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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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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'Master Molecule' May Improve Stem Cell Treatment of Heart Attacks

'Master Molecule' May Improve Stem Cell Treatment of Heart Attacks

Thursday, 21 June 2012

Johns Hopkins researchers have discovered that a single protein molecule may hold the key to turning cardiac stem cells into blood vessels or muscle tissue, a finding that may lead to better ways to treat heart attack patients.

Human heart tissue does not heal well after a heart attack, instead forming debilitating scars. For reasons not completely understood, however, stem cells can assist in this repair process by turning into the cells that make up healthy heart tissue, including heart muscle and blood vessels. Recently, doctors elsewhere have reported promising early results in the use of cardiac stem cells to curb the formation of unhealthy scar tissue after a heart attack. But the discovery of a "master molecule" that guides the destiny of these stem cells could result in even more effective treatments for heart patients, the Johns Hopkins researchers say.

In a study published in the June 5 online edition of the journal Science Signaling, the team reported that tinkering with a protein molecule called p190RhoGAP shaped the development of cardiac stem cells, prodding them to become the building blocks for either blood vessels or heart muscle. The team members said that by altering levels of this protein, they were able to affect the future of these stem cells.

"In biology, finding a central regulator like this is like finding a pot of gold," said Andre Levchenko, a biomedical engineering professor and member of the Johns Hopkins Institute for Cell Engineering, who supervised the research effort.

The lead author of the journal article, Kshitiz, a postdoctoral fellow who uses only his first name, said:

"Our findings greatly enhance our understanding of stem cell biology and suggest innovative new ways to control the behaviour of cardiac stem cells before and after they are transplanted into a patient. This discovery could significantly change the way stem cell therapy is administered in heart patients."

Earlier this year, a medical team at Cedars-Sinai Medical Center in Los Angeles reported initial success in reducing scar tissue in heart attack patients after harvesting some of the patient's own cardiac stem cells, growing more of these cells in a lab and transfusing them back into the patient.

Using the stem cells from the patient's own heart prevented the rejection problems that often occur when tissue is transplanted from another person.

Levchenko's team wanted to figure out what, at the molecular level, causes the stem cells to change into helpful heart tissue. If they could solve this mystery, the researchers hoped the cardiac stem cell technique used by the Los Angeles doctors could be altered to yield even better results.

During their research, the Johns Hopkins team members wondered whether changing the surface where the harvested stem cells grew would affect the cells' development. The researchers were surprised to find that growing the cells on a surface whose rigidity resembled that of heart tissue caused the stem cells to grow faster and to form blood vessels. A cell population boom occurred far less often in the stem cells grown in the glass or plastic dishes typically used in biology labs. This result also suggested why formation of cardiac scar tissue, a structure with very different rigidity, can inhibit stem cells naturally residing there from regenerating the heart.

Looking further into this stem cell differentiation, the Johns Hopkins researchers found that the increased cell growth occurred when there was a decrease in the presence of the protein p190RhoGAP.

"It was the kind of master regulator of this process," Levchenko said.

"And an even bigger surprise was that if we directly forced this molecule to disappear, we no longer needed the special heart-matched surfaces. When the master regulator was missing, the stem cells started to form blood vessels, even on glass."

A final surprise occurred when the team decided to increase the presence of p190RhoGAP, instead of making it disappear.

"The stem cells started to turn into cardiac muscle tissue, instead of blood vessels," Levchenko said.

"This told us that this amazing molecule was the master regulator not only of the blood vessel development, but that it also determined whether cardiac muscles and blood vessels would develop from the same cells, even though these types of tissue are quite different."

But would these lab discoveries make a difference in the treatment of living beings? To find out, the researchers, working on the heart-matching surfaces they had designed, limited the production of p190RhoGAP within the heart cells. The cells that possessed less of this protein integrated more smoothly into an animal's blood vessel networks in the aftermath of a heart attack. In addition, more of these transplanted heart cells survived, compared to what had occurred in earlier cell-growing procedures.

Kshitiz said that the special heart-like surface on which the cardiac stem cells were grown triggers regulation of the master molecule, which then steers the next steps.

"This single protein can control the cells' shape, how fast they divide, how they become blood vessel cells and how they start to form a blood vessel network," he said.

"How it performed all of these myriad tasks that require hundreds of other proteins to act in a complex interplay was an interesting mystery to address, and one that rarely occurs in biology. It was like a molecular symphony being played in time, with each beat placed right at the moment before another melody has to start."

Source: Johns Hopkins University 

Contact: Phil Sneiderman

Reference:

Matrix Rigidity Controls Endothelial Differentiation and Morphogenesis of Cardiac Precursors

Kshitiz, Maimon E. Hubbi, Eun Hyun Ahn, John Downey, Junaid Afzal, Deok-Ho Kim, Sergio Rey, Connie Chang, Arnab Kundu, Gregg L. Semenza, Roselle M. Abraham, and Andre Levchenko
Sci. Signal., 5 June 2012, Vol. 5, Issue 227, p. ra41, DOI: 10.1126/scisignal.2003002

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Stem Cells Help Create Blood Vessel in Life-Saving Surgery

Stem cell technology used in unique surgery of 10-year-old girl Monday, 09 May 2011

A 10-year-old girl with a deadly blood clot underwent a life-saving surgery that showed the power of using stem cells to regenerate healthy organs.

Surgeon and professor Michael

Olausson. Credit: Björn Larsson

Rosvall.

The girl developed a clot in the blood vessel between her intestine and liver during her first year of life, creating the risk of potentially fatal bleeding. Michael Olausson, a surgeon at Sahlgrenska University Hospital at the University of Gothenburg took a blood vessel from a donor, chemically removed tissue and DNA from it, then seeded stem cells from the girl’s bone marrow to create a healthy, living blood vessel.

Surgeon and Professor Michael Olausson were able to create a new connection with the aid of this blood vessel between the liver and the intestines, necessary to cure the girl. The girl is now in good health, and her prognosis is very good. The girl developed during her first year of life a blood clot in the blood vessel that leads blood from the intestines to the liver. This introduced the risk that she would experience life-threatening internal bleeding. The condition can be cured if it is possible to direct the blood along the correct path, back into the liver. In optimal cases, the surgery can be performed using blood vessels from other parts of the patient's body, but a liver transplant may be necessary if the surgery is unsuccessful due to a lack of sufficient blood vessels. A liver transplant will involve subsequent lifelong treatment with immunosuppressive drugs.

Blood vessels from a dead donor were used in the present case. The vessel was then chemically treated to remove all cells RNA and DNA. This left just the supporting tissue. Stem cells were then obtained from the girl's bone marrow and these were added to the supporting tissue. A new blood vessel grew in just under four weeks. This was used during the surgery in order to create the new connection between the liver and the intestines, necessary to cure the girl.

"We carried out the surgery over three months ago now, and the result was very good, with no serious complications. To our knowledge this is the first procedure of this type in the world,” says Michael Olausson.

“The girl is in good health, and we believe that her prognosis is very good. Since the vessel was created with the girl's own stem cells, she does not need to take drugs to prevent rejection", says Michael Olausson at the Transplant Centre, Sahlgrenska University Hospital and professor at the Sahlgrenska Academy.

The procedure shows that it is possible to create new blood vessels from stem cells, using a previous blood vessel as a template. This can lead to the condition that the girl suffered from being treated more easily, and with less risk for the patient. The result of this operation may have implications not only for the condition the girl was suffering from, but also in a number of other fields of research.

"The next step is to intensify research into the recreation of other organs, and to develop methods that can be used for arteries. This can help, among others, patients who need dialysis and those needing surgery for the coronary arteries. It may also help those needing complete organs", says Michael Olausson.

"There may also be major financial benefits for the healthcare system, particularly if it proves possible to produce, for example, complete kidneys by this method, since the consumption of drugs will be dramatically reduced. For the patients, it means that the undesired effects of the drugs that must currently be used will be avoided."

Source: University of Gothenburg
Contact: Michael Olausson
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Stem Cells from Surgery Leftovers Could Repair Damaged Hearts

Stem Cells from Surgery Leftovers Could Repair Damaged Hearts
Thursday, 29 April 2010

Scientists have for the first time succeeded in extracting vital stem cells from sections of vein removed for heart bypass surgery. Researchers funded by the British Heart Foundation (BHF) found that these stem cells can stimulate new blood vessels to grow, which could potentially help repair damaged heart muscle after a heart attack.

The research, by Paolo Madeddu, Professor of Experimental Cardiovascular Medicine and his team in the Bristol Heart Institute (BHI) at the University of Bristol, is published in the leading journal Circulation.

Around 20,000 people each year undergo heart bypass surgery. The procedure involves taking a piece of vein from the person's leg and grafting it onto a diseased coronary artery to divert blood around a blockage or narrowing.

The surgeon normally takes out a longer section of vein than is needed for the bypass. The Bristol team successfully isolated stem cells from leftover veins that patients had agreed to donate.

In tests in mice, the cells proved able to stimulate new blood vessels to grow into injured leg muscles. Professor Madeddu and his team are now beginning to investigate whether the cells can help the heart to recover from a heart attack.

"This is the first time that anyone has been able to extract stem cells from sections of vein left over from heart bypass operations," Professor Madeddu said.

"These cells might make it possible for a person having a bypass to also receive a heart treatment using their body's own stem cells.”

"We can also multiply these cells in the lab to make millions more stem cells, which could potentially be stored in a bank and used to treat thousands of patients."

Professor Peter Weissberg, Medical Director of the BHF, said:

"Repairing a damaged heart is the holy grail for heart patients. The discovery that cells taken from patients' own blood vessels may be able to stimulate new blood vessels to grow in damaged tissues is a very encouraging and important advance. It brings the possibility of 'cell therapy' for damaged hearts one step closer and, importantly, if the chemical messages produced by the cells can be identified, it is possible that drugs could be developed to achieve the same end."
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Scientists Grow Replacement Blood Vessels from Stem Cells

Tissue-engineered grafts composed of adult stem cells could 1 day replace synthetic vascular bypass grafts
Friday, 09 April 2010

Using adult stem cells, researchers have created functional blood vessels that could one day replace synthetic grafts often required in various vascular bypass surgeries, according to research presented at the American Heart Association's Arteriosclerosis, Thrombosis and Vascular Biology Annual Conference 2010.

Bypass surgery is used to open blocked arteries in one part of the body by using a vessel from elsewhere in the body. However, up to 40 percent of patients do not have a vessel suitable for the procedure. In such cases, surgeons use synthetic grafts. But the artificial blood vessels often become clogged within one to three years at a higher rate than natural arteries often leading to serious infections.

"Our grafts have the potential to be used for peripheral artery disease bypass (mostly in the legs), and arteriovenous fistula (a type of vascular access for haemodialysis), and heart bypass surgery," said Stephen E. McIlhenny, Ph.D., lead author of the study and tissue engineer of Thomas Jefferson University Hospital in Philadelphia.

"However, the first uses of the grafts would be for treatment of peripheral artery disease and dialysis access grafting."

McIlhenny and colleagues reported encouraging results from their first pre-clinical tests of the graphs.

"It was our idea to create a more biological conduit that would avoid the problems of synthetic grafts and give patients a better alternative," said McIlhenny.

"The significant finding is that we can build a blood vessel from donor tissue and an animal's own adult stem cells. Potentially, patients requiring bypass surgery could receive optimized grafts that would reduce their future complications."

Researchers grew rabbit adult stem cells on human vein scaffolds in the laboratory. The team removed all cells from sections of human saphenous veins, which left a tube consisting only of the protein scaffolding that supported the cells. Because the scaffolding contains no cells; there is less risk the immune system will reject it.

The researchers derived adult stem cells from the fat cells of each rabbit that would receive the test graft. They grew the stem cells on pieces of scaffolding, and each rabbit received a graft with only its own stem cells on it.

"Fat cells are easily obtained with liposuction," McIlhenny said.

"Bone-marrow-derived stem cells require going into the bone canal to take bone marrow out, which can be painful."

Surgeons cut the abdominal aorta of five male rabbits and inserted stem cell grafts into the large artery. In five other male rabbits, they similarly inserted grafts of the bare protein scaffolding. They examined the animals every two weeks for eight weeks with ultrasound, monitoring whether their inserted grafts were closing up.

After eight weeks, surgeons removed the grafts from the 10 animals. The five bare scaffolding grafts showed significant thickening akin to that seen in human cardiovascular disease, as well as evidence of blood clots.

As for the other five grafts, "we found that using the stem cells as a coating prevented clotting and thickening of the graft wall," McIlhenny said.

"I would say those grafts were significantly better."

Considerably more work remains, including additional animal testing, before studies of the fat-cell-derived grafts can begin in human testing. The team is attempting to grow smooth muscle cells on the outer layer of the scaffolding. In arteries, this layer of cells strengthens the blood vessels and enables them to contract.

The group is also investigating a suspected but unproven function of nitric oxide in the success of the adult stem cell graft. Nitric oxide is known to inhibit platelet aggregation and blood-clot formation in blood vessels.

"We are going to attempt to prove more definitely that this molecule is protecting our graft against wall thickening," McIlhenny said.
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Stem Cells Build New Blood Vessels to Treat Peripheral Arterial Disease

X-ray–visible bone marrow stem cells dramatically improve ability to build new blood vessels. Procedure offering future possibility of reducing or avoiding risk of limb amputation. Tuesday, 16 March 2010 Bone marrow stem cells suspended in X-ray-visible microbubbles dramatically improve the body's ability to build new blood vessels in the upper leg — providing a potential future treatment for those with peripheral arterial disease or PAD, say researchers at the Society of Interventional Radiology's 35th Annual Scientific Meeting in Tampa, Fla. "Bone marrow stem cells, which have the ability to renew themselves, could unlock the door to treat peripheral arterial disease (PAD) with cell-based methods. They offer a future novel method to help PAD patients by increasing the number of blood vessels to replace or augment those choked off by plaque build-up," said Frank Wacker, M.D., an interventional radiologist at Johns Hopkins School of Medicine in Baltimore, Md.. "The future hope is to use adult stem cells extracted from a healthy donor's bone marrow and inject the cells into the patients' legs where circulation problems exist, stimulating the growth of new or more blood vessels in the leg, thus improving circulation," noted veterinary radiologist Dara L. Kraitchman, V.M.D., Ph.D.. "Using an animal model, we found that stem cells in X-ray–visible microbubbles dramatically improve the ability to build new blood vessels when a blood vessel in the upper leg has been suddenly closed or occluded," said Kraitchman, an associate professor at Johns Hopkins School of Medicine. "With this treatment, the body was able to provide a more normal blood supply to the toes — possibly offering the hope of dramatically reducing — or avoiding — amputation. Treatment could also be personalized for individual patients," she added. Using X-ray techniques performed by interventional radiologists, researchers viewed the new blood vessels and validated their results by examining the new vessels that formed via microscope, she said. PAD, which affects about 10 million (mostly older) Americans, is a chronic disease that progressively restricts blood flow causing poor blood circulation (generally in the legs) and — if left untreated — can lead to serious medical complications, including heart attack, stroke, amputation and death. Many people can manage the symptoms of PAD and stop its progression through lifestyle changes. If lifestyle changes are not enough, additional medical treatment may be needed, including prescribed medicine to prevent blood clots, lower blood pressure and cholesterol and control pain. Interventional radiologists treat severe cases of PAD with minimally invasive treatments, including angioplasty and insertion of stents. "About 10 percent of the PAD patients cannot be treated with typical methods to reopen the arteries, such as angioplasty or stents," said Wacker. Because many treatments like stenting are done using X-rays, this microbubble stem cell treatment could be performed when an interventional radiologist is performing a dye study to look at a patient's arteries. Since an interventional radiologist can see where he or she puts the stem cells and whether they remain in the leg, the stem cells could be administered potentially where they can do the most good. The treatment could be repeated, if needed. Hopkins researchers used a technique that encloses stem cells derived from bone marrow (not embryonic stem cells) in an alginate capsule or microbubble made from seaweed that contains stem cells to create factors to recruit the building of new vessels along with an X-ray–visible contrast agent. Tested in a rabbit model, the bubble prevents the body's immune system from reaching and attacking the transplanted cells. "They not only made the stem cells X-ray visible, but they also made the stem cells themselves visible much like the way a firefly's light is visible at night," said Kraitchman. "Because the microbubble protects the stem cells from being destroyed even if they come from another person's bone marrow, there is the potential to provide this therapy similar to blood transfusions. Like blood transfusions, one cannot anticipate which PAD patient will need stem cells, so there may not be time to harvest a person's own stem cells in advance for treatment," said Kraitchman. "We are continuing to test the treatment in animals and attempting to perfect methods using non-invasive imaging, such as magnetic resonance imaging (MRI), ultrasound and blood pressure measurements, which could be used to follow up patients without exposing them to X-rays or needing to enter a blood vessel to inject dye to see the newly formed vessels," said Kraitchman. "We are also fusing the X-ray imaging results with other imaging techniques like MRI to provide a better picture of where to place the stem cells," she added. Reference: Encapsulated, X-ray–visible Mesenchymal Stem Cell Transplants for Rabbit Model of Peripheral Arterial Disease. Estimation of Cell Viability and Efficacy D.A. Kedziorek, Y. Fu, P. Walczak, G. Huang, S. Valdeig, J.W. Bulte, F.K. Wacker, D.L. Kraitchman, T. Ehtiati, Siemens, and L. Hofmann SIR 35th Annual Scientific Meeting March 13th, 2010, Tampa, Fla., Abstract 92 ......... ZenMaster

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Researchers Characterize Stem Cell Function

Researchers Characterize Stem Cell Function Friday, 12 March 2010 The promise of stem cells lies in their unique ability to differentiate into a multitude of different types of cells. But in order to determine how to use stem cells for new therapeutics, scientists and engineers need to answer a fundamental question: if a stem cell changes to look like a certain type of cell, how do we know if it will behave like a certain type of cell? Researchers at Northwestern University's McCormick School of Engineering are the first to fully characterize a special type of stem cell; endothelial progenitor cells (EPCs) that exist in circulating blood, to see if they can behave as endothelial cells in the body when cultured on a bioengineered surface. The results, published online in the journal Stem Cells show promise for a new generation of tissue-engineered vascular grafts, which could improve the success rate of surgery for peripheral arterial disease. Peripheral arterial disease is estimated to affect one in every 20 Americans over the age of 50, a total of 8 to 12 million people. "Normally, stem cells are not studied in the context of improving vascular grafts for bypass surgery. Therefore, we had to develop new tests to assess their use in this application," says Guillermo Ameer, senior author of the paper and associate professor of biomedical engineering and surgery. "We looked at the function of the cells on a citric acid-based polymer, which will be the basis for a new generation of bioengineered vascular grafts." In the study, Josephine Allen, then a graduate student in Ameer's lab, and colleagues isolated endothelial progenitor cells from eight tablespoons of blood. In approximately half of the attempts, the team was able to isolate the EPCs to expand to make millions of endothelial cells that can behave like the cells of a blood vessel. Once the endothelial-like cell colonies were established, the research team performed a battery of tests to examine the properties and functionality of the cell. "These new tests show that these endothelial-like cells can inhibit blood clotting and can prevent platelets from adhering to their surface," says Ameer. "But if you antagonize the cells or stimulate them, they will also respond the same way that an endothelial cell would and will clot blood if needed." The study is an important step in identifying methods to build a tissue-engineered vascular graft. Synthetic grafts, used to treat common diseases such as peripheral arterial disease, have lower success rates when used in small-diameter arteries, such as those found in the leg. "These small-diameter synthetic grafts are more prone to blood clots and other complications, especially over time," Ameer says. "It's thought that a tissue-engineered graft would allow us to preserve many of the body's natural defences against these complications." Reference: Toward Engineering a Human Neoendothelium With Circulating Progenitor Cells J. Allen, S. Khan, K.A. Lapidos, G.A. Ameer Stem Cells, 2010, March ......... ZenMaster

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Functional Blood Vessel Cells Generated From Human Stem Cells

Weill Cornell Medical College study lays groundwork for new treatments for cardiovascular disease and other conditions Thursday, 21 January 2010 In a significant step toward restoring healthy blood circulation to treat a variety of diseases, a team of scientists at Weill Cornell Medical College has developed a new technique and described a novel mechanism for turning human embryonic and pluripotent stem cells into plentiful, functional endothelial cells, which are critical to the formation of blood vessels. Endothelial cells form the interior "lining" of all blood vessels and are the main component of capillaries, the smallest and most abundant vessels. In the near future, the researchers believe, it will be possible to inject these cells into humans to heal damaged organs and tissues. The new approach allows scientists to generate virtually unlimited quantities of durable endothelial cells – more than 40-fold the quantity possible with previous approaches. Based on insights into the genetic mechanisms that regulate how embryonic stem cells form vascular endothelial cells, the approach may also yield new ways to study genetically inherited vascular diseases. The study appears in the advance online issue of Nature Biotechnology. "This technique is the first of its kind with serious potential as a treatment for a diverse array of diseases, especially cardiovascular disease, stroke and vascular complications of diabetes," says Dr. Shahin Rafii, the study's senior author. Dr. Rafii is the Arthur B. Belfer Professor in Genetic Medicine and co-director of the Ansary Stem Cell Institute at Weill Cornell Medical College, and an investigator of the Howard Hughes Medical Institute. In recent years, enormous hopes have been pinned on stem cells as the source of future cures and treatments. Indeed, human embryonic stem cells have the potential to become any one of the more than 200 types of adult cells. However, the factors and pathways that govern their differentiation to abundant derivatives that could be used to repair organs have remained poorly understood. A major challenge for Dr. Rafii's lab has been to improve their understanding, and hence control, of the differentiation process (how stem cells convert to various cell types), and then to generate enough vascular endothelial cells – many millions – so they can be used therapeutically. To meet this challenge, the scientists first screened for molecular factors that come into play when stem cells turn into endothelial cells. Their findings led them to a strategy that significantly boosts the efficiency of producing these cells. Then, the researchers tracked the differentiation process in real-time using a green fluorescent protein marker developed by Dr. Daylon James, the study's first author and assistant research professor in the Department of Reproductive of Medicine at Weill Cornell Medical College. They found that when they exposed stem cells to a compound that blocks TGF-beta (a growth factor involved in cell specialization) at just the right time during cell culturing, the propagation of endothelial cells dramatically increased. Even more striking, they found that the cells worked properly when injected into mice. The cells were quickly assimilated into the animals' circulatory systems, and functioned alongside normal vasculature. To achieve long-lasting clinical benefits, there remain additional hurdles to exploiting endothelial cells generated in vitro. Indeed, a fundamental prerequisite to using vascular cells in regenerative medicine has been the proper assembly in vivo of new blood vessels from stem-cell-derived cells, according to Dr. Sina Rabbany, who is an adjunct professor at Weill Cornell Medical College and professor of bioengineering at Hofstra University. Dr. Rabbany emphasizes that, in addition to manipulating biological factors implicated in endothelial cell differentiation, the impact of blood flow on endothelial cells is critical to engineering durable, vascularised organs. With the plentiful supply of endothelial cells that the new methods provide, Dr. Rabbany's team is working to build biological scaffolds that model the microenvironment of the vasculature, so that the vessels they generate will be functional and long-lasting in patients. Another major obstacle to clinical use of cultured endothelial cells is the potential of immune rejection when the cells are injected into a patient. To address this risk, one approach would be to create a large, genetically diverse bank of human embryonic stem cells that, on demand, could be converted into endothelial cells that are compatible with specific patients. "Given the success rate our group has shown in generating human embryonic stem cells from donated normal and diseased embryos, this new approach has broad implications not only for regenerative medicine, but also for the study of genetic diseases of the vasculature," states Dr. Zev Rosenwaks, who is director and physician-in-chief of the Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine as well as the director of the Tri-Institutional Stem Cell Initiative Derivation Unit at Weill Cornell Medical College. The new endothelial cell culture is currently being validated in ongoing research at Weill Cornell using a number of stem cell "lines," or "families" of stem cells. "Employing a highly sophisticated derivation technology, we have been able to generate 11 normal and diseased human embryonic cell lines from discarded embryos at the Tri-Institute Derivation Unit at Weill Cornell," states Dr. Nikica Zaninovic, an assistant professor at the Department of Reproductive of Medicine who is spearheading the human embryonic stem cell derivation effort. Using the new differentiation methods, several of these new embryonic stem cell lines have been turned into vascular cells. Testing in humans is the next major step in verifying the ability of this breakthrough cell-based approach to restore blood supply to injured organs. Armed with this new technology and under the umbrella of support from the Ansary Stem Cell Institute and Tri-Institutional Stem Cell Initiative (Tri-SCI), this team of scientists is hoping to transfer their methods to the clinic within the next five years. The current study sheds light on the generation of human embryonic vasculature in ways that have not previously been feasible due to obstacles associated with the use of human embryonic tissue. As Dr. James explains: "The unbiased screening technique we used is a major technological advance that opens up possibilities for discovery of how human embryonic stem cells morph into the specific mature cells that compose the brain, liver, pancreas, and so on. Our general approach can be applied to additional human tissues and help other stem cell research groups develop and maintain specialized cell types in the larger effort to understand human development – and to heal many different kinds of human diseases and injuries." The Tri-Institutional Stem Cell Initiative, supported by a generous gift from The Starr Foundation, is a collaborative venture of Memorial Sloan-Kettering Cancer Center, The Rockefeller University, and Weill Cornell Medical College. Ansary Stem Cell Institute The Ansary Stem Cell Institute, established at Weill Cornell Medical College in 2004 through the generous donation of Shahla and Hushang Ansary, brings together a premier team of scientists to focus on stem cells – the primitive, unspecialized cells with an unrivalled capacity to form all types of cells, tissues and organs in the body. The vision of the Ansary Institute is to help lead the way in 21st-century medicine by employing this new field of research with tremendous potential to relieve human suffering. The Institute permits the multidisciplinary collaboration and creativity of Weill Cornell's researchers, as well as helps to attract the best and brightest young researchers in the field. Scientists at the Institute hope to discover the wellspring of adult stem cells in the body and ways to manipulate them to treat human illness. In particular, they hope to understand the regulation of cells that give rise to such essential components as blood vessels, insulin-producing cells in the pancreas (which are damaged in diabetics), and neurons of the brain and nervous system. ......... ZenMaster

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Gene Therapy and Stem Cells save Limb

Gene Therapy and Stem Cells save Limb Wednesday, 09 December 2009 Blood vessel blockage, a common condition in old age or diabetes, leads to low blood flow and results in low oxygen, which can kill cells and tissues. Such blockages can require amputation resulting in loss of limbs. Now, using mice as their model, researchers at Johns Hopkins have developed therapies that increase blood flow, improve movement and decrease tissue death and the need for amputation. The findings, published online last week in the early edition of the Proceedings of the National Academy of Sciences, hold promise for developing clinical therapies. "In a young, healthy individual, hypoxia — low oxygen levels — triggers the body to make factors that help coordinate the growth of new blood vessels but this process doesn't work as well as we age," says Gregg Semenza, M.D., Ph.D., professor of paediatrics and genetic medicine and director of the vascular biology program at the Johns Hopkins Institute for Cell Engineering. "Now, with the help of gene therapy and stem cells we can help reactivate the body's response to hypoxia and save limbs." Previously, Semenza's team generated a virus that carries the gene encoding an active form of the HIF-1 protein, which turns on genes necessary for building new blood vessels. When injected into the hind legs of otherwise healthy mice and rabbits that had been treated to reduce blood flow, the HIF-1 virus treatment partially restored blood flow. People with diabetes have a 40 times higher risk of losing a limb to amputation, says Semenza. To find out if HIF-1 gene therapy could improve blood flow in a diabetic animal, the team then tested the same virus in diabetic and non-diabetic mice that had blood flow cut off to one hind leg. Twenty-one days after treatment, the HIF-1 virus-treated mice had 85 percent recovery of blood flow compared with 24 percent in the mock-treated mice. In addition, treated, diabetic mice had much less tissue damage compared to the untreated diabetic mice. These results were reported in the Nov. 3 issue of the Proceedings of the National Academy of Sciences. In the current study, the team asked if the same gene therapy treatment could improve reduced blood flow associated with advanced age. Comparing 13-month-old mice to 3-month-old mice, blocking the femoral artery in the hind leg causes all older mice to lose their legs while only about a third of younger mice have to lose their legs. The research team treated young and old mice with the HIF-1 virus and examined blood flow and tissue health. They found that while treatment improved young mice, it did not make a noticeable difference in the older mice. However, it was known that when HIF-1 normally activates signals in the body to build new vessels, one of the many types of cells recruited to the site of new vessel growth is a population of stem cells from the bone marrow, which are called bone marrow-derived angiogenic cells. Therefore, the team isolated these cells from mice and grew them under special conditions that would turn on HIF-1 in these cells. When the researchers treated the mice with both the HIF-1 virus and simultaneously injected bone marrow-derived angiogenic cells, treated, older mice were less likely to lose their legs compared to their untreated counterparts. Further study of these mice showed that activating HIF-1 in the cells appeared to turn on a number of genes that help these cells not only home to the ischemic limb, but to stay there once they arrive. To figure out how the cells stay where they're needed, the research team built a tiny micro-fluidic chamber and tested the cells' ability to stay stuck with fluid flowing around them at rates mimicking the flow of blood through vessels in the body. They found that cells under low oxygen conditions were better able to stay stuck only if those same cells had HIF-1 turned on. "Our results are promising because they show that a combination of gene and cell therapy can improve the outcome in the case of critical limb ischemia associated with aging or diabetes," says Semenza. "And that's critical for bringing such treatment to the clinic." ......... ZenMaster

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Adult Stem Cells May Help Repair Hearts Damaged by Heart Attack

Rush University Medical Center enrolling patients for next phase of trial Thursday, 03 December 2009 Adult stem cells may help repair heart tissue damaged by heart attack according to the findings of a new study to be published in the December 8 issue of the Journal of the American College of Cardiology. Results from the Phase I study show stem cells from donor bone marrow appear to help heart attack patients recover better by growing new blood vessels to bring more oxygen to the heart. Rush University Medical Center was the only Illinois site and one of 10 cardiac centres across the country that participated in the 53-patient, double-blind, placebo-controlled Phase I trial. Rush is now currently enrolling patients for the second phase of the study. Researchers say it is the strongest evidence thus far indicating that adult stem cells can actually differentiate, or turn into heart cells to repair damage. Until now, it has been believed that only embryonic stem cells could differentiate into heart or other organ cells. "The results point to a promising new treatment for heart attack patients that could reduce mortality and lessen the need for heart transplants," said Dr. Gary Schaer, head of the Rush Cardiac Catheterization Laboratory and study principal investigator at Rush. In phase I of the study, a group of 53 patients who had heart attacks in the previous ten days received adult mesenchymal stem cells and were kept under close study for two years. The mesenchymal stem cells (MSC) were harvested from the bone marrow of healthy adult donors. These cells have the potential to develop into mature heart cells and new blood vessels. Similar to Blood Type O, mesenchymal stem cells have the advantage that they can be taken from the bone marrow of an unrelated donor without needing to be matched by blood type. After the stem cells were extracted, they were purified by drug manufacturer Osiris Therapeutics into a formulation for intravenous delivery called Prochymal. Patients were administered an infusion of either Prochymal or placebo as an injection into a vein in the arm or leg. To prevent bias, neither the patient nor the physician knew who received the stem cell treatment and who received the placebo. In the study, patients who received the adult stem cells were compared to similar patients who received inert placebo injections. MRI and echocardiogram followed both. After six months, patients who received the adult stem cells were four times as likely to have improved overall condition, were able to pump more blood with each heartbeat than untreated patients, had only one-quarter as many dangerous heart arrhythmias, and suffered no toxicity or other serious adverse side effects from the treatment. "It is suspected that these stem cells may take part in the growth of new blood vessels to bring more oxygen to the heart and help reduce the scarring from a heart attack," said Schaer. Echocardiograms showed patients had improved heart function, particularly in those patients with large amounts of cardiac damage. Patients also have improvements in lung function. According to Schaer, one reason the study results are so promising is that these stem cells can be used without tissue typing and do not trigger an immune response, and are available for every patient. A unique benefit of the stem cell product is that it is given to patients through a standard intravenous (IV) line which is simple and easy for the patient compared to other therapies that require delivery to the site of the disease through catheterization or open surgical procedures, Adult stem cells are designed by nature to perform tissue repair in a mature adult. It is believed that these cells can be used in patients unrelated to the donor, without rejection, eliminating the need for donor matching and recipient immune suppression. Once transplanted, the cells promote healing of damaged or diseased tissues. "It is possible that in the future, hospitals might be able to keep frozen adult stem cells on hand for speedy use in treating heart attacks," said Schaer. "This study suggests that adult bone marrow derived stem cells are more flexible than previously thought," said Schaer. "If the benefits and safety are confirmed in the ongoing Phase II trial, we may soon have a remarkable new therapy for patients with a large heart." ......... ZenMaster

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Major Improvements Made in Engineering Heart Repair Patches from Stem Cells

Pre-formed blood vessels in patches connect to rodents' heart circulation Monday, 12 October 2009 University of Washington (UW) researchers have succeeded in engineering human tissue patches free of some problems that have stymied stem-cell repair for damaged hearts. The disk-shaped patches can be fabricated in sizes ranging from less than a millimetre to a half-inch in diameter. Until now, engineering tissue for heart repair has been hampered by cells dying at the transplant core, because nutrients and oxygen reached the edges of the patch but not the centre. To make matters worse, the scaffolding materials to position the cells often proved to be harmful. Heart tissue patches composed only of heart muscle cells could not grow big enough or survive long enough to take hold after they were implanted in rodents, the researchers noted in their article, published last month in the Proceedings of the National Academy of Sciences. The researchers decided to look at the possibility of building new tissue with supply lines for the oxygen and nutrients that living cells require. The scientists testing this idea are from the UW Center for Cardiovascular Biology and the UW Institute for Stem Cell and Regenerative Medicine, under the guidance of senior author Dr. Charles "Chuck" Murry, professor of pathology and bioengineering. The lead author is Dr. Kelly R. Stevens, a UW doctoral student in bioengineering who came up with solutions to the problems observed in previous grafts. The study is part of a collaborative tissue engineering effort called BEAT (Biological Engineering of Allogeneic Tissue). Stevens and her fellow researchers added two other types of cells to the heart muscle cell mixture. These were cells similar to those that line the inside of blood vessels and cells that provide the vessel's muscular support. All of the heart muscle cells were derived from embryonic stem cells, while the vascular cells were derived from embryonic stem cells or a variety of more mature sources such as the umbilical cord. The resulting cell mixture began forming a tissue containing tiny blood vessels. "These were rudimentary blood vessel networks like those seen early in embryonic development," Murry said. In contrast to the heart muscle cell-only tissue, which failed to survive transplantation and which remained apart from the rat's heart circulatory system, the pre-formed vessels in the mixed-cell tissue joined with the rat's heart circulatory system and delivered rat blood to the transplanted graft. "The viability of the transplanted graft was remarkably improved," Murry observed. "We think the gain in viability is due to the ability for the tissue to form blood vessels." Equally as exciting, the scientists observed that the patches of engineered tissue actively contracted. Moreover, these contractions could be electronically paced, up to what would translate to 120 beats per minute. Beyond that point, the tissue patch did not relax fully and the contractions weakened. However, the average resting adult heart pulses about 70 beats per minute. This suggests that the engineered tissue could, within limits, theoretically keep pace with typical adult heart muscle, according to the study authors. Another physical quality that made the mixed-cell tissue patches superior to heart muscle-cell patches was their mechanical stiffness, which more closely resembled human heart muscle. This was probably due to the addition of supporting cells, which created connective tissues. Passive stiffness allows the heart to fill properly with blood before it contracts. When the researchers implanted these mixed celled, pre-vascularised tissue patches into rodents, the patches grew into cell grafts that were ten times larger than the too-small results from tissue composed of heart muscle cells only. The rodents were bred without an immune system that rejects tissue transplants. Murry noted that these results have significance beyond their contribution to the ongoing search for ways to treat heart attack damage by regenerating heart tissue with stem cells. The study findings, he observed, suggest that researchers consider including blood vessel-generating and vascular-supporting elements when designing human tissues for certain other types of regenerative therapies unrelated to heart disease. One of the major obstacles still to be overcome is the likelihood that people's immune systems would reject the stem transplant unless they take medications for the rest of their lives to suppress this reaction. Murry hopes someday that scientists would be able to create new tissues from a person's own cells. "Researchers can currently turn human skin cells back to stem cells, and then move them forward again into other types of cells, such as heart muscle and blood vessel cells," Murry said. "We hope this will allow us to build tissues that the body will recognize as 'self.'" While the clinical application of tissues engineered from stem cells in treating hearts damaged from heart attacks or birth defects is still in the future, the researchers believe progress has been made. This study showed that researchers could create the first entirely human heart tissue patch from human embryonic cell-derived heart muscle cells, blood vessel lining cells and fibre-producing cells, and successfully engraft the tissue into an animal. Future studies will try to move heart cell regeneration closer toward clinical usefulness, according to Murry and his research team. They forecast that such research would include testing other sources of human cells and developing techniques to create bigger patches for treating larger animals through surgical transplantation or through catheter delivered injections. Lastly, they concluded, researchers would need to test whether tissue patches actually improve physical functioning after implantation in damaged hearts. ......... ZenMaster

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Major Genetic Differences between Blood and Tissue Cells Revealed

Genetic research based only on blood samples called into question Wednesday, 15 July 2009 Research by a group of Montreal scientists at McGill University calls into question one of the most basic assumptions of human genetics: that when it comes to DNA, every cell in the body is essentially identical to every other cell. Their results appear in the July issue of the journal Human Mutation. This discovery may undercut the rationale behind numerous large-scale genetic studies conducted over the last 15 years, studies that were supposed to isolate the causes of scores of human diseases. Except for cancer, samples of diseased tissue are difficult or even impossible to take from living patients. Thus, the vast majority of genetic samples used in large-scale studies come in the form of blood. However, if it turns out that blood and tissue cells do not match genetically, these ambitious and expensive genome-wide association studies may prove to have been essentially flawed from the outset. This discovery sprang from an investigation into the underlying genetic causes of abdominal aortic aneurysms (AAA) led by Dr. Morris Schweitzer, Dr. Bruce Gottlieb, Dr. Lorraine Chalifour and colleagues at McGill University and the affiliated Lady Davis Institute for Medical Research at Montreal's Jewish General Hospital. The researchers focused on BAK, a gene that controls cell death. What they found surprised them. AAA is one of the rare vascular diseases where tissue samples are removed as part of patient therapy. When they compared them, the researchers discovered major differences between BAK genes in blood cells and tissue cells coming from the same individuals, with the suspected disease "trigger" residing only in the tissue. Moreover, the same differences were later evident in samples derived from healthy individuals. "In multi-factorial diseases other than cancer, usually we can only look at the blood," explained Gottlieb, a geneticist with McGill's Centre for Translational Research in Cancer. "Traditionally when we have looked for genetic risk factors for, say, heart disease, we have assumed that the blood will tell us what's happening in the tissue. It now seems this is simply not the case." "From a genetic perspective, therapeutic implications aside, the observation that not all cells are the same is extremely important. That's the bottom line," he added. "Genome-wide association studies were introduced with enormous hype several years ago, and people expected tremendous breakthroughs. They were going to draw blood samples from thousands or hundreds of thousands of individuals, and find the genes responsible for disease.” "Unfortunately, the reality of these studies has been very disappointing, and our discovery certainly could explain at least one of the reasons why." AAA is a localized widening and weakening of the abdominal aorta, and primarily affects elderly Caucasian men who smoke, have high blood pressure and high cholesterol levels. It often has no symptoms, but can lead to aortic ruptures, which are fatal in 90 per cent of cases. If the mutations discovered in the tissue cells actually predispose for AAA, they present an ideal target for new therapies, and may have even wider therapeutic implications. "This will probably have repercussions for vascular disease in general," said Schweitzer, of McGill's Department of Medicine. "We have not yet looked at coronary or cerebral arteries, but I would suspect that this mutation may be present across the board." Schweitzer is optimistic that this discovery may lead to new treatments for vascular disease in the near to medium term. "The timeline might be five to 10 years," he said. "We have to do in vitro cell culture experiments first, prove it in an animal model, and then develop a molecule or protein which will affect the mutated gene product. This is the first step, but it's an important step." Reference: BAK1 gene variation and abdominal aortic aneurysms Bruce Gottlieb, Lorraine E. Chalifour, Benjamin Mitmaker, Nathan Sheiner, Daniel Obrand, Cherrie Abraham, Melissa Meilleur, Tomoko Sugahara, Ghassan Bkaily, Morris Schweitzer Human Mutation, Volume 30 Issue 7, pp.1043 – 1047, DOI: 10.1002/humu.21046 ......... ZenMaster

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Genetic Factors That Hold Promise for Treatment of Vascular Diseases

microRNAs turns stem cells into vascular smooth muscle cells Sunday, 05 July 2009 Researchers at the Gladstone Institute of Cardiovascular Disease (GICD) have discovered a key switch that makes stem cells turn into the type of muscle cells that reside in the wall of blood vessels. The same switch might be used in the future to limit growth of vascular muscle cells that cause narrowing of arteries leading to heart attacks and strokes, limit formation of blood vessels that feed cancers, or make new blood vessels for organs that are not getting enough blood flow. In a study published in the current issue of the journal Nature, the researchers found that a tiny RNA molecule, called microRNA-145 (miR-145), not only had all the information necessary to turn a stem cell into a vascular smooth muscle cell (VSMC), but could also affect VSMCs in the adult artery. VSMCs have the unique property that they can start dividing when an artery is injured or during atherosclerosis, ultimately causing narrowing of the vessel leading to occlusion. miR-145 and its sister microRNA, miR-143, work together to stop the pathologic division of VSMCs. In the setting of vessel disease, their activity was turned down, allowing the VSMCs to divide and clog up the artery. microRNAs are small RNA molecules that do not make protein, but instead affect that amount of protein synthesized by the cell from their target mRNAs — the blueprints for translating the genetic code into proteins. miR-145 and miR-143 together controlled the synthesis of a network of "master regulators" that control VSMCs, and thereby were able to function as a central "switch" for the behaviour of these important cells. "The ability of miR-145 to efficiently direct the cell fate of vascular smooth muscle cells from stem cells represents the power of these tiny microRNAs to exert major effects on cells," said Deepak Srivastava, MD, GICD director and senior author of the study. "We hope that we can use this knowledge to control when the body makes or does not make new blood vessels," he added. Previously, GICD researchers had shown that miR-143 is highly enriched when embryonic stem cells turned into cardiac stem cells. Here they found that miR-143 and miR-145 were both present as the heart was forming in mice, but became localized to the smooth muscle of blood vessels and of the gut after birth. Further analysis revealed that miR-143 and miR-145 are directly controlled by a protein called myocardin, which itself is sufficient to "reprogram" an adult non-muscle cell into a VSMC. Furthermore, the activation of these microRNAs by myocardin was a necessary event for myocardin to induce the VSMC fate. In one type of stem cell, miR-145 by itself was enough to completely push the stem cell into a functioning VSMC. These findings suggested that miR-143 and miR-145 are involved in the switch between the differentiation and proliferation of VSMCs — and thus contribute to vessel narrowing in heart disease. In a mouse model of this switch generated by collaborator Joseph Miano, PhD, a professor at the Cardiovascular Research Institute of the University of Rochester, expression of miR-143 and miR-145 was markedly reduced in injured arteries containing proliferating, less differentiated smooth muscle cells. Interestingly, miR-145 mRNA was also reduced to almost undetectable levels in atherosclerotic blood vessels with thickened walls. "miR-145 was necessary and sufficient for differentiation of VSMCs, so it is possible that restoring its activity could prevent the vessel narrowing in atherosclerosis," said Kimberly Cordes, PhD, a postdoctoral fellow in the Srivastava lab and lead author of the study. Since the effects of miRNAs depend on their mRNA targets, the researchers looked for mRNA targets of miR-143 and miR-145. They found that miR-143 and miR-145 cooperate in targeting a network of transcription factors, including Klf4, myocardin, and Elk-1, to promote the differentiation and repress proliferation of smooth muscle cells. "The multiple targets we identified for miR-143 and miR-145 reveal an elegant mechanism by which these miRNAs promote differentiation and simultaneously repress proliferation of VSMCs" said Dr. Srivastava. The targets miR-145 and miR-143 regulate are not only major regulators of VSMCs, but also control whether cells divide excessively in conditions such as cancer. According to Dr. Cordes, "the down regulation of miR-145 in numerous cancers and our findings in this study raise the possibility that miR-145 could function as a pro-differentiation factor in cancers also and could be a new therapeutic target." "Our findings in this study offer insights into regulatory mechanisms that govern the differentiation and proliferation of smooth muscle," said Dr. Srivastava. "They have fundamental implications for the treatment of vessel diseases like atherosclerosis and also may be important for cancer." About the Gladstone Institutes The J. David Gladstone Institutes, an independent, non-profit biomedical research organization, affiliated with the University of California, San Francisco, is dedicated to the health and welfare of humankind through research into the causes and prevention of some of the world's most devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology and the Gladstone Institute of Neurological Disease. Reference: miR-145 and miR-143 regulate smooth muscle cell fate and plasticity Kimberly R. Cordes, Neil T. Sheehy, Mark P. White, Emily C. Berry, Sarah U. Morton, Alecia N. Muth, Ting-Hein Lee, Joseph M. Miano, Kathryn N. Ivey & Deepak Srivastava Nature advance online publication 5 July 2009, doi:10.1038/nature08195 ......... ZenMaster

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