How a Silly Putty ingredient could advance stem cell therapies

By Nicole Casal Moore
Michigan Engineering

The sponginess of the environment where human embryonic stem cells are growing affects the type of specialized cells they eventually become, a University of Michigan study shows.

The researchers coaxed human embryonic stem cells to turn into working spinal cord cells more efficiently by growing the cells on a soft, utrafine carpet made of a key ingredient in Silly Putty. Their study is published online at Nature Materials on April 13.

This research is the first to directly link physical, as opposed to chemical, signals to human embryonic stem cell differentiation. Differentiation is the process of the source cells morphing into the body’s more than 200 cell types that become muscle, bone, nerves and organs, for example.

Jianping Fu, U-M assistant professor of mechanical engineering, says the findings raise the possibility of a more efficient way to guide stem cells to differentiate and potentially provide therapies for diseases such as amyotrophic lateral sclerosis (Lou Gehrig’s disease), Huntington’s or Alzheimer’s.

In the specially engineered growth system—the ‘carpets’ Fu and his colleagues designed—microscopic posts of the Silly Putty component polydimethylsiloxane serve as the threads. By varying the post height, the researchers can adjust the stiffness of the surface they grow cells on. Shorter posts are more rigid—like an industrial carpet. Taller ones are softer—more plush.

The team found that stem cells they grew on the tall, softer micropost carpets turned into nerve cells much faster and more often than those they grew on the stiffer surfaces. After 23 days, the colonies of spinal cord cells—motor neurons that control how muscles move—that grew on the softer micropost carpets were four times more pure and 10 times larger than those growing on either traditional plates or rigid carpets.

“This is extremely exciting,” Fu said. “To realize promising clinical applications of human embryonic stem cells, we need a better culture system that can reliably produce more target cells that function well. Our approach is a big step in that direction, by using synthetic microengineered surfaces to control mechanical environmental signals.”

Fu is collaborating with doctors at the U-M Medical School. Eva Feldman, the Russell N. DeJong Professor of Neurology, studies amyotrophic lateral sclerosis, or ALS. It paralyzes patients as it kills motor neurons in the brain and spinal cord.

Researchers like Feldman believe stem cell therapies—both from embryonic and adult varieties—might help patients grow new nerve cells. She’s using Fu’s technique to try to make fresh neurons from patients’ own cells. At this point, they’re examining how and whether the process could work, and they hope to try it in humans in the future.

“Professor Fu and colleagues have developed an innovative method of generating high-yield and high-purity motor neurons from stem cells,” Feldman said. “For ALS, discoveries like this provide tools for modeling disease in the laboratory and for developing cell-replacement therapies.”

Fu’s findings go deeper than cell counts. The researchers verified that the new motor neurons they obtained on soft micropost carpets showed electrical behaviors comparable to those of neurons in the human body. They also identified a signaling pathway involved in regulating the mechanically sensitive behaviors. A signaling pathway is a route through which proteins ferry chemical messages from the cell’s borders to deep inside it. The pathway they zeroed in on, called Hippo/YAP, is also involved in controlling organ size and both causing and preventing tumor growth.

Fu says his findings could also provide insights into how embryonic stem cells differentiate in the body.

“Our work suggests that physical signals in the cell environment are important in neural patterning, a process where nerve cells become specialized for their specific functions based on their physical location in the body,” he said.

The paper is titled “Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells.” Fu collaborated with researchers at the School of Dentistry and the Department of Molecular, Cellular and Developmental Biology in the U-M College of Literature, Science, and the Arts.

Fu is also a U-M assistant professor of biomedical engineering.

Regenerative medicine: Injectable stem cell incubator

By Kate McAlpine
Michigan Engineering

Some tissue damage is too extensive for the body to heal well, such as a bad slipped disc or the muscle death that follows a heart attack, so researchers are looking for ways to bridge the gaps. The most promising avenue is stem cell therapy, but these cells take their cues about what kind of cell to be from their surroundings.

Now, a team of researchers from the University of Michigan has developed microscopic particles that can be tailored for different parts of the body, loaded with stem cells, and injected straight into damaged tissues.

The new “microspheres” amount to an injectable kind of scaffolding that could hold the stem cells in place while they assumed their new roles, replacing dead tissue.

To make them, the researchers tweaked two aspects of the biodegradable molecules that make up the spheres, enabling the control of their solidity and fibrousness.

By simulating the structure of the target tissue, the cells can better integrate with the body, says Peter Ma, the Richard H. Kingery Collegiate Professor who led the development of the spheres.

“The degree of the hollowness and the smallness of the fibers that make up the microspheres can dramatically facilitate tissue or organ regeneration,” said Ma. “Currently, these novel microspheres are being used to regenerate intervertebral disc and heart tissues in our lab and our collaborators’ labs. If successful, we will potentially develop ways to cure low back pain and rescue patients suffering from heart attacks.”

His group previously showed that microspheres loaded with stem cells could help heal cartilage in the knees of rabbits when injected into the damaged area.

Ma’s team worked with the group of Sharon Glotzer, the Stuart W. Churchill Professor of Chemical Engineering, to discover a way to control the structure of the microspheres so that they can effectively support the repair of other types of tissue.

The microspheres are made of lactic acid, a product of metabolism, arranged into star-shaped polymers – so the human body can easily break them down. The team first mixed the polymers into glycerol, which is commonly used as sweetener. In the solution, the star-shaped polymers assembled into microspheres about 0.06 millimeters in diameter.

Then, the team tailored the nanoscale structure of the microspheres, which must mimic the network of proteins and other molecules that surround cells in the body, by plunging the mixture into liquid nitrogen at a temperature of about -321°F. If the lactic acid arms of the star-shaped polymers were too short, they formed solid microspheres. But if the arms were over about 150 to 200 lactic acids long, the polymers crystallized into fibers.

The trick to controlling the cell-scale porousness is in the hydroxyl groups, composed of a hydrogen atom and an oxygen atom stuck together, which are tacked onto the polymer. It doesn’t take many – less than one for every hundred lactic acid groups – but the hydroxyl allows the glycerol to support the microsphere structure. As a result, the polymer stars don’t feel the need to fill in every gap, leaving room for stem cells.

“Through manipulating the structure of star-shaped molecules, we can simultaneously control their assembly at both the nano- and microscales,” said Zhanpeng Zhang, who recently earned his doctorate in biomedical engineering, in part for his experimental work with the microspheres.

After washing out the glycerol, the team freeze-dried the microspheres for later use.

Computer simulations from Glotzer’s team confirmed that the interactions between the hydroxyl groups and the glycerol led to spongy or hollow the microspheres.

“Because the droplet assembly is so varied, we can also envision other applications such as drug delivery or catalysis, which in turn might require different kinds of nano- or micro-structure,” said Ryan Marson, who recently earned his doctorate in materials science and engineering, in part for his work on the simulations.

Other potential applications include electronic displays, self-healing materials and artificial photosynthesis.

Ma is also a professor of dentistry, biomedical engineering, macromolecular science and engineering and materials science and engineering. Glotzer is also a professor of materials science and engineering, macromolecular science and engineering, physics, and applied physics.

The research was supported by the National Institutes of Health, the Department of Defense, the National Science Foundation and the U.S. Army Research Office.