Matrix Stiffness is an Essential Tool in Stem Cell Differentiation

Matrix Stiffness is an Essential Tool in Stem Cell Differentiation

Sunday, 10 August 2014

Cells grown on hydrogels of the same stiffness 

all display fat cell markers and deform the

underlying matrix material the same way.

Credit: Adam Engler, UC San Diego Jacobs

School of Engineering. 

Bioengineers at the University of California, San Diego have proven that when it comes to guiding stem cells into a specific cell type, the stiffness of the extracellular matrix used to culture them really does matter. When placed in a dish of a very stiff material, or hydrogel, most stem cells become bone-like cells. By comparison, soft materials tend to steer stem cells into soft tissues such as neurons and fat cells. The research team, led by bioengineering professor Adam Engler, also found that a protein binding the stem cell to the hydrogel is not a factor in the differentiation of the stem cell as previously suggested. The protein layer is merely an adhesive, the team reported Aug. 10 in the advance online edition of the journal Nature Materials.

Their findings affirm Engler's prior work on the relationship between matrix stiffness and stem cell differentiations.

Cells grown on three hydrogels of the same

stiffness all display fat cell markers and deform

the underlying matrix material in the same way.

Credit: Adam Engler, UC San Diego Jacobs

School of Engineering. 

"What's remarkable is that you can see that the cells have made the first decisions to become bone cells, with just this one cue. That's why this is important for tissue engineering," said Engler, a professor at the UC San Diego Jacobs School of Engineering.

Engler's team, which includes bioengineering graduate student researchers Ludovic Vincent and Jessica Wen, found that the stem cell differentiation is a response to the mechanical deformation of the hydrogel from the force exerted by the cell. In a series of experiments, the team found that this happens whether the protein tethering the cell to the matrix is tight, loose or non-existent. To illustrate the concept, Vincent described the pores in the matrix as holes in a sponge covered with ropes of protein fibres. Imagine that a rope is draped over a number of these holes, tethered loosely with only a few anchors or tightly with many anchors. Across multiple samples using a stiff matrix, while varying the degree of tethering, the researchers found no difference in the rate at which stem cells showed signs of turning into bone-like cells. The team also found that the size of the pores in the matrix also had no effect on the differentiation of the stem cells as long as the stiffness of the hydrogel remained the same.

Cells grown on three hydrogels of the same

stiffness all display fat cell markers and deform

the underlying matrix material in the same way.

Credit: Adam Engler/UC San Diego Jacobs

School of Engineering. 

"We made the stiffness the same and changed how the protein is presented to the cells (by varying the size of the pores and tethering) and ask whether or not the cells change their behaviour," Vincent said.

"Do they respond only to the stiffness? Neither the tethering nor the pore size changed the cells."

"We're only giving them one cue out of dozens that are important in stem cell differentiation," said Engler.

"That doesn't mean the other cues are irrelevant; they may still push the cells into a specific cell type. We have just ruled out porosity and tethering, and further emphasized stiffness in this process."

Source: University of California - San Diego 

Contact: Catherine Hockmuth

Reference:

Interplay of matrix stiffness and protein tethering in stem cell differentiation

Jessica H. Wen, Ludovic G. Vincent, Alexander Fuhrmann, Yu Suk Choi, Kolin Hribar, Hermes Taylor-Weiner, Shaochen Chen and Adam J. Engler

Nature Materials, Aug. 10 in the Advance online edition, Aug. 10 2014.

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Signal Gradients in 3D Guide Stem Cell Behaviour

System can help discern recipes for tissue and organ repair and replacements

Wednesday, 18 September 2013

Scientists know that physical and biochemical signals can guide cells to make, for example, muscle, blood vessels or bone. But the exact recipes to produce the desired tissues have proved elusive.

Now, researchers at Case Western Reserve University have taken a step toward identifying that mix by developing an easy and versatile way of forming physical and biochemical gradients in three dimensions.

Ultimately, one of their goals is to engineer systems to manipulate stem cells to repair or replace damaged tissues and organs.

"If we can control the spatial presentation of signals, we may be able to have more control over cell behaviour and enhance the rate and quality of tissue formation," said Eben Alsberg, an associate professor of biomedical engineering and orthopaedic surgery at Case Western Reserve and senior author of the research.

"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, postdoctoral scholar Oju Jeon and graduate student Daniel S. Alt of Case Western Reserve, and Stephen W. Linderman, a visiting undergraduate on a National Science Foundation Research Experience for Undergraduates summer fellowship, tested their system on mesenchymal stem cells, turning them toward bone or cartilage cells. They report their findings in Advanced Materials.

Regulating the presentation of certain signals in three-dimensional space may be a key to engineering complex tissues, such as repairing osteochondral defects, damaged cartilage and bone in osteoarthritic joints, Alsberg said.

"There must be a transition from bone to cartilage," he said, "and that may require control over multiple signals to induce the stem cells to change into the different kinds of cells to form tissues where you need them."

In their first test, the researchers found that stem cells changed into cartilage or bone cells in the directions of two opposing soluble growth factor gradients: one that promotes cartilage, called TGF-beta 1, and another that promotes bone, called BMP-2. The stem cells were placed in a solution of modified alginate, a material derived from seaweed that can form a jelly-like material called a hydrogel when exposed to low level ultraviolet light.

The solution was divided between two computer-controlled syringe pumps, with BMP-2 in one syringe and TGF-beta 1 in the other. By controlling the rate of injection with the pumps and using a mixing unit, a hydrogel with a BMP-2 gradient starting with a large amount and tapering to nearly none and an opposing TGF-beta 1 gradient from low-to-high was formed.

The hydrogels were further modified in such a way that the growth factors were retained for a longer period of time. This enabled prolonged exposure of stem cells to the growth factors and further control over their differentiation into bone or cartilage cells.

The researchers then modified the hydrogel with a gradient of adhesion ligands, molecular strings that allow the stem cells to attach to the hydrogel itself. After two weeks of culturing the cells, they found the highest number of cells in the hydrogel region where the concentration of ligands was highest.

In a third test, they created a gradient of crosslink density within the hydrogels. Crosslinks provide structure to the gels. The lower the density, the more flexible the hydrogel; the higher, the stiffer the gel will become.

After two weeks, more cells were found in the most flexible gel regions within the gradient. The flexibility may allow for more free movement of nutrients and removal of waste products, Alsberg explained.

"This is exciting," Alsberg said.

"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 behaviour 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 behaviour."

If the technology enables them to unravel recipes that generate complex tissues, the biodegradable hydrogel mix could be implanted or injected at the site of an injury, the researchers say. The recipe would guide cell behaviour until new tissue is formed, restoring function.

Source: Case Western Reserve University 

Contact: Kevin Mayhood

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Fingernails Reveal Clues to Limb Regeneration

Researchers discover biochemical pathway that links nail growth to fingertip regeneration

Wednesday, 12 June 2013

Mammals possess the remarkable ability to regenerate a lost fingertip, including the nail, nerves and even bone. In humans, an amputated fingertip can sprout back in as little as two months, a phenomenon that has remained poorly understood until now. In a paper published today in the journal Nature, researchers at NYU Langone Medical Center shed light on this rare regenerative power in mammals, using genetically engineered mice to document for the first time the biochemical chain of events that unfolds in the wake of a fingertip amputation. The findings hold promise for amputees who may one day be able to benefit from therapies that help the body regenerate lost limbs.

"Everyone knows that fingernails keep growing, but no one really knows why," says lead author Mayumi Ito, PhD, assistant professor of dermatology in the Ronald O. Perelman Department of Dermatology at NYU School of Medicine. Nor is much understood about the link between nail growth and the regenerative ability of the bone and tissue beneath the nail.

Now, Dr. Ito and team have discovered an important clue in this process: a population of self-renewing stem cells in the nail matrix, a part of the nail bed rich in nerve endings and blood vessels that stimulate nail growth. Moreover, the scientists have found that these stem cells depend upon a family of proteins known as the "Wnt signalling network" — the same proteins that play a crucial role in hair and tissue regeneration — to regenerate bone in the fingertip.

"When we blocked the Wnt-signalling pathway in mice with amputated fingertips, the nail and bone did not grow back as they normally would," says Dr. Ito. Even more intriguing, the researchers found that they could manipulate the Wnt pathway to stimulate regeneration in bone and tissue just beyond the fingertip.

"Amputations of this magnitude ordinarily do not grow back," says Dr. Ito.

These findings suggest that Wnt signalling is essential for fingertip regeneration, and point the way to therapies that could help people regenerate lost limbs. An estimated 1.7 million people in the U.S. live with amputations.

The team's next step is to zoom in on the molecular mechanisms that control how the Wnt signalling pathway interacts with the nail stem cells to influence bone and nail growth.

Source: New York University School of Medicine 

Contact: Christopher Rucas

Reference:

Wnt activation in nail epithelium couples nail growth to digit regeneration

Makoto Takeo, Wei Chin Chou, Qi Sun, Wendy Lee, Piul Rabbani, Cynthia Loomis, M. Mark Taketo & Mayumi Ito

Nature 12 June 2013 (2013), doi:10.1038/nature12214

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Scientists Create Personalized Bone Substitutes from Skin Cells

For treatment of large bone defects and traumatic injuries

Tuesday, 07 May 2013

A team of New York Stem Cell Foundation (NYSCF) Research Institute scientists report today the generation of patient-specific bone substitutes from skin cells for repair of large bone defects. The study, led by Darja Marolt, PhD, a NYSCF-Helmsley Investigator and Giuseppe Maria de Peppo, PhD, a NYSCF Research Fellow, and published in the Proceedings of the National Academy of Sciences of the USA, represents a major advance in personalized reconstructive treatments for patients with bone defects resulting from disease or trauma.

This advance will facilitate the development of customizable, three-dimensional bone grafts on-demand, matched to fit the exact needs and immune profile of a patient. Taking skin cells, the NYSCF scientists utilized an advanced technique called "reprogramming" to revert adult cells into an embryonic-like state. These induced pluripotent stem (iPS) cells carry the same genetic information as the patient and they can become any of the body's cell types.

The NYSCF team guided these iPS cells to become bone-forming progenitors and seeded the cells onto a scaffold for three-dimensional bone formation. They then placed the constructs into a device called a bioreactor, which provides nutrients, removes waste, and stimulates maturation, mimicking a natural developmental environment.

"Bone is more than a hard mineral composite, it is an active organ that constantly remodels. Blood vessels shuttle important nutrients to healthy cells and remove waste; nerves provide connection to the brain; and, bone marrow cells form new blood and immune cells," said Marolt.

Previous studies have demonstrated the bone-forming potential from other cell sources, yet serious caveats for clinical translation remain. A patient's own bone marrow stem cells can form bone and cartilaginous tissue, not the underlying vasculature and nerve compartments; and, embryonic stem cell derived bone may prompt an immune rejection. The NYSCF scientists chose to work with iPS cells to overcome these limitations, comparing iPS sources with embryonic stem cells and bone marrow derived cells.

"No other research group has published work on creating fully-viable, functional, three-dimensional bone substitutes from human iPS cells. These results bring us closer to achieving our ultimate goal, to develop the most promising treatments for patients," said de Peppo.

While severity varies, bone defects and injuries are currently treated with bone grafts, taken either from another part of the patient's body or a donor bone bank, or with synthetic substitutes. None of these permit complex reconstruction, and they may elicit immune rejection or fail to integrate with surrounding connective tissues. For trauma patients, suffering from shrapnel wounds or vehicular injury, these traditional treatments provide limited functional and cosmetic improvement.

After a comprehensive in vitro analysis of the generated bone, the NYSCF team assessed stability when transplanted in an animal model to address a major concern for iPS-based cell therapies. Undifferentiated iPS cells can form teratomas, a type of tumour. The iPS cell-derived bone substitutes were implanted under the skin of immune compromised mice. After 12 weeks, the explanted constructs matured and showed no malignancies but complete maturation of bone tissue, while blood vessel cells began to integrate along the grafts. These results indicate the stability of the bone substitutes.

The scientists caution that although these results represent a major advance, further research is necessary before skin cell-derived bone grafts reach patients. Next steps include protocol optimization and the successful growth of blood vessels within the bone.

"Following from these findings, we will be able to create tailored bone grafts, on demand, for patients without any immune rejection issues," said Susan L. Solomon, CEO of NYSCF.

"This is not a good approach, it is the best approach to repair devastating damage or defects."

Beyond potential therapeutic relevance, these adaptive bone substitutes may be implemented to model bone development and different pathologies. Analysis could enrich current understanding and identify potential drug targets.

Source: New York Stem Cell Foundation 

Contact: David McKeon

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Stem Cell Proliferation and Differentiation Observed Within Hydrogel

CWRU research developing technique with promise to guide formation of complex tissues

Friday, 12 April 2013

Stem cells can be coaxed to grow into new bone or new cartilage better and faster when given the right molecular cues and room inside a water-loving gel, researchers at Case Western Reserve University show.

By creating a three-dimensional checkerboard — one with alternating highly connected and less connected spaces within the hydrogel — the team found adjusting the size of the micro-pattern could affect stem cell behaviours, such as proliferation and differentiation.

Inducing how and where stem cells grow — and into the right kind of cell in three dimensions — has proven a challenge to creating useful stem cell therapies. This technique holds promise for studying how physical, chemical and other influences affect cell behaviour in three-dimensions, and, ultimately, as a method to grow tissues for regenerative medicine applications.

"We think that control over local biomaterial properties may allow us to guide the formation of complex tissues," said Eben Alsberg, an associate professor of Biomedical Engineering at Case Western Reserve.

"With this system, we can regulate cell proliferation and cell-specific differentiation into, for example, bone-like or cartilage-like cells."

Oju Jeon, PhD, a postdoctoral researcher in Biomedical Engineering, pursued this work with Alsberg. Their work is described April 11, 2013 in the online edition of Advanced Functional Materials.

Hydrogels are hydrophilic three-dimensional networks of water-soluble polymers bonded, or cross-linked, to one another. Crosslinks increase rigidity and alter the porous structure inside the gel.

Alsberg and Jeon used a hydrogel of oxidized methacrylated alginate and an 8-arm poly(ethylene glycol) amine. A chemical reaction between the alginate and the poly(ethylene glycol) creates crosslinks that provide structure within the gel.

They tweaked the mix so that a second set of crosslinks forms when exposed to light. They used checkerboard masks to create patterns of alternating singly and doubly cross-linked spaces.

The spaces, which varied in size at 25, 50, 100 and 200 micrometres across, were evenly singly and doubly cross-linked.

Human stem cells isolated from fat tissue were encapsulated in the singly and doubly cross-linked regions. The doubly cross-linked spaces are comparatively cluttered with structures. The cells grew into clusters in the singly cross-linked regions, but remained mostly isolated in the doubly cross-linked regions.

The larger the spaces in the checkerboard, the larger the clusters grew.

Cells were cultivated in media that promote differentiation into either bone or cartilage.

In both the singly and doubly cross-linked spaces, stem cells increasingly differentiated according to the media composition as the space size increased. The results were more dramatic in the singly cross-linked spaces.

"Potentially, what's happening is the single cross-linked regions allow better nutrient transport and provide more space for cells to interact and, because it's less restrictive, there's space for new cells and matrix production," Alsberg said.

"Cluster formation, in turn, may influence proliferation and differentiation. Differences in mechanical properties between regions likely also regulate the cell behaviours."

The researchers are continuing to use micro-patterning to understand the influences of biomaterials on stem cell fate decisions. This approach may permit local control over cell behaviour and, ultimately, allow the engineering of complex tissues comprised of multiple cell types using a single stem cell source.

Source: Case Western Reserve University 

Contact: Kevin Mayhood

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Molecular Beacons Light Up Stem Cell Transformation

Molecular Beacons Light Up Stem Cell Transformation

Wednesday, 12 September 2012

A novel set of custom-designed "molecular beacons" allows scientists to monitor gene expression in living populations of stem cells as they turn into a specific tissue in real-time. The technology, which Brown University researchers describe in a new study, provides tissue engineers with a potentially powerful tool to discover what it may take to make stem cells transform into desired tissue cells more often and more quickly. That's a key goal in improving regenerative medicine treatments.

"We're not the inventors of molecular beacons but we have used it in a way that hasn't been done before, which is to do this in long-term culture and watch the same population change in a reliable and harmless way," said graduate student Hetal Desai, lead author of the paper published online Sept. 5, 2012, in the journal Tissue Engineering Part A.

A population of fat-derived stem cells

expresses the bone-specific COL1A1 gene.

Green fluorescence from a “molecular

beacon” shows increasing expression from

day nine, left, through day 11 and day 14.

By day 16, right, expression begins to wane.

Credit: Darling Lab/Brown University.

In their research, Desai and corresponding author Eric Darling, assistant professor of biology in the Department of Molecular Pharmacology, Physiology, and Biotechnology, designed their beacons to fluoresce when they bind to mRNA from three specific genes in fat-derived stem cells that are expressed only when the stem cells are transforming into bone cells.

Throughout 21 days of their development, the cells in the experiments remained alive and unfettered, Desai said, except that some populations received a chemical inducement toward becoming bone and others did not. Over those three weeks, the team watched the populations for the fluorescence of the beacons to see how many stem cells within each population were becoming bone and the timing of each gene expression milestone.

The beacons' fluorescence made it easy to see a distinct pattern in that timing. Expression of the gene ALPL peaked first in more than 90 percent of induced stem cells on day four, followed by about 85 percent expressing the gene COL1A1 on day 14. The last few days of the experiments saw an unmistakably sharp rise in expression of the gene BGLAP in more than 80 percent of the induced stem cells.

Each successive episode of gene expression ramped up from zero to the peak more quickly, the researchers noted, leading to a new hypothesis that the pace of the stem cell transformation, or "differentiation" in stem cell parlance, may become more synchronized in a population over time.

"If you could find a way to get them on this track earlier, you could get the differentiation faster," Darling said.

Meanwhile the stem cell populations that were not induced with bone-promoting chemicals, showed virtually no beacon fluorescence or expression of the genes, indicating that the beacons were truly indicators of steps along the transformation from stem cell to bone.

Beacons don't affect cells

Desai said the team took extra care to design beacons that would not alter the cells' development or functioning in any way. While the beacons do bind to messenger RNA produced in gene expression, for example, they do not require adding any genes to the stem cells' DNA, or expressing any special proteins, as many other fluorescence techniques do.

The team performed several experiments using the beacons in conventionally developing bone cells to make sure that they developed normally even as the beacons were in operation. While some scientists design RNA-based probes to purposely interfere with gene expression, this team had the opposite intent.

"You know that song 'Hold on Loosely but Don't Let Go?'" Desai said.

"That's sort of the theme song for this. There's a set of rules for interference RNA, and we essentially did the opposite of what those rules said you should do."

Toward quicker healing

Now that the beacons' performance in indicating milestones of stem cell differentiation has been demonstrated, Darling said, the technology can be applied to studying the process in a wide variety of cells and under a variety of other experimental conditions.

In the case of tissue engineering, he said, the beacons can aid experiments seeking to determine what conditions (inducing chemicals or otherwise) are most effective in converting the most stem cells to desired tissues most quickly. They could help tissue engineers learn the best timing for adding an inducing chemical. They might also provide a way for tissue engineers to identify and harvest only those cells that are converting to the desired tissue.

"They are becoming bone cells and if you enrich for them and you get rid of all the ones that aren't becoming bone cells, it stands to reason that you will have a better product at the end," Darling said.

More broadly, Darling added, molecular beacons are proving useful in a wide variety of gene expression studies.

"The reason we are looking at this technique is that we wanted something we could use on any cell, look at any gene and not affect that cell while we are looking at it," Darling said.

"If this is acting as we believe it is, we can really look at any gene that we want. It seems like a very versatile tool."

Source: Brown University 

Contact: David Orenstein

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A Better Way to Grow Bone

Fresh, purified fat stem cells grow bone faster and better

Tuesday, 12 June 2012

UCLA stem cell scientists purified a subset of stem cells found in fat tissue and made from them bone that was formed faster and was of higher quality than bone grown using traditional methods, a finding that may one day eliminate the need for painful bone grafts that use material taken from the patient during invasive procedures.

Adipose, or fat, tissue is thought to be an ideal source of cells called mesenchymal stem cells - capable of developing into bone, cartilage, muscle and other tissues - because they are plentiful and easily attained through procedures such as liposuction, said Dr. Chia Soo, vice chair for research at UCLA Plastic and Reconstructive Surgery. The co-senior authors on the project, Soo and Bruno Péault, are members of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA.

Traditionally, cells taken from fat had to be cultured for weeks to isolate the stem cells which could become bone, and their expansion increases risk of infection and genetic instability. A fresh, non-cultured cell composition called stromal vascular fraction (SVF) also is used to grow bone. However, SVF cells taken from adipose tissue are a highly heterogeneous population that includes cells that aren't capable of becoming bone.

Péault and Soo's team used a cell sorting machine to isolate and purify human perivascular stem cells (hPSC) from adipose tissue and showed that those cells worked far better than SVF cells in creating bone. They also showed that a growth factor called NELL-1, discovered by Dr. Kang Ting of the UCLA School of Dentistry, enhanced the bone formation in their animal model.

"People have shown that culture-derived cells could grow bone, but these are a fresh cell population and we didn't have to go through the culture process, which can take weeks," Soo said.

"The best bone graft is still your own bone, but that is in limited supply and sometimes not of good quality. What we show here is a faster and better way to create bone that could have clinical applications."

The study appears June 11, 2012 in the early online edition of the peer-reviewed journal Stem Cells Translational Medicine, a new journal that seeks to bridge stem cell research and clinical trials.

In the animal model, Soo and Péault's team put the hPSCs with NELL-1 in a muscle pouch, a place where bone is not normally grown. They then used X-rays to determine that the cells did indeed become bone.

"The purified human hPSCs formed significantly more bone in comparison to the SVF by all parameters," Soo said.

"And these cells are plentiful enough that patients with not much excess body fat can donate their own fat tissue."

Soo said if everything goes well, patients may one day have rapid access to high quality bone graft material by which doctors get their fat tissue, purify that into hPSCs and replace their own stem cells with NELL-1 back into the area where bone is required. The hPSC with NELL-1 could grow into bone inside the patient, eliminating the need for painful bone graft harvestings. The goal is for the process to isolate the hPSCs and add the NELL-1 with a matrix or scaffold to aid cell adhesion to take less than an hour, Soo said.

"Excitingly, recent studies have already demonstrated the utility of perivascular stem cells for regeneration of disparate tissue types, including skeletal muscle, lung and even myocardium," said Péault, a professor of orthopedic surgery

"Further studies will extend our findings and apply the robust osteogenic potential of hPSCs to the healing of bone defects."

Source: UCLA Health Science

Contact: Kim Irwin

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New York Stem Cell Foundation Scientist Grows Bone from Human Embryonic Stem Cells

New York Stem Cell Foundation Scientist Grows Bone from Human Embryonic Stem Cells

Monday, 14 May 2012

Dr. Darja Marolt, an Investigator at The New York Stem Cell Foundation (NYSCF) Laboratory, is lead author on a study showing that human embryonic stem cells can be used to grow bone tissue grafts for use in research and potential therapeutic application. Dr. Marolt conducted this research as a post-doctoral NYSCF – Druckenmiller Fellow at Columbia University in the laboratory of Dr. Gordana Vunjak-Novakovic.

The study is the first example of using bone cell progenitors derived from human embryonic stem cells to grow compact bone tissue in quantities large enough to repair centimetre-sized defects. When implanted in mice and studied over time, the implanted bone tissue supported blood vessel ingrowth, and continued development of normal bone structure, without demonstrating any incidence of tumour growth.

Dr. Marolt's work is a significant step forward in using pluripotent stem cells to repair and replace bone tissue in patients. Bone replacement therapies are relevant in treating patients with a variety of conditions, including wounded military personnel, patients with birth defects, or patients who have suffered other traumatic injury.

Since conducting this work as proof of principle at Columbia University, Dr. Marolt has continued to build upon this research as an Investigator in the NYSCF Laboratory, developing bone grafts from induced pluripotent stem (iPS) cells. iPS cells are similar to embryonic stem cells in that they can also give rise to nearly any type of cell in the body, but iPS cells are produced from adult cells and as such are individualized to each patient. By using iPS cells rather than embryonic stem cells to engineer tissue, Dr. Marolt hopes to develop personalized bone grafts that will avoid immune rejection and other implant complications.

Source: New York Stem Cell Foundation

Contact: David McKeon

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Influencing Stem Cell Fate

New screening method helps scientists identify key information rapidly 
Tuesday, 06 March 2012

Northwestern University scientists have developed a powerful analytical method that they have used to direct stem cell differentiation. Out of millions of possibilities, they rapidly identified the chemical and physical structures that can cue stem cells to become osteocytes, cells found in mature bone.

Researchers can use the method, called nanocombinatorics, to build enormous libraries of physical structures varying in size from a few nanometers to many micrometers for addressing problems within and outside biology.

Those in the fields of chemistry, materials engineering and nanotechnology could use this invaluable tool to assess which chemical and physical structures — including size, shape and composition — work best for a desired process or function.

Nanocombinatorics holds promise for screening catalysts for energy conversion, understanding properties conferred by nanostructures, identifying active molecules for drug discovery or even optimizing materials for tissue regeneration, among other applications.

Details of the method and proof of concept is published in the Proceedings of the National Academy of Sciences.

"With further development, researchers might be able to use this approach to prepare cells of any lineage on command," said Chad A. Mirkin, who led the work.

"Insight into such a process is important for understanding cancer development and for developing novel cancer treatment methodologies."

Mirkin is the George B. Rathmann Professor of Chemistry in the Weinberg College of Arts and Sciences and professor of medicine, chemical and biological engineering, biomedical engineering and materials science and engineering. He also is the director of Northwestern's International Institute for Nanotechnology (IIN).

The new analytical method utilizes a technique invented at Northwestern called polymer pen lithography, where basically a rubber stamp having as many as 11 million sharp pyramids is mounted on a transparent glass backing and precisely controlled by an atomic force microscope to generate desired patterns on a surface. Each pyramid — a polymeric pen — is coated with molecules for a particular purpose.

In this work, the researchers used molecules that bind proteins found in the natural cell environment, such as fibronectin, which could then be attached onto a substrate in various patterns. (Fibronectin is a protein that mediates cell adhesion.) The team rapidly prepared millions of textured features over a large area, which they call a library. The library consisted of approximately 10,000 fibronectin patterns having as many as 25 million features ranging in size from a couple hundred nanometers to several micrometers.

To make these surfaces, they intentionally tilt the stamp and its array of pens as the stamp is brought down onto the substrate, each pen delivering a spot of molecules that could then bind fibronectin. The tilt results in different amounts of pressure on the polymeric pens, which dictates the feature size of each spot. Because the pressure varies across a broad range, so does the feature size.

The researchers then introduced mesenchymal stem cells, or MSCs, to the library of millions of fibronectin features. (MSCs are multipotent stem cells that can differentiate into a variety of other cell types.)

"We let the cells sample the library and watched what happened," Mirkin said.

He and his team found areas with stem cell differentiation and areas with none. Nano-scale features, particularly protein spots that were 300 nanometers in diameter, were more likely to lead to bone-like cells than larger micron-scale features.

The researchers next built a library made up of only 300-nanometer dots and introduced stem cells. Almost all of the cells became bone-like.

"We want to make stem cells go down a predetermined path — to make bone cells instead of nerve or muscle cells," Mirkin said.

"Starting with millions of possibilities, we quickly zeroed in on the pattern of protein features that best directed the cells to become osteocytes."

This stem cell differentiation was accomplished without the use of additional chemical cues (beyond the proteins in the patterns). The transition from stem cell to osteocyte was dictated solely by the physical cues of the patterned structures. And the researchers demonstrated better control over stem cell differentiation than chemical reagent methods currently used.

"It doesn't stop with stem cells," Mirkin said.

"Scientists can use nanocombinatorics to build libraries of structures that vary in shape, size and distance between particles and determine the best structures for controlling important events, like speeding up a catalytic reaction."

Source: Northwestern University

Contact: Megan Fellman
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New Method for Producing Precursor of Neurons, Bone and Other Important Tissues from Stem Cells

New Method for Producing Precursor of Neurons, Bone and Other Important Tissues from Stem Cells
Tuesday, 15 November 2011

In principle, stem cells offer scientists the opportunity to create specific cell types — such as nerve or heart cells — to replace tissues damaged by age or disease. In reality, coaxing stem cells to become the desired cell type can be challenging, to say the least.

University of Georgia Researcher Stephen

Dalton has developed a method that – in a

single step – directs undifferentiated, or

pluripotent, stem cells to become neural

crest cells, which are the precursors of

bone cells, smooth muscle cells and

neurons. Credit: University of Georgia.

In a paper published this week in the journal Proceedings of the National Academy of Sciences, however, scientists at the University of Georgia describe a method that — in a single step — directs undifferentiated, or pluripotent, stem cells to become neural crest cells, which are the precursors of bone cells, smooth muscle cells and neurons.

"Now that we have methods for efficiently making neural crest stem cells, we can start to use them to better understand human diseases," said lead author Stephen Dalton, Georgia Research Alliance Eminent Scholar of Molecular Biology and professor of cellular biology in the UGA Franklin College of Arts and Sciences.

"The cells can be also used in drug discovery and potentially in cell therapy, which involves the transplantation of cells."

The process by which a pluripotent stem cell, which has the ability to become any type of cell in the body, becomes a specific cell type is orchestrated by signaling molecules that activate specific "decision" pathways within cells. As a stem cell divides, various combinations of these molecules at different points during its development narrow its possible outcomes so that it ultimately becomes one type of cell, a skin cell, for example, instead of, say, a muscle cell.

Until now, creating neural crest cells relied on a mix of science and serendipity. Scientists would take undifferentiated stem cells and direct them to become a related but different cell type known as neural progenitor cells. The neural crest cells they really wanted would often show up as contaminants, which scientists would then isolate and use for their studies. Not surprisingly, the process was laborious, time consuming, expensive and sub-optimal for clinical applications.

The method developed by Dalton and a post-doctoral researcher in his laboratory, Laura Menendez, involves bathing cells in a solution of small molecules that suppress one pathway, known as Smad, and amplify another, known as Wnt. The inhibition of Smad is used in the process that creates the related neural progenitor cells, which suggested that the pathway could also play a role in the development of neural crest cells. Observing that the Wnt pathway is highly active in the formation of the neural crest in developing organisms led Dalton and his team to suspect that activating the pathway could give them the cells they needed. After testing various concentrations of the signaling molecules and determining the optimal time to deliver them, the scientists discovered that they could create neural crest cells with little or no contamination of other cell types.

The new method cuts the amount of time required to generate the cells by approximately one-half. Dalton said another benefit is that instead of using costly large-molecule compounds known as growth factors and cytokines to direct the differentiation of cells, his method uses inexpensive small molecules that have a much higher degree of consistency.

With their newly developed ability to create neural crest cells, Dalton and his team are working to gain a deeper understanding of normal development — as well as what goes wrong in devastating diseases that are associated with neural crest defects, such as Hirschsprung's disease, DiGeorge syndrome and Treacher-Collins syndrome.

The cells that Dalton and his team have created are self-renewing, which means that multiple additional cells can be created from an initial batch. Having large numbers of cells that can easily be stored is essential for drug testing as well as for cell transplantation, the holy grail of stem cell science.

"Now that we've worked out ways for making the cells, we've greatly enhanced their potential in disease modeling and regenerative medicine," Dalton said.

Source: University of Georgia

Contact: Stephen Dalton
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

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Synthetic Bone Graft Recruits Stem Cells for Faster Bone Healing

Synthetic Bone Graft Recruits Stem Cells for Faster Bone Healing
Tuesday, 03 August 2010

Scientists have developed a material for bone grafts that could one day replace the 'gold standard' natural bone implants.

A new study shows how particles of a ceramic called calcium phosphate have the ability to stimulate promising bone regrowth by attracting stem cells and 'growth factors' to promote healing and the integration of the grafted tissue.

"The rate of bone repair we see with these materials rivals that of traditional grafts using a patients' own bone," said Professor Joost de Bruijn from the School of Engineering and Materials Science at Queen Mary, University of London.

"And what sets it apart from other synthetic graft substitutes is its ability to attract stem cells and the body's natural growth factors, which coincide to form new, strong, natural bone around an artificial graft."

The researchers tested natural bone grafts against ceramic particles with varied structural and chemical properties. They found that micro-porous ceramic particles composed of calcium phosphate, the primary component of bone ash, induced stem cells to develop into bone cells in the test tube and stimulated bone growth in live tissue in mice, dogs and sheep.

Bone injuries packed with the ceramic particles healed similarly to implants constructed from the animals' own bone, reports Professor de Bruijn along with collaborators from the University of Twente, Netherlands, in the journal Proceedings of the National Academy of Sciences. The study also shows how it also matches a commercially available product that contains artificial growth factors and has the undesirable side-effect of causing bone fragments to form in nearby soft tissue, such as muscle.

Although the researchers have not yet identified the mechanism that drives bone growth in the synthetic implants, they note that variations in the ceramic material's chemistry, micro-porosity, microstructure, and degradation influence the graft's performance.

The study suggests that biomaterials-based bone grafts can manipulate cell behaviour in order to repair injury, and one day may be used to repair bone injuries in humans.

Source: Queen Mary, University of London
Contact: Simon Levey

Reference:
Osteoinductive ceramics as a synthetic alternative to autologous bone grafting
Huipin Yuan, Hugo Fernandes, Pamela Habibovic, Jan de Boer, Ana Barradas, Ad de Ruiter, William Walsh, Clemens van Blitterswijk, and Joost de Bruijn
PNAS doi: 10.1073/pnas.1003600107
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

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