How do you get stem cells to survive after they have been transplanted? You can pre-condition them, but research from Johns Hopkins University has capitalized on a different strategy. The Hopkins team used hydrogel to protect and feed the stem cells that had been implanted into the heart.
They utilized a rat model system for this work. Rats that had been given heart attacks were given stem cell implants encased in a hydrogel. The hydrogel supported stem cells survival and also kept the stem cells at the site of their implantation where they re-muscularized the damaged heart muscle. 73% of the stem cells embedded in hydrogel survived whereas only 12% of the non-hydrogel-embedded stem cells survived after injection into the heart.
Previously, stem cell injections have been shown to aid damaged heart tissue, but the vast majority of the injected cells die or are washed from the heart into other tissues. Hydrogel, which mostly consists of water, allows the cells to live and grow while they integrate into the surrounding tissue and initiate healing.
Heart-damaged rats injected with hydrogel-loaded stem cells saw a 15% increase in pumping efficiency for the treated ventricle, compared with just 8% for regular stem cell therapies. Hydrogen can support both adult and embryonic stem cells, and if it’s not put inside a living being, the hydrogel can actually maintain 100% of the stem cells embedded in them.
Hydrogels are useful in biology because they are safe for use in living organisms. In fact, this study found that injecting the hydrogel alone, with no stem cells at all, had a mild benefit all its own by promoting new blood vessel growth.
These are the sorts of breakthroughs that will allow the stem cell technologies of today to become the amazing stem cell technologies of tomorrow.
Regenerative medicine relies upon the ability to isolate, manipulate, and exploit stem cells from our own bodies or from the bodies of stem cell donors. A present obstacle to present therapeutic strategies is the poor survival of implanted stem cells. There are also worries of about properly directing the differentiation of transplanted stem cells. After all, if implanted stem cells do not differentiate into the terminal cell types you want to be replaced, the use of such cells seems pointless.
To address this problem, David Mooney from the Wyss Institute and his colleagues have designed a three-dimensional system that might keep transplanted stem cells alive and happy, ready to heal.
Mooney’s group has adopted a strategy based on the concept of stem cell “niches.”. In our bodies, stem cells have particular places where they live. These stem cell-specific microenvironments provide unique support systems for stem cells and typically include extracellular matrix molecules to which stem cells attach.
Mooney and others have identified chemical and physical cues that act in concert to promote stem cell growth and survival. The chemical cues found in stem cell niches are relatively well-known but the physical and mechanical properties are less well understood at the present time.
Stem cells in places like bone, cartilage, or muscle, when cultured in the laboratory, display particular mechanical sensitivities and they must rest on a substrate with a defined elasticity and stiffness in order to proliferate and mature. As you might guess, reproducing the right physical properties in the laboratory is no mean feat. However, several laboratories have used hydrogels to generate the right combination of chemical and physical properties.
Mooney and his colleagues have made two hydrogels with very different properties. A stable, “bulk” gel is filled with small bubbles of a pore-making molecule called a “porogen,” which degrades quickly and leaves porous cavities in its wake. When the bulk hydrogel is combined with extracellular matrix molecules from stem cell niches and filled with tissue-specific stem cells, and the porogen, Mooney and his team can make an artificial bone-forming stem cell niche. The porous cavities in the hydrogel, in combination with the chemical signals, drive the stem cells to grow, and divide while expanding into the open spaces in the gel. Then the cell move from the hydrogel to form mineralized bone.
In small animal experiments, Mooney and his colleagues showed that a porous hydrogel with the correct chemical and elastic properties provides better bone regeneration than transplanting stem cells alone. The maturing stem cells deployed by the hydrogel also induce neighboring stem cell populations to contribute to the bone repair, which further amplifies their regenerative effects.
This study provides the first demonstration that adjusting the physical properties of a biomaterial can not only help deliver stem cells but also tune the behavior of those cells in a living organism. Even though Mooney has primarily focused on bone formation, he and his group believe that the hydrogel concept can be broadly applied to other regenerative process as well.
This work was published in Nature Materials 2015; DOI: 10.1038/nmat4407.
Healing the heart after a heart attack is a tough venture. Stem cell treatments have shown definite glimmers to success, but a lack of consistency is a persistent problem. Kick-starting the resident stem cell population in the heart is also a possibility but no single strategy has emerged as a tried and true method to treat a sick heart. Tissue engineering remains an engaging possibility and in the laboratory of Amit Patel at the University of Utah, the possibilities push the boundaries on your imagination.
Patel and his colleagues have been hammering at this problem for decades. The problem is how you replace dead tissue in a beating heart with live tissue that can beat in sync with the rest of the tissue. Unfortunately, you cannot ask the heart to take a vacation to help heal itself. Presently, Patel said that “The doctors say, ‘We’ll give you the beta blocker and the aspirin and the Lipitor and we can just hope to maintain you. But short of them getting worse or getting a heart transplant, there’s [sic] not too many options.”
Patel’s work, however, might change all that. He is presently leading trials on an experimental technology that might repair scarred heart tissue and even arrest or, perhaps, reverse heart failure.
His procedure is in a Phase 1 FDA clinical trial. The trial is designed to mix a powder that consists of a mixture of proteins and molecules isolated from heart muscle with saline or water, inject this mixture into the dead portions of the patient’s heart by means of a catheter, and then wait three to six months to determine if the patient’s heart muscle regenerates.
“Heart disease is the most common cause of death in the world, and the most prominent problem is heart failure,” said Tim Henry, the director of cardiology at the Cedars-Sinai Heart Institute. “Effectively, it’s basically one of the biggest problems in the U.S.” Curing the heart with stem cells is, according to Henry, “within our reach,” and Patel, is, to Henry’s thinking, “is clearly one of the most experienced stem cell people in the country”
After a heart attack, the dead regions of the heart form a scar that does not contract, does not conduct electrical impulses, and the rest of the heart has to work around. Reviving the heart scar, shrinking it or reprogramming it to live again has been the dream of stem cell therapy and gene therapy research. However, according to Patel, these venues have not proven to be very good at regenerating dead scar tissue.
Patel, however, noted that “endocardial matrix therapy” would probably be cheaper than stem cell or gene therapy, since it requires an off-the-shelf product that has the advantage of being mass-produced, is easily delivered clinically speaking, and can be easily commercialized and marketed.
This leads to a new question: “What is “extracellular matrix therapy?”
The extracellular matrix is a foundational material upon which cells sit. Extracellular matrix or ECM also provides the glue that attaches cells to each other, layers of cells to each other, and binds tissues together. In Patel’s rendering, ECM consists of everything in our tissues and organs except the cells. If you were to break down the ECM to its parts, you would end up with a concoction of proteins, minerals and a whole cadre of small molecules that can provide a scaffold for cells, nerves and vessels to attach.
To emphasize the importance of the ECM for the heart, Patel said: “A heart without scaffolding is just a bag of cells.” That pretty well nails it.
The ECM also plays a very important signaling role, since it acts as a repository for important signaling molecules that tell cells to grow and develop or divide and heal. The ECM is the milieu in which cells live and grow.
The foundational importance of the ECM gave Patel a revolutionary thought: to heal the heart the matrix has to come first before the cells can follow.
The powder form of heart-specific ECM was developed by scientists at the University of California, San Diego. This group removed the heart muscle from pig hearts, washed away all the cells, and then freeze-dried the remaining ECM into a powder. Using this work as their template, Patel and his team have also devised a protocol to make ECM power from human heart muscle.
When you add water or saline to this ECM powder, it forms a gooey substance called a “hydrogel.” This hydrogel has been called “VentriGel” and it is as flexible as native tissue. Hydrogels are the mainstay of tissue engineering experiments. VentriGel and hydrogels like it can mimic the molecular environment in which cells normally grow and develop. Fortunately, VentriGel has already been shown to successfully reduce scar tissue in the hearts of rats and pigs. To test VentriGel in human patients, Patel and his co-workers can come to the forefront.
Patel recruited a Utah woman who had suffered a heart attack six months ago. This episode reduced her overall heart blood pumping ability from 60 percent (normal) to less than 45 percent (well below normal). Patel and his colleagues made a virtual model of the inside of the patient’s heart to determine where her dead heart muscle resided. Then they marked out 18 different injection sites, and used a catheter to inject the matrix into her heart. The matrix injection procedure took less than two hours.
“This first patient was able to be done awake and safe and she’s already back to work,” Patel said. “She went home the next day.”
Patel plans to treat up to eighteen patients with his experimental procedure. Additionally, cardiologists at the Minneapolis Heart Institute in Minnesota, the only other site approved to test the new technology, performed the procedure on a second patient on Tuesday.
The risks of this procedure are well-known: When hydrogels are directly injected into the heart muscle, they can unintentionally interrupt the electrical conduction of the heart and cause irregular heartbeats. Also, the injected matrix can travel to other parts of the body where it can form a clot that could lead to a stroke. Clots in other parts of the body can also cause the patient’s blood vessels could collapse.
“If you go through all the bad things that could happen, you’d be so depressed, you’d be like, ‘Really? You found somebody to go through this?'” Patel said. “The key is that the team that we have here, and many of my collaborators, we’re all at that same level of healthy enthusiasm mixed with extreme paranoia.”
All patients will be examined three and six months after the procedure out for evidence of muscle regrowth and revived heart function.
“We want to treat this before it ends up leading to permanent damage,” Patel said.
If the trial returns positive results, it will represent another step forward in a long journey to eradicate heart disease. Patel estimates, that if everything goes smoothly, the technology could become approved for clinical use within five to seven years.
The growth of new blood vessels in culture received in new boost from researchers at Rice University and Texas Children’s Hospital who used stem cells from amniotic fluid to promote the growth of robust, functional blood vessels in healing hydrogels.
These results were published in the Journal of Biomedical Materials Research Part A.
Engineer Jeffrey Jacot thinks that amniotic fluid stem cells are valuable for regenerative medicine because of their ability to differentiate into many other types of cells, including endothelial cells that form blood vessels. Amniotic fluid stem cells are taken from the discarded membranes in which babies are encased in before birth. Jacot and others combined these cells with an injectable hydrogel that acted as a scaffold.
In previous experiments, Jacot and his colleagues used amniotic fluid cells from pregnant women to help heal infants born with congenital heart defects. Amniotic fluids, drawn during standard tests, are generally discarded but show promise for implants made from a baby’s own genetically matched material.
“The main thing we’ve figured out is how to get a vascularized device: laboratory-grown tissue that is made entirely from amniotic fluid cells,” Jacot said. “We showed it’s possible to use only cells derived from amniotic fluid.”
Researchers from Rice, Texas Children’s Hospital and Baylor College of Medicine combined amniotic fluid stem cells with a hydrogel made from polyethylene glycol and fibrin. Fibrin is the proteins formed during blood clots, but it is also used for cellular-matrix interactions, wound healing and angiogenesis (the process by which new vessels are made). Fibrin is widely used as a bioscaffold but it suffers from low mechanical stiffness and is degraded rapidly in the body. When fibrin was combined with polyethylene glycol, the hydrogel became much more robust, according to Jacot.
Additionally, these groups used a growth factor called vascular endothelial growth factor to induce the stem cells to differentiate into endothelial cells. Furthermore, when induced in the presence of fibrin, these cells infiltrated the native vasculature from neighboring tissue to make additional blood vessels.
When mice were injected with fibrin-only hydrogels, thin fibril structures formed. However if those same hydrogels were infused with amniotic fluid stem cells that had been induced with vascular endothelial growth factor, the cell/fibrin hydrogel concoctions showed far more robust vasculature.
In similar experiments with hydrogels seeded with bone marrow-derived mesenchymal cells, once again, vascular growth was observed, but these vessels did not have the guarantee of a tissue match. Interestingly, seeding with endothelial cells didn’t work as well as the researchers expected, he said.
Jacot and others will continue to study the use of amniotic stem cells to build biocompatible patches for the hearts of infants born with birth defects and for other procedures.
Stem cell-based treatments for bone injuries have made some remarkable strides in the past few years. Unfortunately, a common pitfall of bone-making stem cells is the tendency of these cells to wander away from the site of injury. This “wander lust” among stem cells can inhibit healing and reduce stem cell efficacy. How to keep the cells home? The answer seems to be encasing them in a water-retaining gel that keeps them in place, but degrades once the cells have done their job.
Cartilage production has benefitted from the use of these so-called “hydrogels” that encase cells and keep them at the site of injury. However, hydrogels have yet to be tried with bone regeneration.
Assistant Professor of Biomedical Engineering, Danielle Benoit, said, “For example, we should not be able to pinpoint repairs within the periosteum, or outer membrane of bone material.”
The hydrogels used by Benoit and her colleagues mimic the body’s natural tissues, but they also are biodegradable and disappear before the immune system recognizes them as foreign substances.
Benoit believes that the special properties of hydrogels could direct bone-making mesenchymal stem cells to make bone mad repair bone fractures at the site of injury, and then leave the site once the cells have completed their mission.
In previous work (M.D. Hoffman, and others, Biomaterials, 34 (35) (2013), pp. 8887–8898), ) Benoit and her co-workers transplanted hydrogel-encased stem cells onto the surface of mouse bone grafts. However, Benoit’s group not only closely observed the behavior of these implanted cells in the animal, but also in culture dishes outside the animal.
In these experiments, Michael Hoffman and others grafted decellularized bone into the long bones of mice. Because these grafts had all their living material removed, all the bone healing that occurred would be solely due to the implanted stem cells.
Then stem cells that had been genetically engineered to glow a fluorescent green color. The bone material was also coated with hydrogels to keep the stem cells at the site of the bone graft. Then Benoit’s group monitored the bone regeneration process to determine the loss or retention of stem cells at the site of the bone graft in the presence or absence of hydrogels. They used the amount of fluorescence to ascertain the number of cells present at the site of repair. Strangely, Benoit and her colleagues were unable to demonstrate the ability of the PEG hydrogels to control spatiotemporal MSC localization. Therefore, it seemed to be due to the hydrogels and their properties.
As it turns out, depending on how the hydrogels are made, they have different rates of degradation. Benoit, therefore, decided to synthesize gel fibers that underwent biodegradation at different rates. Once the hydrogel began to experience degradation, the spaces between the hydrogels fibers increased and this allowed cells to exit the hydrogel.
In a series of experiments Hoffman, Van Hover and Benoit showed that the faster the rates of hydrogels degradation, the poorer the retention of the cells within the hydrogels. Retention rates were directly proportional to the hydrogels rate of degradation, since longer-lived hydrogels showed higher levels of cell retention and shorter-lived gels showed shorter retention times. In the words of Benoit and her colleagues: “cell localization at allograft surfaces decays in close agreement with network degradation kinetics both in vitro and in vivo.
Such hydrogels with variable degradation rates show promise in not only in bone regeneration, but also in heart attacks in which the initiation of healing might be instigated without invasive surgical procedures that can greatly weaken an already incredibly sick patient.
Scientists from Case Western Reserve in Cleveland, Ohio have used hydrogels (jello-like materials) to make three-dimensional structures that direct stem cell behavior.
Physical and biochemical signals guide stem cell behavior and directs them to differentiate and make tissues like muscle, blood vessels, or bone. The exact recipes to produce each particular tissue remains unknown, but the Case Western Reserve team has provided a way to discover these recipes.
Ultimately, scientists would like to manipulate stem cells in order to repair or replace damaged tissues. They would also like to engineer new tissues and organs.
Eben Alsberg. associate professor of biomedical engineering and orthopedic surgery at Case Western Reserve, who was also the senior author on this research said, “If we can control the spatial preservation of signals, we have be able to have more control over cell behavior and enhance the rate and quality of tissue formation. Many tissues form during development and healing processes at least in part due to gradients of signals: gradients of growth factors, gradients of physical triggers.”
Alsberg and his colleagues have tested their system on mesenchymal stem cells, and in doing so have turned them into bone or cartilage cells. Regulating the presentation of certain signals in three-dimensional space may be a key to engineering complex tissues; such tissues as bone and cartilage. For example, if we want to convert cartilage-making cells into bone-making cells or visa-verse, several different signals are required to induce the stem cells to change into different cell types in order to form the tissues you need.
To test their ideas, Alsberg and coworkers two different growth factors directed the stem cells to differentiate into either bone or cartilage. One of these growth factors, transforming growth factor-beta (TGF-beta) promotes cartilage formation while a different growth factor, bone morphogen protein-2 (BMP-2). Alsberg and his crew placed mesenchymal stem cells into an alginate hydrogel with varying concentrations of these growth factors. Alginate comes from seaweed and when you hit it with ultraviolet light, it crosslinks to form a jello-like material called a hydrogel. To create gradients of these growth factors, Alsberg developed a very inventive method in which they loaded a syringes with these growth factors and hooked them to a computer controlled pump that released lots of BMP-2 and a little TGF-1beta and tapered the levels of BMP-2 and then gradually increased the levels of TGF-1beta (see panel A below).
The result has an alginate hydrogen with mesenchymal stem cell embedded in it that had a high concentration of BMP-2 at one end and a high concentration of TGF-1beta at the other end. Alsberg also modified the hydrogel by attached RGD peptides to it so that the stem cells would bind the hydrogel. The peptide RGD (arginine-glycine-aspartic acid) binds to the integrin receptors, which happen to be one of the main cell adhesion protein on the surfaces of these cells. This modification increases the exposure of the mesenchymal stem cells to the growth factors. After culturing mesenchymal stem cells in the hydrogel, they discovered that the majority of the cells were in the areas of the hydrogel that had the highest concentration of RDG peptides.
In another other experiment Alsberg and others varied the crosslinks in the hydrogel. They used hydrogels with few crosslinks that were more flexible and hydrogels that have quite a few crosslinks and were stiffer. The stem cells clearly preferred the more flexible hydrogels. Alsberg thinks that the more flexible hydrogels might show better diffusion of the growth factors and better waste removal.
“This is exciting,” gushed Alsberg. “We can look at this work as a proof of principle. Using this approach, you can use any growth factor or any adhesion ligand that influences cell behavior and study the role of gradient presentation. We can also examine multiple different parameters in one system to investigate the role of these gradients in combination on cell behavior.”
This technology might also be a platform for testing different recipes that would direct stem cells to become fat, cartilage, bone, or other tissues. Also, since this hydrogel is also biodegradable, stem cells grown in the hydrogel could be implanted into patients. Since the cells would be in the process of forming the desired tissue, their implantation might restore function and promote healing. Clearly Alsberg is on to something.
Johns Hopkins University scientists have directed stem cells to form networks of new blood vessels, and successfully transplanted those laboratory-made blood vessels into laboratory mice.
The stem cells in this experiment were made by reprogramming ordinary cells. Thus this new technique could potentially be used to make blood vessels that are genetically matched to individual patients and have a very low chance of being rejected by the patient’s immune system.
“In demonstrating the ability to rebuild a microvascular bed in a clinically relevant manner, we have made an important step toward the construction of blood vessels for therapeutic use,” said Sharon Gerecht, associate professor in the Johns Hopkins University Department of Chemical and Biomolecular Engineering, Physical Sciences-Oncology Center and Institute for NanoBioTechnology. “Our findings could yield more effective treatments for patients afflicted with burns, diabetic complications and other conditions in which vasculature function is compromised.”
Gerecht’s research group and others have previously grown blood vessels in the laboratory using stem cells, but there are problems with using these blood vessels in human patients. For example, in a paper published by Gerecht’s group in Stem Cells Translational Medicine earlier this year (Stem Cells Trans Med April 2013 vol. 2 no. 4 297-306), ECFCs or endothelial colony-forming cells from human umbilical cords were grown and used to make networks of blood vessels in culture. Those blood vessels were then embedded in blocks of “hyaluronic acid.” Hyaluronic acid is a component of human connective tissue, and when the ECFCs were embedded into it, they were then placed on the skin of mice that had received third-degree burns. On day 3 following transplantation, white blood cells called macrophages degraded the hyaluronic acid gel rather quickly. Between days 5–7, the mouse’s blood vessels infiltrated the implant and connected with the human blood vessels in the wound area. The growth of the human blood vessels peaked at day 7 and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the blood vessels, including the transplanted human vessels generally regressed, and a few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 square micrometers (including the human ones), remained in the healed wound.
This is a fascinating experiment, but making blood vessels this way is a heck of a lot of work, and even though the umbilical cord ECFCs are less likely to be rejected by the immune system of the patient, the chances of immune system rejection are still present. is there a better way?
In this current study, Gerecht and her team tried to streamline the new growth process. Where other experiments used chemical cues to differentiate stem cells into the desired cell type, Sravanti Kusuma in Gerecht’s laboratory devised a way to instruct stem cells to exclusively form the two cell types required for blood vessel construction (smooth muscle cells and endothelial cells).
According to Kusuma, “It makes the process quicker and more robust if you don’t have to sort through a lot of cells you don’t need to find the ones you do, or grow two batches of cells,”
Another difference from previous experiments was the use of induced pluripotent stem cells rather than bone marrow-derived endothelial precursor cells or umbilical cord-derived endothelial colony-forming cells. Gerecht’s team collaborated with Linzhao Cheng from the Institute for Cell Engineering to co-opt her expertise with induced pluripotent stem cells (iPSCs), which are made from adult cells and are de-differentiated through genetic engineering techniques to become embryonic-like stem cells.
Cheng said that this experiment is “an elegant use of human induced pluripotent stem cells that can form multiple cell types within one kind of tissue or organ and have the same genetic background [as the patient].” Cheng continued” “In addition to being able to form blood cells and neural cells as previously shown, blood-derived human induced pluripotent stem cells can also form multiple types of vascular network cells.”
To grow blood vessels, Cheng, Gerecht and others placed the stem cells into a scaffolding made of hydrogel (hyaluronic acid and water). This hydrogel was full of chemical cues that directed the cells to differentiate in to endothelial and smooth muscle cells and form a network of blood vessels. This constitutes the first time human blood vessels had been made from human pluripotent stem cells in a synthetic material.
While these networks of blood vessels looked like the real thing, would they work within a living creature? The answer that question, Gerecht and her group transplanted them into mice. After two weeks the lab-grown blood vessels had integrated with the mouse’s own blood vessels and the hydrogel had dissolved and been degraded. “That these vessels survive and function inside a living animal is a crucial step in getting them to medical application,” Kusama said.
An important follow-up to these experiments is to examine the three-dimensional structure of these blood vessels to determine if truly have all the characteristics of human blood vessels that can deliver blood to damaged tissues and help those tissues recover from injury or trauma.