Stanford scientists are growing brain stem cells in bulk using 3D hydrogels

This blog is the final installment in our #MonthofCIRM series. Be sure to check out our other blogs highlighting important advances in CIRM-funded research and initiatives.

Neural stem cells from the brain have promising potential as cell-based therapies for treating neurological disorders such as Alzheimer’s disease, Parkinson’s, and spinal cord injury. A limiting factor preventing these brain stem cells from reaching the clinic is quantity. Scientists have a difficult time growing large populations of brain stem cells in an efficient, cost-effective manner while also maintaining the cells in a stem cell state (a condition referred to as “stemness”).

CIRM-funded scientists from Stanford University are working on a solution to this problem. Dr. Sarah Heilshorn, an associate professor of Materials Science and Engineering at Stanford, and her team are engineering 3D hydrogel technologies to make it easier and cheaper to expand high-quality neural stem cells (NSCs) for clinical applications. Their research was published yesterday in the journal Nature Materials.

Stem Cells in 3D

Similar to how moviegoers prefer to watch the latest Star Wars installment in 3D, compared to the regular screen, scientists are turning to 3D materials called hydrogels to grow large numbers of stem cells. Such an environment offers more space for the stem cells to proliferate and expand their numbers while keeping them happy in their stem cell state.

To find the ideal conditions to grow NSCs in 3D, Heilshorn’s team tested two important properties of hydrogels: stiffness and degradability (or how easy it is to remodel the structure of the hydrogel material). They designed a range of hydrogels, made from proteins with elastic qualities, that varied in these two properties. Interestingly, they found that the stiffness of the material did not have a profound effect on the “stemness” of NSCs. This result contrasts with other types of adult stem cells like muscle stem cells, which quickly differentiate into mature muscle cells when exposed to stiffer materials.

On the other hand, the researchers found that it was crucial for the NSCs to be able to remodel their 3D environment. NSCs maintained their stemness by secreting enzymes that broke down and rearranged the molecules in the hydrogels. If this enzymatic activity was blocked, or if the cells were grown in hydrogels that couldn’t be remodeled easily, NSCs lost their stemness and stopped proliferating. The team tested two other hydrogel materials and found the same results. As long as the NSCs were in a 3D environment they could remodel, they were able to maintain their stemness.

NSCs maintain their stemness in hydrogels that can be remodeled easily. Nestin (green) and Sox2 (red) are markers that indicate “high-quality” NSCs. (Image courtesy of Chris Madl, Stanford)

Caption: NSCs maintain their stemness in hydrogels that can be remodeled easily. Nestin (green) and Sox2 (red) are markers that indicate “high-quality” NSCs. (Images courtesy of Chris Madl)

Christopher Madl, a PhD student in the Heilshorn lab and the first author on the study, explained how remodeling their 3D environment allows NSCs to grow robustly in an interview with the Stem Cellar:

Chris Madl

“In this study, we identified that the ability of the neural stem cells to dynamically remodel the material was critical to maintaining the correct stem cell state. Being able to remodel (or rearrange) the material permitted the cells to contact each other.  This cell-cell contact is responsible for maintaining signals that allow the stem cells to stay in a stem-like state. Our findings allow expansion of neural stem cells from relatively low-density cultures (aiding scale-up) without the use of expensive chemicals that would otherwise be required to maintain the correct stem cell behavior (potentially decreasing cost).”

To 3D and Beyond

When asked what’s next on the research horizon, Heilshorn said two things:

Sarah Heilshorn

“First, we want to see if other stem cell types – for example, pluripotent stem cells – are also sensitive to the “remodel-ability” of materials. Second, we plan to use our discovery to create a low-cost, reproducible material for efficient expansion of stem cells for clinical applications. In particular, we’d like to explore the use of a single material platform that is injectable, so that the same material could be used to expand the stem cells and then transplant them.”

Heilshorn is planning to apply the latter idea to advance another study that her team is currently working on. The research, which is funded by a CIRM Tools and Technologies grant, aims to develop injectable hydrogels containing NSCs derived from human induced pluripotent stem cells to treat mice, and hopefully one day humans, with spinal cord injury. Heilshorn explained,

“In our CIRM-funded studies, we learned a lot about how neural stem cells interact with materials. This lead us to realize that there’s another critical bottleneck that occurs even before the stage of transplantation: being able to generate a large enough number of high-quality stem cells for transplantation. We are developing materials to improve the transplantation of stem cell-derived therapies to patients with spinal cord injuries. Unfortunately, during the transplantation process, a lot of cells can get damaged. We are now creating injectable materials that prevent this cell damage during transplantation and improve the survival and engraftment of NSCs.”

An injectable material that promotes the expansion of large populations of clinical grade stem cells that can also differentiate into mature cells is highly desired by scientists pursuing the development of cell replacement therapies. Heilshorn and her team at Stanford have made significant progress on this front and are hoping that in time, this technology will prove effective enough to reach the clinic.

Lights, Camera, Stem Cells! How photo-responsive hydrogels can improve stem cell therapies

Watching a movie in IMAX 3D.

These days, going to the movie theater is like riding the wildest rollercoaster at your local theme park. It can be an IMAX 3D, surround sound, vibrating seat experience that makes you feel like you’re living the actual movie.

As the entertainment industry evolves towards more intense, realistic cinematic experiences, scientists are following a similar path towards 3D technologies that will improve stem cell-based therapies for biomedical applications. One such technology is called a hydrogel. Hydrogels are biological materials made of either synthetic polymers or natural molecules that scientists use to simulate the native environment in which cells and tissues develop.

Growing stem cells on a flat surface, such as a culture dish, is like watching a movie in a standard, less immersive 2D theater – the stem cells aren’t in their typical 3D environment where they receive biochemical and physical cues to develop into the appropriate cell types of the tissue they are destined to become.

With hydrogels, scientists can more closely mimic a stem cell’s natural environment, or what is called the “stem cell niche”. A lot of research has been dedicated towards fine-tuning hydrogels in a way that can control how stem cells behave and mature. We’ve blogged on this topic previously, and today we bring you an update on a new type of hydrogel that improves upon current technologies.

Scientists from The Hong Kong University of Science and Technology created photo-responsive or light-sensitive hydrogels that they used to grow human mesenchymal stem cells in 3D cultures. These hydrogels contain a vitamin B12-dependent, photo-responsive protein called CarHC. In the dark, coenzyme B12 binds to CarHC and triggers the protein to self-assemble into polymers that create an elastic hydrogel structure. When exposed to light, B12 is absorbed and can no longer bind CarHC, causing the hydrogel structure to dissolve into a liquid solution.

A hydrogel containing mesenchymal stem cells. (Image courtesy of Harvard Paulson School).

This photo-responsive hydrogel is the equivalent of a light-sensing switch that allows the scientists to capture or release stem cells without damaging them or affecting their viability. Senior author on the study, Dr. Fei Sun, elaborated in an interview with,

“The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-solultion transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability.”

Sun’s team is one of the first to report the development of photo-sensitive “smart” hydrogels for stem cell research applications. Looking forward, Sun believes that their technology will be useful for making “tunable materials” that will aid in the development of stem cell-based therapies.

He concluded,

“Given the growing demand for creating stimuli-responsive “smart” hydrogels, the direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.”

Tunable hydrogels guide stem cell differentiation

Differentiating stem cells into mature cells of adult tissue involves many intricate steps to get them to develop into the right cell types. You could compare the process to the careful adjustments you make when tuning a guitar.

In the body, stem cells receive cues from their surrounding environment to mature into specific types of cells. These cues can be biochemical – molecules like lipids, growth factors and metabolites (products of cell metabolism) – or they can be physical – the stiffness of surrounding tissue. But these molecules and structures aren’t always present when scientists attempt to differentiate stem cells outside the body, say in a cell culture dish.

One way researchers are improving the methods for differentiating stem cells outside the body is by using biomaterials such as hydrogels that mimic properties of the structures and molecules found naturally in various stem cell niches that aid in their maturation to adult cell types.

A CIRM-funded study published last week in the journal Chem, has developed “tunable hydrogels” that direct stem cells to differentiate into brain, cartilage and bone cells based on adjustments to the hydrogel’s stiffness and metabolite profile. The work was a collaboration between scientists in New York and in Scotland and one of the co-authors, Bruno Péault, was a CIRM-funded scientist in California during the time of the study.

Hydrogels with different stiffness' direct stem cells to differentiate into different types of tissue. (Chem)

Hydrogels with different stiffness’ direct stem cells to differentiate into different types of tissue. (Chem)

Tuning gels to differentiate stem cells

The scientists started with hydrogels composed of nanofibers that varied in stiffness and observed that perivascular stem cells (from the connective tissue surrounding blood vessels) grown in more flexible gels turned into brain cells and those that were grown in stiffer gels turned into bone cells. The stiffness of these different hydrogels was comparable to that of actual brain and bone tissue, which indicated that stiffness is important for stem cell fate.

But stiffness alone isn’t responsible for directing stem cells into different cell fates – biochemical metabolites are also key to this process. The authors also analyzed the metabolite profiles of the different hydrogels to determine which metabolites were important for stem cell differentiation. They tested different concentrations of over 600 metabolites in the hydrogels during stem cell differentiation and found that certain lipids like lysophosphatidic acid and cholesterol sulfate were essential for differentiation into cartilage and bone tissue respectively. When these specific lipids were added to regular stem cell cultures (without hydrogels), the stem cells differentiated towards cartilage and bone cells. Thus they concluded that both the metabolite profile and the stiffness of hydrogels are important for directing stem cell differentiation.

Interestingly, the authors also showed how metabolites like cholesterol sulfate could influence and activate transcription factors – proteins that also direct stem cell differentiation – which controlled the activation of bone-related genes. This finding suggests a relationship between metabolite expression and transcription factor activity and offers a simpler way to activate transcription factors important for stem cell fate.

Big picture of tunable hydrogels

Looking at the big picture, this study offers a useful strategy to identify molecules that promote formation of specific tissue types from stem cells. These molecules could be potential drug candidates that could aid in regenerating bone and cartilage tissue for patients with osteoporosis or osteoarthritis.

Co-senior author on the study and professor at the University of Glasgow, Matthew Dalby, who was interviewed by Science Magazine elaborated on the importance of their study:

Matthew Dalby

Matthew Dalby

“Our ambition is to simplify drug discovery — by using the cell’s own metabolites as drug candidates. For example, cholesterol sulfate, which our rigid gel revealed as critical to bone cell differentiation, could be a safer solution (e.g., minimal off-target effects) for treating osteoporosis, spinal fusion, and other bone-related conditions. Presently, growth factors are used, but these can lead to unwanted collateral damage, and government agencies in the UK and US have published warnings against their use.”

Rein Ulijn, co-senior author with Dalby and professor at the City University of New York and University of Strathclyde, concluded by emphasizing how the metabolites they identified could be potential drug candidates and would pass regulatory approval if shown to be safe and effective:

Rein Ulijn

Rein Ulijn

“That you can use simple metabolites like cholesterol sulfate, which is readily available, to induce differentiation is in my view very powerful if you think about this as a potential drug candidate. These metabolites are inherently biocompatible, so the hurdles to approval are going to be much lower compared to those associated with completely new chemical entities.”

In the future, both teams plan to further “tune” their hydrogels to mimic more complex tissue environments that incorporate additional properties besides stiffness in hopes of creating more relevant 3D micro-environments to model the stem cell niche.

Stem cell stories that caught our eye: growing better bone, synthetic diaphragms, nerves that make music

Here are some stem cell stories that caught our eye this past week. Some are groundbreaking science, others are of personal interest to us, and still others are just fun.

Image of the hydrogel containing mesenchymal stem cells.

Image of the hydrogel containing mesenchymal stem cells. Credit Harvard SEAS/Wyss Institute

A better way to grow bone.  The term hydrogel gets tossed around a lot in tissue engineering discussions. The porous, generally pliable materials used to hold stem cells in place when creating new tissue are far from uniform. They have highly variable properties. Making a version that mimics the natural environment where bone grows allowed a Harvard team to grow better quality bone and more of it than prior methods.

It turns out stem cells prefer to turn into bone when grown in an environment that readily relaxes in response to stress.  Think of Silly Putty instead of hard rubber.  When the Harvard team grew stem cells on a fast relaxing hydrogel, they saw an increased number of stem cells turn into bone and those cells continued to create more bone for weeks.

“This work both provides new insight into the biology of regeneration, and is allowing us to design materials that actively promote tissue regeneration,” said David Mooney, who led the team.

In a press release from the university picked up by Science Newsline, the researchers speculated that the findings should both enable better bone repair grafts, but also generate more research into how mechanical properties influence cell behavior.

Replacement diaphragms.  Paolo Macchiarini, the Italian scientist based at Sweden’s Karolinska Institute who created much news and a bit of controversy with surgeries to give patients lab-made windpipes, burst back into the news this week. This time with replacement diaphragms, that muscle in the abdomen critical for breathing. The tireless muscle is much more complex than the static windpipe, or trachea, and Macchiarini readily noted that his current work in rats is not nearly ready for patients.

When it is, it could be a life changer and maybe life saver for the one in 2,500 babies born with defects in their diaphragm. Using a technique similar to his work with the trachea, his team took stem cells from bone marrow and grew them on an artificial polymer scaffold.  When they transplanted sections of the synthetic diaphragm into a damaged diaphragm in the animals the sections of muscle beat in synchrony with the rest of the rat’s existing diaphragm. But Macchiarini notes they have no idea why this happened and until they do, the procedure will not be ready for the clinic.

“If you ask me why it happens, to be very honest I don’t know,” he told Alice Park writing for Time. “I can just say that we saw many proteins, extracellular matrix components that belong to the nervous system. So probably via this, the muscle was able to contract again.”

The team wants to refine the process by determining which of the stem cells are destined to become muscle.  The university put a bit more detail in a press release.

Video on synthetic windpipe.  Since Macchiarini’s early reports of giving patients new windpipes, or tracheas, several teams around the world have tried to refine the procedure. The East Coast TV station WFMZ did an easy-to-understand segment on one team’s efforts at Mount Sinai in New York City. So far, their work remains confined to lab animals, but hey hope to treat patients within 18 months.


Lab grown trachea. Credit University College London.

Neural music synthesizer.  On first read, this one sounds a little far fetched. The headline says “world’s first neural synthesizer.” And even crazier, the artist did it with iPSC-type stem cells reprogrammed from his own skin.

The web magazine Fact ran a short piece in its music section. This is how the writer there described the synthesizer:

“Music is fed into the neurons as electrical stimulations and the neurons respond by controlling the synthesizer, creating an improvised post-human sound piece.”

Image from Guy Ben-Ary website.

Image from Guy Ben-Ary website.

It provides a bit more description and notes that the project by artist Guy Ben-Ary is supported by a creative Australia Fellowship award to develop a biologic self-portrait. The article does provide a link to Ben-Ary’s web site, which goes into great detail on every aspect of the project called “cellF.” He describes everything from the procedure for making the stem cells in Barcelona to how they are grown into nerve cells in special plates that can both send and receive signals to respond to the natural electro physiology of nerves.  He explains the special lab plates in this way:

“The dishes that host my ‘external brain’ (neural networks) consist of a grid of electrodes that can record the electric signals that the neurons produce and at the same time send stimulation to the neurons – essentially a read-and-write interface.”

The first concert using the synthesizer occurred October 4. A guest musician, a drummer from Tokyo, provided the sound that was converted to electrical stimulus for the nerves. The nerves responded by controlling the music synthesizer. The video documenting the performance is due to be posted later this month.