Researchers Grow Cardiac Tissue on 'Spider Silk' Substrate

Researchers Grow Cardiac Tissue on 'Spider Silk' Substrate

Saturday, 11 April 2015

Genetically engineered fibres of the protein spidroin, which is the construction material for spider webs, has proven to be a perfect substrate for cultivating heart tissue cells, Moscow Institute of Physics and Technology researchers found. They discuss their findings in an article that has recently come out in the journal PLOS ONE.

These are heart tissue cells grown on a matrix,

stained with fluorescent markers. Credit: ©

Alexander Teplenin et al./PLOS ONE.

The cultivation of organs and tissues from a patient's cells is the leading edge of medical research - regenerative methods can solve the problem of transplant rejection. However, it’s quite a challenge to find a suitable frame, or substrate, to grow cells on. The material should be non-toxic and elastic and should not be rejected by the body or impede cell growth. A group of researchers led by Professor Konstantin Agladze, who heads the Laboratory of the Biophysics of Excitable Systems at MIPT, works on cardiac tissue engineering. The group has been cultivating fully functional cardiac tissues, able to contract and conduct excitation waves, from cells called cardiomyocytes. Previously, the group used synthetic polymeric nano-fibres but recently decided to assay another material – electro-spun fibres of spidroin, the cobweb protein. Cobweb strands are incredibly light and durable. They're five times stronger than steel, twice more elastic than nylon, and are capable of stretching a third of their length. The structure of spidroin molecules that make up cobweb drag lines is similar to that of the silk protein, fibroin, but is much more durable.

This is a spidroin fibre matrix captured with a

microscope. Credit: Alexander Teplenin et al./

PLOS ONE.

Researchers would normally use artificial spidroin fibre matrices as a substrate to grow implants like bones, tendons and cartilages, as well as dressings. Professor Agladze's team decided to find out whether a spidroin substrate derived from genetically modified yeast cells can serve to grow cardiac cells.

For this purpose, they seeded isolated neonatal rat cardiomyocytes on fibre matrices. During the experiment, the researchers monitored the growth of the cells and tested their contractibility and the ability to conduct electric impulses, which are the main features of normal cardiac tissue.

The monitoring, carried out with the help of a microscope and fluorescent markers, showed that within three to five days a layer of cells formed on the substrate that were able to contract synchronously and conduct electrical impulses just like the tissue of a living heart would.

"We can answer positively all questions we put at the beginning of this research project," Professor Agladze says.

"Cardiac tissue cells successfully adhere to the substrate of recombinant spidroin; they grow forming layers and are fully functional, which means they can contract co-ordinately."

Source: Moscow Institute of Physics and Technology

Contact: Stanislav Goryachev

Reference:

Functional Analysis of the Engineered Cardiac Tissue Grown on Recombinant Spidroin Fiber Meshes

Alexander Teplenin, Anna Krasheninnikova, Nadezhda Agladze, Konstantin Sidoruk, Olga Agapova, Igor Agapov, Vladimir Bogush, Konstantin Agladze

PLOS ONE, March 23, 2015, DOI:10.1371/journal.pone.0121155

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Researchers Grow Functional Tissue-engineered Intestine from Human Cells

Regenerative medicine technique brings surgeons one step closer to helping patients

Thursday, 08 January 2015

This is the principal investigator Tracy C.

Grikscheit of The Saban Research Institute of

Children's Hospital Los Angeles. Credit:

Children's Hospital Los Angeles. 

A new study by researchers at Children's Hospital Los Angeles has shown that tissue-engineered small intestine grown from human cells replicates key aspects of a functioning human intestine. The tissue-engineered small intestine they developed contains important elements of the mucosal lining and support structures, including the ability to absorb sugars, and even tiny or ultra-structural components like cellular connections.

Published online January 8 by the American Journal of Physiology: GI & Liver, the work brings surgeons one step closer to helping human patients using this regenerative medicine technique.

Tissue-engineered small intestine (TESI) grows from stem cells contained in the intestine and offers a promising treatment for short bowel syndrome (SBS), a major cause of intestinal failure, particularly in premature babies and newborns with congenital intestinal anomalies. TESI may one day offer a therapeutic alternative to the current standard treatment, which is intestinal transplantation, and could potentially solve its largest challenges - donor shortage and the need for lifelong immunosuppression.

Tracy C. Grikscheit, MD, a principal investigator in The Saban Research Institute of CHLA and its Developmental Biology and Regenerative Medicine program, is also a pediatric surgeon at Children's Hospital Los Angeles and an assistant professor of surgery at the Keck School of Medicine of the University of Southern California.

Grikscheit aims to help her most vulnerable young patients, including babies who are born prematurely and develop a devastating disease called necrotizing enterocolitis (NEC), where life-threatening intestinal damage requires removal of large portions of the small intestine. Without enough intestinal length, the babies are dependent on intravenous feeding, which is costly and may cause liver damage. NEC and other contributors to intestinal failure occur in 24.5 out of 100,000 live births, and the incidence of SBS is increasing. Nearly a third of patients die within five years.

CHLA scientists had previously shown that TESI could be generated from human small intestine donor tissue implanted into immunocompromised mice. However, in those initial studies - published in July 2011 in the biomedical journal Tissue Engineering, Part A - only basic components of the intestine were identified. For clinical relevance, it remained necessary to more fully investigate intact components of function such as the ability to form a healthy barrier while still absorbing nutrition or specific mechanisms of electrolyte exchange.

The new study determined that mouse TESI is highly similar to the TESI derived from human cells, and that both contain important building blocks such as the stem and progenitor cells that will continue to regenerate the intestine as a living tissue replacement. And these cells are found within the engineered tissue in specific locations and in close proximity to other specialized cells that are known to be necessary in healthy human intestine for a fully functioning organ.

"We have shown that we can grow tissue-engineered small intestine that is more complex than other stem cell or progenitor cell models that are currently used to study intestinal regeneration and disease, and proven it to be fully functional as it develops from human cells," said Grikscheit.

"Demonstrating the functional capacity of this tissue-engineered intestine is a necessary milestone on our path toward one day helping patients with intestinal failure."

Source: Children's Hospital Los Angeles

Contact: Debra Kain

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Researchers Coax Human Embryonic Stem Cells to Organize Themselves Using Geometry

Using Geometry, Researchers Coax Human Embryonic Stem Cells to Organize Themselves

Tuesday, 01 July 2014

About seven days after conception, something remarkable occurs in the clump of cells that will eventually become a new human being. They start to specialize. They take on characteristics that begin to hint at their ultimate fate as part of the skin, brain, muscle or any of the roughly 200 cell types that exist in people, and they start to form distinct layers.

Forty-two hours after they began to differentiate,

embryonic cells are clearly segregating into

endoderm (red), mesoderm (blue) and ectoderm

(black). Researchers say the key to achieving this

patterning in culture is confining the colonies

geometrically. Credit: Brivanlou Lab, the

Rockefeller University. 

Although scientists have studied this process in animals, and have tried to coax human embryonic stem cells into taking shape by flooding them with chemical signals, until now the process has not been successfully replicated in the lab. But researchers led by Ali Brivanlou, Robert and Harriet Heilbrunn Professor and head of the Laboratory of Stem Cell Biology and Molecular Embryology at The Rockefeller University, have done it, and it turns out that the missing ingredient is geometrical, not chemical.

"Understanding what happens in this moment, when individual members of this mass of embryonic stem cells begin to specialize for the very first time and organize themselves into layers, will be a key to harnessing the promise of regenerative medicine," Brivanlou says.

"It brings us closer to the possibility of replacement organs grown in petri dishes and wounds that can be swiftly healed."

In the uterus, human embryonic stem cells receive chemical cues from the surrounding tissue that signal them to begin forming layers — a process called gastrulation. Cells in the centre begin to form ectoderm, the brain and skin of the embryo, while those migrating to the outside become mesoderm and endoderm, destined to become muscle and blood and many of the major organs, respectively.

Brivanlou and his colleagues, including postdocs Aryeh Warmflash and Benoit Sorre as well as Eric Siggia, Viola Ward Brinning and Elbert Calhoun Brinning Professor and head of the Laboratory of Theoretical Condensed Matter Physics, confined human embryonic stem cells originally derived at Rockefeller to tiny circular patterns on glass plates that had been chemically treated to form "micropatterns" that prevent the colonies from expanding outside a specific radius. When the researchers introduced chemical signals spurring the cells to begin gastrulation, they found the colonies that were geometrically confined in this way proceeded to form endoderm, mesoderm and ectoderm and began to organize themselves just as they would have under natural conditions. Cells that were not confined did not.

By monitoring specific molecular pathways the human cells use to communicate with one another to form patterns during gastrulation — something that was not previously possible because of the lack of a suitable laboratory model — the researchers also learned how specific inhibitory signals generated in response to the initial chemical cues function to prevent the cells within a colony from all following the same developmental path.

"At the fundamental level, what we have developed is a new model to explore how human embryonic stem cells first differentiate into separate populations with a very reproducible spatial order just as in an embryo," says Warmflash.

"We can now follow individual cells in real time in order to find out what makes them specialize, and we can begin to ask questions about the underlying genetics of this process."

The research also has direct implications for biologists working to create "pure" populations of specific cells, or engineered tissues consisting of multiple cell types, for use in medical treatments.

"These cells have a powerful intrinsic tendency to form patterns as they develop," Warmflash says.

"Varying the geometry of the colonies may turn out to be an important tool that can be used to guide stem cells to form specific cell types or tissues."

Source: Rockefeller University 

Contact: Zach Veilleux

Reference:

A method to recapitulate early embryonic spatial patterning in human embryonic stem cells

Aryeh Warmflash, Benoit Sorre, Fred Etoc, Eric D. Siggia and Ali H. Brivanlou

Nature Methods online: June 29, 2014, doi:10.1038/nmeth.3016

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An Essential Step Toward Printing Living Tissues

New method enables scientists to print tissue constructs with blood vessels

Wednesday, 19 February 2014

A new 3-D printing method developed by Wyss

Core Faculty member Jennifer Lewis and her

team uses multiple print heads and customized

"inks" to create complex living tissue constructs,

complete with tiny blood vessels. To see how it

works, visit http://wyss.harvard.edu/. Credit: 

Wyss Institute and Harvard School of Engineering

and Applied Sciences. 

A new bio-printing method developed at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard School of Engineering and Applied Sciences (SEAS) creates intricately patterned 3D-tissue constructs with multiple types of cells and tiny blood vessels. The work represents a major step toward a longstanding goal of tissue engineers: creating human tissue constructs realistic enough to test drug safety and effectiveness.

The method also represents an early but important step toward building fully functional replacements for injured or diseased tissue that can be designed from CAT scan data using computer-aided design (CAD), printed in 3D at the push of a button, and used by surgeons to repair or replace damaged tissue.

"This is the foundational step toward creating 3D living tissue," said Jennifer Lewis, Ph.D., senior author of the study, who is a Core Faculty Member of the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard SEAS. Along with lead author David Kolesky, a graduate student in SEAS and the Wyss Institute, her team reported the results February 18 in the journal Advanced Materials.

Tissue engineers have tried for years to produce lab-grown vascularized human tissues robust enough to serve as replacements for damaged human tissue. Others have printed human tissue before, but they have been limited to thin slices of tissue about a third as thick as a dime. When scientists try to print thicker layers of tissue, cells on the interior starve for oxygen and nutrients, and have no good way of removing carbon dioxide and other waste. So they suffocate and die.

Nature gets around this problem by permeating tissue with a network of tiny, thin-walled blood vessels that nourish the tissue and remove waste, so Kolesky and Lewis set out to mimic this key function.

3D printing excels at creating intricately detailed 3D structures, typically from inert materials like plastic or metal. In the past, Lewis and her team have pioneered a broad range of novel inks that solidify into materials with useful electrical and mechanical properties. These inks enable 3D printing to go beyond form to embed functionality.

To print 3D tissue constructs with a predefined pattern, the researchers needed functional inks with useful biological properties, so they developed several "bio-inks" — tissue-friendly inks containing key ingredients of living tissues. One ink contained extracellular matrix, the biological material that knits cells into tissues. A second ink contained both extracellular matrix and living cells.

To create blood vessels, they developed a third ink with an unusual property: it melts as it cools, rather than as it warms. This allowed the scientists to first print an interconnected network of filaments, then melt them by chilling the material and suction the liquid out to create a network of hollow tubes, or vessels.

The Harvard team then road-tested the method to assess its power and versatility. They printed 3D tissue constructs with a variety of architectures, culminating in an intricately patterned construct containing blood vessels and three different types of cells – a structure approaching the complexity of solid tissues.

Moreover, when they injected human endothelial cells into the vascular network, those cells regrew the blood-vessel lining. Keeping cells alive and growing in the tissue construct represents an important step toward printing human tissues.

"Ideally, we want biology to do as much of the job of as possible," Lewis said.

Lewis and her team are now focused on creating functional 3D tissues that are realistic enough to screen drugs for safety and effectiveness.

"That's where the immediate potential for impact is," Lewis said.

Scientists could also use the printed tissue constructs to shed light on activities of living tissue that require complex architecture, such as wound healing, blood vessel growth, or tumour development.

"Tissue engineers have been waiting for a method like this," said Don Ingber, M.D., Ph.D., Wyss Institute Founding Director.

"The ability to form functional vascular networks in 3D tissues before they are implanted not only enables thicker tissues to be formed, it also raises the possibility of surgically connecting these networks to the natural vasculature to promote immediate perfusion of the implanted tissue, which should greatly increase their engraftment and survival".

Source: Wyss Institute for Biologically Inspired Engineering at Harvard 

Contact: Dan Ferber

Reference:

3D Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs

David B. Kolesky, Ryan L. Truby, A. Sydney Gladman, Travis A. Busbee, Kimberly A. Homan and Jennifer A. Lewis

Advanced Materials, 18 FEB 2014, DOI: 10.1002/adma.201305506

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New 3D Method used to Grow Miniature Pancreas

New 3D Method used to Grow Miniature Pancreas

Tuesday, 15 October 2013

An international team of researchers from the University of Copenhagen have successfully developed an innovative 3D method to grow miniature pancreas from progenitor cells. The future goal is to use this model to help in the fight against diabetes. The research results have just been published in the scientific journal Development.

The new method allows the cell material from

mice to grow vividly in picturesque tree-like

structures. Credit: The Danish Stem Cell Center. 

Professor Anne Grapin-Botton and her team at the Danish Stem Cell Centre have developed a three-dimensional culture method which enables the efficient expansion of pancreatic cells. The new method allows the cell material from mice to grow vividly in picturesque tree-like structures. The method offers huge long term potential in producing miniature human pancreas from human stem cells. These human miniature organs would be valuable as models to test new drugs fast and effective – and without the use of animal models.

"The new method allows the cell material to take a three-dimensional shape enabling them to multiply more freely. It’s like a plant where you use effective fertilizer, think of the laboratory like a garden and the scientist being the gardener,” says Anne Grapin-Botton.

Social cells

The cells do not thrive and develop if they are alone, and a minimum of four pancreatic cells close together is required for subsequent organoid development.

“We found that the cells of the pancreas develop better in a gel in three-dimensions than when they are attached and flattened at the bottom of a culture plate. Under optimal conditions, the initial clusters of a few cells have proliferated into 40,000 cells within a week. After growing a lot, they transform into cells that make either digestive enzymes or hormones like insulin and they self-organize into branched pancreatic organoids that are amazingly similar to the pancreas,” adds Anne Grapin-Botton.

The scientists used this system to discover that the cells of the pancreas are sensitive to their physical environment such as the stiffness of the gel and to contact with other cells.

Pancreas and diabetes connection

An effective cellular therapy for diabetes is dependent on the production of sufficient quantities of functional beta-cells. Recent studies have enabled the production of pancreatic precursors but efforts to expand these cells and differentiate them into insulin-producing beta-cells have proved a challenge.

“We think this is an important step towards the production of cells for diabetes therapy, both to produce mini-organs for drug testing and insulin-producing cells as spare parts. We show that the pancreatic cells care not only about how you feed them but need to be grown in the right physical environment. We are now trying to adapt this method to human stem cells,” adds Anne Grapin-Botton.

Source: The Danish Stem Cell Center (DanStem)

Contact: Stine Rasmussen

Reference:

Artificial three-dimensional niches deconstruct pancreas development in vitro 

Chiara Greggio, Filippo De Franceschi, Manuel Figueiredo-Larsen,Samy Gobaa, Adrian Ranga, Henrik Semb, Matthias Lutolf and Anne Grapin-Botton

Development, October 15, 2013, Vol. 140, Issue 21, 4452-4462, doi: 10.1242/dev.096628

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An Organized Approach to 3-D Tissue Engineering

IBN's novel technique brings researchers closer to viable organ implants

Wednesday, 21 August 2013

Researchers at the Institute of Bioengineering and Nanotechnology (IBN) have developed a simple method of organizing cells and their microenvironments in hydrogel fibres. Their unique technology provides a feasible template for assembling complex structures, such as liver and fat tissues, as described in their recent publication in Nature Communications1.

According to IBN Executive Director Professor Jackie Y. Ying:

"Our tissue engineering approach gives researchers great control and flexibility over the arrangement of individual cell types, making it possible to engineer pre-vascularized tissue constructs easily. This innovation brings us a step closer toward developing viable tissue or organ replacements."

IBN Team Leader and Principal Research Scientist, Dr Andrew Wan, elaborated:

"Critical to the success of an implant is its ability to rapidly integrate with the patient's circulatory system. This is essential for the survival of cells within the implant, as it would ensure timely access to oxygen and essential nutrients, as well as the removal of metabolic waste products. Integration would also facilitate signalling between the cells and blood vessels, which is important for tissue development."

Tissues designed with pre-formed vascular networks are known to promote rapid vascular integration with the host. Generally, pre-vascularization has been achieved by seeding or encapsulating endothelial cells, which line the interior surfaces of blood vessels, with other cell types. In many of these approaches, the eventual distribution of vessels within a thick structure is reliant on in vitro cellular infiltration and self-organization of the cell mixture. These are slow processes, often leading to a non-uniform network of vessels within the tissue. As vascular self-assembly requires a large concentration of endothelial cells, this method also severely restricts the number of other cells that may be co-cultured.

Alternatively, scientists have attempted to direct the distribution of newly formed vessels via three-dimensional (3D) co-patterning of endothelial cells with other cell types in a hydrogel. This approach allows large concentrations of endothelial cells to be positioned in specific regions within the tissue, leaving the rest of the construct available for other cell types. The hydrogel also acts as a reservoir of nutrients for the encapsulated cells. However, co-patterning multiple cell types within a hydrogel is not easy. Conventional techniques, such as micro-moulding and organ printing, are limited by slow cell assembly, large volumes of cell suspension, complicated multi-step processes and expensive instruments. These factors also make it difficult to scale up the production of implantable 3D cell-patterned constructs. To date, these approaches have been unsuccessful in achieving vascularization and mass transport through thick engineered tissues.

To overcome these limitations, IBN researchers have used interfacial polyelectrolyte complexion (IPC) fibre assembly, a unique cell patterning technology patented by IBN, to produce cell-laden hydrogel fibres under aqueous conditions at room temperature. Unlike other methods, IBN's novel technique allows researchers to incorporate different cell types separately into different fibres, and these cell-laden fibres may then be assembled into more complex constructs with hierarchical tissue structures. In addition, IBN researchers are able to tailor the microenvironment for each cell type for optimal functionality by incorporating the appropriate factors, e.g. proteins, into the fibres. Using IPC fibre assembly, the researchers have engineered an endothelial vessel network, as well as cell-patterned fat and liver tissue constructs, which have successfully integrated with the host circulatory system in a mouse model and produced vascularized tissues.

The IBN researchers are now working on applying and further developing their technology toward engineering functional tissues and clinical applications.

Source: Agency for Science, Technology and Research (A*STAR), Singapore

Contact: Nidyah Sani

Reference:

Patterned Prevascularized Tissue Constructs by Assembly of Polyelectrolyte Hydrogel Fibers 

M. F. Leong, J. K. C. Toh, D. Chan, K. Narayanan, H. F. Lu, T. C. Lim, A. C. A. Wan and J. Y. Ying

Nature Communications, (2013) DOI: 10.1038/ncomms3353.

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Researchers Create the Inner Ear from Stem Cells

Researchers Create the Inner Ear from Stem Cells

Thursday, 11 July 2013

Indiana University scientists have transformed mouse embryonic stem cells into key structures of the inner ear. The discovery provides new insights into the sensory organ's developmental process and sets the stage for laboratory models of disease, drug discovery and potential treatments for hearing loss and balance disorders.

A research team led by Eri Hashino, Ph.D., Ruth C. Holton Professor of Otolaryngology at Indiana University School of Medicine, reported that by using a three-dimensional cell culture method, they were able to coax stem cells to develop into inner-ear sensory epithelia - containing hair cells, supporting cells and neurons - that detect sound, head movements and gravity. The research was reportedly online Wednesday in the journal Nature.

Stem cell-derived sensory hair cells are in red

with hair bundles in green. Cellular nuclei are

shown in blue. Credit: IU Communications.

Previous attempts to "grow" inner-ear hair cells in standard cell culture systems have worked poorly in part because necessary cues to develop hair bundles - a hallmark of sensory hair cells and a structure critically important for detecting auditory or vestibular signals - are lacking in the flat cell-culture dish. But, Dr. Hashino said, the team determined that the cells needed to be suspended as aggregates in a specialized culture medium, which provided an environment more like that found in the body during early development.

The team mimicked the early development process with a precisely timed use of several small molecules that prompted the stem cells to differentiate, from one stage to the next, into precursors of the inner ear. But the three-dimensional suspension also provided important mechanical cues, such as the tension from the pull of cells on each other, said Karl R. Koehler, B.A., the paper's first author and a graduate student in the medical neuroscience graduate program at the IU School of Medicine.

"The three-dimensional culture allows the cells to self-organize into complex tissues using mechanical cues that are found during embryonic development," Koehler said.

"We were surprised to see that once stem cells are guided to become inner-ear precursors and placed in 3-D culture, these cells behave as if they knew not only how to become different cell types in the inner ear, but also how to self-organize into a pattern remarkably similar to the native inner ear," Dr. Hashino said.

Karl Koehler and Eri Hashino, Ph.D. Credit:

IU Communications.

"Our initial goal was to make inner-ear precursors in culture, but when we did testing we found thousands of hair cells in a culture dish."

Electrophysiology testing further proved that those hair cells generated from stem cells were functional, and were the type that sense gravity and motion. Moreover, neurons like those that normally link the inner-ear cells to the brain had also developed in the cell culture and were connected to the hair cells.

Additional research is needed to determine how inner-ear cells involved in auditory sensing might be developed, as well as how these processes can be applied to develop human inner-ear cells, the researchers said.

However, the work opens a door to better understanding of the inner-ear development process as well as creation of models for new drug development or cellular therapy to treat inner-ear disorders, they said.

Source: Indiana University

Contact: Eric Schoch

Reference:

Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture

Karl R. Koehler, Andrew M. Mikosz, Andrei I. Molosh, Dharmeshkumar Patel & Eri Hashino

Nature (2013), doi:10.1038/nature12298

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Micro-particles Create Localized Control of Stem Cell Differentiation

Engineering stem cells

Wednesday, 10 July 2013

Before scientists and engineers can realize the dream of using stem cells to create replacements for worn out organs and battle damaged body parts, they'll have to develop ways to grow complex three-dimensional structures in large volumes and at costs that won't bankrupt health care systems.

Researchers are now reporting advances in these areas by using gelatine-based microparticles to deliver growth factors to specific areas of embryoid bodies, aggregates of differentiating stem cells. The localized delivery technique provides spatial control of cell differentiation within the cultures, potentially enabling the creation of complex three-dimensional tissues. The local control also dramatically reduces the amount of growth factor required, an important cost consideration for manufacturing stem cells for therapeutic applications.

Georgia Tech/Emory University Associate

Professor Todd McDevitt and graduate student

Anh Nguyen make microparticles to be used

for delivering growth factors to stem cells.

Credit: Georgia Tech Photo: Rob Felt.

The microparticle technique, which was demonstrated in pluripotent mouse embryonic cells, also offers better control over the kinetics of cell differentiation by delivering molecules that can either promote or inhibit the process. Based on research sponsored by the National Institutes of Health and the National Science Foundation, the developments were reported online July 1 in the journal Biomaterials and were presented at the 11th Annual International Society for Stem Cell Research meeting held in Boston June 12-15, 2013 .

"By trapping these growth factors within microparticle materials first, we are concentrating the signal they provide to the stem cells," said Todd McDevitt, an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

"We can then put the microparticle materials physically inside the multicellular aggregate system that we use for differentiation of the stem cells. We have good evidence that this technique can work, and that we can use it to provide advantages in several different areas."

The differentiation of stem cells is largely controlled by external cues, including morphogenic growth factors, in the three-dimensional environment that surrounds the cells. Most stem cell researchers currently deliver the growth factors into liquid solutions surrounding the stem cell cultures with a goal of creating homogenous cultures of cells. Delivering the growth factors from microparticles, however, provides better control of the spatial and temporal presentation of the molecules that govern the growth and differentiation of the stem cells, potentially allowing formation of heterogeneous structures formed from different cells.

Groups of stem cells stick together as they develop, forming multicellular aggregates that form spheroids as they grow. The researchers took advantage of that by driving microparticles containing growth factor BMP4 or noggin – which inhibits BMP4 signalling – into layers of stem cells using centrifugation. When the cell aggregates formed, the microparticles became trapped inside.

The researchers used confocal imaging and flow cytometry to observe the differentiation process and found that growth factors in the microparticles directed the cells toward mesoderm and ectoderm tissues just as they do in solution-based techniques. But because the BMP4 and noggin molecules were directly in contact with the cells, much less growth factor was needed to spur the differentiation – approximately 12 times less than what would be required by conventional solution-based techniques.

"One of the major advantages, in a practical sense, is that we are using much less growth factor," said McDevitt, who is also director of the Stem Cell Engineering Center at Georgia Tech.

"From a bioprocessing standpoint, a lot of the cost involved in making stem cell products is related to the cost of the molecules that must be added to make the stem cells differentiate."

Beyond more focused signalling, the microparticles also provided a localized control not available through any other technique. That allowed the researchers to create spatial differences in the aggregates – a possible first step toward forming more complex structures with different tissue types such as vasculature and stromal cells.

Andres Bratt-Leal, a former graduate student

in the laboratory of Todd McDevitt, analyzes

stem cells using a microscope. Credit: Georgia

Tech Photo: Rob Felt.

"To build tissues, we need to be able to take stem cells and use them to make many different cell types which are grouped together in particular spatial patterns," explained Andres M. Bratt-Leal, the paper's first author and a former graduate student in McDevitt's lab.

"This spatial patterning is what gives tissues the ability to perform higher order functions."

After creating stem cell aggregates with microparticles containing different growth factors, the researchers observed a hemispherical organization of cells for several days, with the different cells remaining spatially segregated.

"We can see the microparticles had effects on one population that were different from the population that didn't have the particles," McDevitt said.

"This may allow us to emulate aspects of how development occurs. We can ask questions about how tissues are naturally patterned. With this material incorporation, we have the ability to better control the environment in which these cells develop."

The microparticles could also provide better control over the kinetics of cell differentiation. Including different amounts of molecules – one the growth factor and the other its antagonist – could vary the rate at which the stem cell differentiation proceeds.

While the research reported in this paper manipulated pluripotent mouse cells, the researchers have moved ahead in performing similar studies with human stem cells and achieved comparable types of results with the microparticle delivery approaches.

The developments not only help move stem cell technologies closer to the clinic, but also provide a new tool for research.

"Our findings will provide a significant new tool for tissue engineering, bioprocessing of stem cells and also for better studying early development processes such as axis formation in embryos," said Bratt-Leal.

"During development, particular tissues are formed by gradients of signalling molecules. We can now better mimic these signal gradients using our system."

Source: Georgia Institute of Technology 

Contact: John Toon

Reference:

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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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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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A Step Closer to Artificial Liver

A Step Closer to Artificial Liver

Monday, 03 June 2013

Prometheus, the mythological figure who stole fire from the gods, was punished for this theft by being bound to a rock. Each day, an eagle swept down and fed on his liver, which then grew back to be eaten again the next day.

Modern scientists know there is a grain of truth to the tale, says MIT engineer Sangeeta Bhatia: The liver can indeed regenerate itself if part of it is removed. However, researchers trying to exploit that ability in hopes of producing artificial liver tissue for transplantation have repeatedly been stymied: Mature liver cells, known as hepatocytes, quickly lose their normal function when removed from the body.

"It's a paradox because we know liver cells are capable of growing, but somehow we can't get them to grow" outside the body, says Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Technology and Electrical Engineering and Computer Science at MIT, a senior associate member of the Broad Institute and a member of MIT's Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science.

Now, Bhatia and colleagues have taken a step toward that goal. In a paper appearing in the June 2 issue of Nature Chemical Biology, they have identified a dozen chemical compounds that can help liver cells not only maintain their normal function while grown in a lab dish, but also multiply to produce new tissue.

Cells grown this way could help researchers develop engineered tissue to treat many of the 500 million people suffering from chronic liver diseases such as hepatitis C, according to the researchers.

Lead author of the paper is Jing (Meghan) Shan, a graduate student in the Harvard-MIT Division of Health Sciences and Technology. Members of Bhatia's lab collaborated with researchers from the Broad Institute, Harvard Medical School and the University of Wisconsin.

Large-scale screen

Bhatia has previously developed a way to temporarily maintain normal liver-cell function after those cells are removed from the body, by precisely intermingling them with mouse fibroblast cells. For this study, funded by the National Institutes of Health and Howard Hughes Medical Institute, the research team adapted the system so that the liver cells could grow, in layers with the fibroblast cells, in small depressions in a lab dish. This allowed the researchers to perform large-scale, rapid studies of how 12,500 different chemicals affect liver-cell growth and function.

The liver has about 500 functions, divided into four general categories: drug detoxification, energy metabolism, protein synthesis and bile production. David Thomas, an associate researcher working with Todd Golub at the Broad Institute, measured expression levels of 83 liver enzymes representing some of the most finicky functions to maintain.

After screening thousands of liver cells from eight different tissue donors, the researchers identified 12 compounds that helped the cells maintain those functions, promoted liver cell division, or both.

MIT researchers have generated mature liver cells 

from induced pluripotent stem cells. In this image, 

the cell nuclei are stained blue. The green stain 

identifies liver cells, and the red stain identifies cells 

that are actively dividing. Image: Shan et al., Nature 

Chemical Biology, 2013.

Two of those compounds seemed to work especially well in cells from younger donors, so the researchers — including Robert Schwartz, an IMES postdoc, and Stephen Duncan, a professor of human and molecular genetics at the University of Wisconsin — also tested them in liver cells generated from induced pluripotent stem cells (iPSCs). Scientists have tried to create hepatocytes from iPSCs before, but such cells don't usually reach a fully mature state. However, when treated with those two compounds, the cells matured more completely.

Bhatia and her team wonder whether these compounds might launch a universal maturation program that could influence other types of cells as well. Other researchers are now testing them in a variety of cell types generated from iPSCs.

In future studies, the MIT team plans to embed the treated liver cells on polymer tissue scaffolds and implant them in mice, to test whether they could be used as replacement liver tissues. They are also pursuing the possibility of developing the compounds as drugs to help regenerate patients' own liver tissues, working with Trista North and Wolfram Goessling of Harvard Medical School.

Making connections

Bhatia and colleagues have also recently made progress toward solving another challenge of engineering liver tissue, which is getting the recipient's body to grow blood vessels to supply the new tissue with oxygen and nutrients. In a paper published in the Proceedings of the National Academy of Sciences in April, Bhatia and Christopher Chen, a professor at the University of Pennsylvania, showed that if preformed cords of endothelial cells are embedded into the tissue, they will rapidly grow into arrays of blood vessels after the tissue is implanted.

To achieve this, Kelly Stevens in the Bhatia lab worked with Peter Zandstra at the University of Toronto to design a new system that allows them to create 3-D engineered tissue and precisely control the placement of different cell types within the tissue. This approach, described in the journal Nature Communications in May, allows the engineered tissue to function better with the host tissue.

"Together, these papers offer a path forward to solve two of the longstanding challenges in liver tissue engineering — growing a large supply of liver cells outside the body and getting the tissues to graft to the transplant recipient," Bhatia says.

Source: Massachusetts Institute of Technology 

Contact: Anne Trafton, MIT News Office

Reference:

Identification of small molecules for human hepatocyte expansion and iPS differentiation

Jing Shan, Robert E Schwartz, Nathan T Ross, David J Logan, David Thomas, Stephen A Duncan, Trista E North, Wolfram Goessling, Anne E Carpenter & Sangeeta N Bhatia

Nature Chemical Biology, 02 June 2013, doi:10.1038/nchembio.1270

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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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