistance Drug-resistant bugs are on the rise and new approaches are needed.

Health officials at the U.S. Centers for Disease Control and Prevention earlier this month said they are seeing rising cases of “nightmare bacteria” that show strong resistance to antibiotics. More than 200 cases were reported in the last year alone, and across every state in the U.S.

“Unusual resistance germs—which are resistant to all or most antibiotics tested and are uncommon or carry special resistance genes—are constantly developing and spreading,” the CDC said.

A particular concern is the number of cases that crop up in hospitals and nursing homes where IVs, catheters and medical implants—all particularly susceptible to infection—are common.

“Antibiotic resistance is one of the most important public health problems of the 21st century,” said Angela Violi, professor of mechanical engineering and chemical engineering at U-M.

Violi is one of many researchers at Michigan Engineering who are are tackling this issue from a variety of angles. Some are exploring new ways to combine antibiotics to stay one step ahead of the bugs. Others are looking beyond antibiotics—to nanoparticles.

Nicholas Kotov, the Joseph B. and Florence V. Celka professor of chemical engineering, is part of a team researching the use of nanoparticles as a new form of antibiotics. Nanoparticles can be shaped specifically to get past a bacterium’s defenses and shut down processes essential to its survival. Nanoparticles can also be used to coat medical implants in order to prevent infection from drug resistant bacteria.

“New methods of suppressing or otherwise diminishing the health impact of antibiotic resistant bacteria are needed,” Kotov said. “Molecular and nanoscale engineering of inorganic nanoparticles offers this opportunity by utilizing the latest experimental and computational tools targeting the bacteria where it does not expect.”

Violi helps identify the best pathways for utilizing nanoparticles to attack antibiotic resistant bacteria.

“Potentially, all it takes is a single mutated bacterium to render an antibiotic useless for that infection,” she said. “When that mutant cell replicates, it will pass on its resistant phenotype to its daughter cells, and so on.

“At that point part of the replicating bacteria will be drug resistant: the drug will kill only those cells that do not have the newly evolved drug-resistance capacity. Eventually, the entire bacterial population will become resistant to the prescribed antibiotic.

“It is only when antibiotics are used that drug-resistant phenotypes have a selective advantage and survive.

“Nano and chemical engineering approaches provide unparalleled flexibility to control the composition, size, shape, surface chemistry, and functionality of nanostructures that can be used to develop a new generation of modified materials or to coat existing solid surfaces to fight bacteria.”

Professor working on a computer in the lab
Sriram Chandrasekaran, an assistant professor of biomedical engineering, uses computer simulations to develop strategies for using current antibiotics in combination as well as roadmaps for creating new classes of antibiotics. Photo by Joseph Xu

Sriram Chandrasekaran, an assistant professor of biomedical engineering, approaches drug resistant bacteria from a different angle. He and his team study proteins and analyze their behaviors via computer simulations to develop strategies for using current antibiotics in combination as well as roadmaps for creating new classes of antibiotics.

“In addition to better stewardship of antibiotics, we also need to come up with smarter treatment strategies that can reduce the rise of resistance,” Chandrasekaran said.

“For example, our lab and others are designing combinations of antibiotics that are more effective in retarding the evolution of drug resistance compared to using drugs individually. Such combinations of FDA approved drugs can also reach the clinic faster than developing new drugs from scratch.

“We are also developing computer algorithms that can identify the most optimal combination of drugs for a specific strain of pathogen. Overall, what we can learn from this crisis is that we cannot take antibiotics for granted. We have to keep investing on new treatments as bacteria will always eventually evolve resistance to whatever new drug we throw at it.”

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No sponge left behind: tags for surgical equipment A simple, easy-to-implement technology could prevent the debilitating injuries that can occur when organs are damaged by surgical tools left in the body.

Items left behind in patients after surgery can have an enormous personal cost when organs and tissues are damaged. Surgical sponges are among the worst offenders – difficult to see in post-surgical X-rays and yet capable of causing holes when the intestines grow around them, for example. These rare cases, estimated around one in 3,000 surgeries that carry a risk, add up to around $1.5 billion in costs per year.

X-ray image showing scissors inside a cadaver
Marentis took about 2,800 X-ray images of the tag to train and test the software.

The current method of accounting for surgical tools involves counting them before and after surgery and performing an X-ray if there’s a mismatch. Without the metal bands inside them, the gauze sponges wouldn’t appear at all, but they are still difficult to see. A new, unmistakeable tag could change that – and its signature is so clear that computers can also detect it.

The tag, which is about the same size and shape as an acetaminophen tablet, contains four metal spheres, arranged at the points of a tetrahedron. This simple shape can be recognized by the computer no matter how it is turned. With human radiologists having a first look at the X-rays and then comparing their findings with a computer, over 98 percent of the tags can be seen. In contrast, as many as half of surgical sponges are missed in X-rays today.

The research team has formed the company Kalyspo, and they are building partnerships with surgical sponge manufacturers and hospitals in an effort to make the tag and software a standard part of surgical procedures, keeping patients safer.

Nikolaos Chronis, an associate professor of mechanical engineering at U-M, led the development of the tag. Theodore Marentis, then a radiology resident at U-M, identified the need for such a tag and worked with Chronis to develop and test it. Lubomir Hadjiyski, a professor of radiology at U-M, led the development of the software that locates the tags.

Chronis is also an associate professor of biomedical engineering and macromolecular science and engineering. Marentis is now a radiologist at the Mercy Medical Center in Mt. Shasta, CA.

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Closest look yet at killer T-cell activity could yield new approach to tackling antibiotic resistance An in-depth look at the work of T-cells, the body's bacteria killers, could provide a roadmap to effective drug treatments.

In a study that could provide a roadmap for combatting the rising threat of drug-resistant pathogens, researchers have discovered the specific mechanism the body’s T-Cells use to kill bacteria.

University of Michigan researchers, in collaboration with colleagues at Harvard University, have discovered a key difference between the way immune cells attack bacteria and the way antibiotics do. Where drugs typically attack a single process within bacteria, T-Cells attack a host of processes at the same time.

On Thursday, the journal Cell published findings from a team headed by U-M’s Sriram Chandrasekaran and Harvard’s Judy Lieberman. It’s a study with potential implications for drug-resistant pathogens—a problem projected to kill as many as 10 million people annually across the globe by the year 2050.

“We have a huge crisis of antibiotic resistance right now in that most drugs that treat diseases like tuberculosis or listeria, or pathogens like E.coli, are not effective,” said Chandrasekaran, an assistant professor of biomedical engineering. “So there is a huge need for figuring out how the immune system does its work. We hope to design a drug that goes after bacteria in a similar way.”

We’ve reached a point where we take what antibiotics can do for granted, and we can’t do that anymore.Sriram Chandrasekaran

Killer T-Cells, formally known as cytotoxic lymphocytes, attack infected cells by producing the enzyme granzyme B. How this enzyme triggers death in bacteria has not been well understood, Chandrasekaran said.

Proteomics – a technique that measures protein levels in a cell—and computer modeling, allowed researchers to see granzyme B’s multi-pronged attack targeting multiple processes.

Chandrasekaran and his team monitored how T-Cells deal with three different threats: E. coli, listeria and tuberculosis.

“When exposed to granzyme B, the bacteria were unable to develop resistance to the multi-pronged attack, even after exposure over multiple generations,” Chandrasekaran said. “This enzyme breaks down multiple proteins that are essential for the bacteria to survive.

“It’s essentially killing several birds with one stone.”

The possible applications of the new findings on T-Cells run the gamut from the creation of new medications to the re-purposing of previously-approved drugs in combination to fight infections by mimicking granzyme B.

Chandrasekaran’s team is now looking at how bacteria hide to avoid T-Cell attacks.

And the need for a new approach in some form is dire. World Health Organization officials describe antibiotic resistance as “one of the biggest threats to global health, food security and development today.”

Sriram Chandrasekaran, Assistant Professor of Biomedical Engineering, shows a computer model of a pathway for a potential disease or infection. Photo: Joseph Xu

Each year, an estimated 700,000 deaths are linked to antibiotic-resistant bacteria, according to the World Health Organization. Projections show that number skyrocketing to 10 million by 2050.

England’s top health official, Sally Davies, recently said the lost effectiveness of antibiotics would mean “the end of modern medicine.”

“We really are facing—if we don’t take action now—a dreadful post-antibiotic apocalypse,” she was quoted saying earlier this month. “I don’t want to say to my children that I didn’t do my best to protect them and their children.”

Of particular concern is the fact that there are few new antibiotics in the pipeline. The heyday of new antibiotics occurred the 1940s through the 1960s, with releases eventually grinding almost to a halt by the end of the twentieth century.

“We’ve reached a point where we take what antibiotics can do for granted, and we can’t do that anymore,” Chandrasekaran said. “We’re taking inspiration from the human immune system, which has been fighting infections for thousands of years.”

The paper is titled, “Granzyme B disrupts central metabolism and protein synthesis in bacteria to promote an immune cell death program.” The research is funded by the National Institutes of Health, Harvard University and the University of Michigan.

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Bionic heart tissue: U-Michigan part of $20M center Scar tissue left over from heart attacks creates dead zones that don’t beat. Bioengineered patches could fix that.

The University of Michigan is partnering on an ambitious $20 million project to grow new heart tissue for cardiac patients. The new research center has been awarded to Boston University (BU), with strong partnership from U-M and Florida International University (FIU).

“A heart attack creates scar tissue, and the heart never returns to full function. But for every person, we could create a living patch that a surgeon could stitch in,” said Stephen Forrest, who leads the nanotechnology aspect of the project and is U-M’s Peter A. Franken Distinguished University Professor of Engineering. “It’s very audacious.”

The project is a National Science Foundation Engineering Research Center. These 5-year grants are typically renewed for another 5 years, so the researchers are looking at a 10-year timeline to go from the current state of tissue engineering to working, implantable heart tissue.

A heart attack creates scar tissue, but we could create a living patch that a surgeon could stitch in.Steve Forrest

“Heart disease is one of the biggest problems we face,” said David Bishop, director of the new center and a BU professor of electrical and computer engineering and physics. “This grant gives us the opportunity to define a societal problem, and then create the industry to solve it.”

The living patches the researchers are developing would consist of heart muscle cells, blood vessels to carry nutrients in and waste out, and optical circuitry to make the heart muscle cells beat in synchrony. Already, researchers in the lab have been developing ways to structure cells in scaffolds that mimic particular organs and grow blood vessels into artificial tissues. But typically, working implants have been static, biodegradable materials such as artificial windpipes that the body gradually replaces with tissue. Working tissue, like heart muscle, would need to be responsive as soon as it was implanted.

Engineering Research Center grants are extremely competitive, with only four of more than 200 applicants receiving an award in 2017. These centers are designed to work directly with industry to translate breakthroughs along the way out of the lab and into healthcare. Just producing a more true-to-life “heart on a chip” could aid the pharmaceutical industry in developing better treatments for problems such as arrhythmia.

Ramcharan and her colleagues in Lahann’s lab will help design and produce a polymer-protein construct that mimics the 3D matrix connecting the cells in human heart muscle. Heart muscle cells moving into this environment will then be able to link up into a single tissue. Photo: Joseph Xu, Michigan Engineering Communications & Marketing.

In order to produce the heart tissue, the team intends to start with an artificial scaffold that mimics the 3D structure of heart tissue. Joerg Lahann, a U-M professor of chemical engineering, will work with the team building the flexible polymer scaffold, as well as on the attachment and monitoring of cells within that framework.

“Michigan is pleased to lend expertise to the development of implantable heart tissue, which could improve and extend so many lives,” said Alec D. Gallimore, the Robert J. Vlasic Dean of Engineering. “Our faculty members are leaders in nanotechnology and in developing materials that support and interact with living cells and tissues, two areas that are critical to the project’s success.”

The 3D scaffold will initially be peppered with nanometer-sized gold patches that act as attachment points for protein fragments, called peptides, which will then serve as anchors for the cells. They will be printed onto the gold patches using a technique developed by Forrest and Max Shtein, a U-M associate professor of materials science and engineering. This method, called organic vapor jet printing, was initially invented for mass-producing electronic devices.

“The adaptation of this technology to biological systems represents a radically new step,” said Forrest. U-M will receive $2.8 million for these contributions.

Christopher Chen, the center’s director of cellular engineering and a BU professor of biomedical engineering, will lead the effort to grow heart muscle cells on the scaffold and infuse the tissue with blood vessels. Meanwhile, Alice White, director of nanomechanics and chair of the BU mechanical engineering department will work closely with Arvind Agarwal, an FIU professor of mechanical and materials engineering, to produce an artificial nervous system that uses light to synchronize the heartbeat in the tissue.

Stacy Ramcharan, a doctoral student in chemical engineering, uses a computerized system to layer polymer fibers, forming a scaffold for growing cells into artificial tissues. Photo: Joseph Xu, Michigan Engineering Communications & Marketing.

“It’s humbling to have the opportunity to work on something that could really be a game changer,” says Bishop. “If we succeed, we’ll save a lot of lives and add meaningful years for many people.”

In addition to the technical thrusts led by Forrest, Chen and White, Thomas Bifano, a professor of mechanical engineering and director of BU’s Photonics Center, will direct imaging.

Along with the core partners, Harvard Medical School, Columbia University, the Wyss Institute at Harvard, Argonne National Laboratory, the École Polytechnique Fédérale de Lausanne in Switzerland, and the Centro Atómico in Argentina will offer expertise in bioengineering, nanotechnology, and other areas.

Forrest is also the Paul G. Goebel Professor of Engineering, and a professor of electrical engineering and computer science, material science and engineering, and physics. Lahann is also a professor of material science and engineering, biomedical engineering, and macromolecular science and engineering. Shtein is also an associate professor of chemical engineering, macromolecular science and engineering, and art and design. Gallimore is also the Richard F. and Eleanor A. Towner Professor, an Arthur F. Thurnau Professor, and a professor of aerospace engineering.

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