June 20, 2005
Replacement Human Muscles With Blood Vessels Grown In Rodents

Using human embryonic stem cells and a mixture of tissue types a group of researchers has found a way to grow replacement muscle tissue which has blood vessels.

CAMBRIDGE, Mass.--For years, a major obstacle has dashed the hopes of creating "replacement parts" for the human body: the lack of an internal, nourishing blood system in engineered tissues. Without it, thicker tissues can't thrive, which has confined tissue engineering's practical application to thin skin, which can recruit blood vessels from underlying tissue.

Now, researchers in Institute Professor Robert Langer's lab at MIT have used a novel cocktail of cells to coax muscle tissue to develop its own vascular network, a process called pre-vascularization. When implanted in living mice and rats, these tissues integrated more robustly with the body's own tissues than similar implants without blood vessels.

This approach should work with other tissue types.

"What's even more exciting than being able to make skeletal muscles for reconstructive surgery or to repair congenitally defective muscles, for instance, is that this a generic approach that can be applied towards making other complex tissues. It could allow us to do really wonderful things," says collaborator Daniel Kohane, an affiliate at MIT and assistant professor of pediatrics at Harvard Medical School.

The researchers published their work in Nature Biotechnology, available online in advance on June 19, 2005. An accompanying News and Views commentary says this "landmark paper" provides "a compelling demonstration of the benefits of pre-vascularization for engineering larger pieces of tissue."

"When I came to work with Bob Langer for my postdoc, it was my dream to vascularize a tissue," recalls first author Shulamit Levenberg, who is now on the faculty of the biomedical engineering department at Technion in Haifa, Israel where she completed these studies. She chose to tackle muscles, since they depend on blood vessels interspersed with muscle fibers and also serve as a model for highly vascularized organs such as the liver, heart, and lung.

The researchers used three cell types: myoblasts, endothelial cells, and fibroblasts. Some of the endothelial cells formed the needed blood vessels.

Levenberg theorized she needed to combine three cell types: myoblasts that form muscle fibers; endothelial cells that independently self-organize into vessel tubes; and fibroblasts that are the precursors for the smooth muscle cells that stabilize the vessel amidst the tissue's gooey extracellular matrix. "No one had tried a 3-D tri-culture scaffold before. It's hard enough to work with one cell type, let alone three!" explains senior author Langer, who is a pioneer in tissue engineering.

The VEGF mentioned here is a Vascular Endothelial Growth Factor, a hormone that causes blood vessels to form. The process of blood vessel formation is called angiogenesis. Angiogenesis has come to be well understood as a result of Harvard cancer researcher Judah Folkman's decades of pursuit of anti-angiogenesis compounds as an approach to stopping cancer tumor growth. The field of tissue engineering therefore benefits from insights developed by cancer researchers.

In vitro experiments validated Levenberg's hypothesis: "The endothelial cells formed vessels, recruited the fibroblasts, and differentiated them into smooth muscle cells," she says. "The differentiated fibroblasts expressed the angiogenic growth factor, VEGF, which further stimulated vessel growth." The constructs measured 5mm by 5mm by 1mm.

Note the use of human embryonic stem cells to make endothelial cells. My guess is that work was done in Israel.

For implantation in living animals, the lab used immunodeficient mice and rats that would not reject the human-derived endothelial cells. At the beginning of the project, Levenberg had isolated endothelial cells from human embryonic stem cells a first. Human derivation is key for clinical use to avoid an immune rejection.

The animal studies progressed in stages. First, the researchers implanted a muscle construct under the skin, then inserted one within a leg muscle, and finally replaced a piece of a rat's abdominal muscle with a construct, simulating a situation applicable to trauma victims, for instance. Later tissue staining showed that the implants' vessels grew into the host tissue and the host's vessels grew into the constructs.

But what good are blood vessels if they don't deliver the goods blood? Using two non-invasive live imaging techniques (labeled lectin injected into the tail vein and a luminescent luciferase-based system), the researchers could watch the host's blood flow into the engineered vessels. About 41% of the constructed vessels became perfused with the hosts' blood, meaning they functioned in the living body. "That's pretty good for a first try," Levenberg asserts.

Importantly, twice as many of the cells survived in the tri-culture implants compared to implants without endothelial cells. "The myoblasts also became even longer tubes when implanted, and they began to align themselves with the host's muscle fibers," Levenberg recounts.

"This tri-culture system shows a whole new way of creating a vascular network in the tissue," summarizes Langer. "We've also demonstrated another powerful use of human embryonic stem cells."

In addition to Kohane, Levenberg and Langer collaborated with Patricia D'Amore and Diane Darland at The Schepens Eye Research Institute, Evan Garfein at Brigham and Women's Hospital, Robert Martin of MIT's Division of Comparative Medicine, Richard Mulligan of Children's Hospital and Harvard Medical School, Clemens van Blitterswijk at Twente University in the Netherlands, and present and former MIT graduate students Mara Macdonald Jeroen Rouwkema.

The discovery of an approach which causes the growth of blood vessels in bioengineered organs lifts a major obstacle in the way of tissue engineering. If tissue engineers can cause cells to build vascular networks then the construction of larger three dimensional pieces of tissue becomes possible.

One of the 7 Strategies for Engineered Negligible Senescence (SENS) is the introduction of replacement cells. I think the category should be worded a bit more broadly since we have parts that are not even cells (e.g. heart valves). Also, while some of those replacement parts will be delivered as cell types injected into the body for many organ failure problems we will need to grow replacement organs. That requires the development of an additional set of capabilities for doing tissue engineering to create three dimensional structures. This latest result is a very helpful step in that direction.

Share |      Randall Parker, 2005 June 20 10:22 AM  Biotech Organ Replacement

Brendan Bartlett said at June 21, 2005 10:54 AM:

I can just see the next big sports flap when this technology is further developed. They'll learn how to adjust the level of fast vs slow twitch muscles and you'll see professional athletes having their muscles 'tuned' to their sport. It'll be the new steroids!

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