A previously overlooked group of cells has been shown to be essential in helping adult stem cells multiply and revitalize damaged tissue.

In a pair of studies that has the potential to change the way researchers think about regenerative medicine, scientists have shown that a previously overlooked group of cells—the endothelial layer of blood vessels—is essential in helping adult stem cells multiply and revitalize damaged tissue.

The endothelium is the innermost layer of blood vessels, made up of cells that had largely been assumed to function primarily as delivery vehicles for oxygen and nutrients. But earlier this year, Howard Hughes Medical Institute investigator Shahin Rafii figured out that these endothelial cells also release growth factors that direct bone marrow stem cells to multiply and differentiate into different types of blood cells.

To regenerate long-lasting liver, we may need to transplant hepatocytes with the properly activated endothelium, which produces the right growth factors for the hepatocytes to attach, grow, and connect with other parts of the liver.

Shahin Rafii

Now, the researchers have shown that such ability is not limited to bone marrow but exists in the endothelium of the liver, and that it can be activated to initiate and sustain liver regeneration in adult mice. In two papers published October 24, 2010, in Nature Cell Biology and November 11, 2010, in Nature, Rafii and his colleagues show that by altering the activation state of the endothelium in liver and bone marrow, they could induce adult hepatocytes and blood stem cells to divide and regenerate lost tissue.

During development, embryonic stem cells can differentiate into just about any tissue in the body. But adult stem cells, which are in short supply, are more tissue specific and employed mostly for maintenance and repair of the types of tissue they reside in. Being able to prompt these adult stem cells to proliferate and differentiate on command could have a profound impact on the field of regenerative medicine: Injury from therapeutic chemotherapy and radiation could be repaired; heart muscle damaged during a heart attack could be restored, and a failing liver could be rescued and rebuilt.

The idea that endothelial cells might play a role in tissue regeneration was an unorthodox one, but Rafii and his team noticed that evidence was beginning to build in support of that theory. His and other labs had observed that adult stem cells in a number of body parts—bone marrow, brain, muscle, testes, even fat—clustered near the endothelium.

“Some people would say, ‘Of course stem cells like to hang out with blood vessels, because they can conveniently extract oxygen, glucose, and nutrients very well that way,’” says Rafii, a professor of genetic and regenerative medicine and co-director of the Ansary Stem Cell Institute at Weill Cornell Medical College in New York.

But he and his colleagues noticed something else. After endothelial cells in bone marrow had been damaged by chemotherapy or radiation, the blood vessels still delivered oxygen but the blood stem cells weren’t dividing. “I hypothesized that, in addition to delivering nutrients, maybe endothelial cells are also releasing growth factors,” Rafii says.

Rafii’s earlier research, published in March of this year, showed that endothelial cells in bone marrow express stem cell-active growth factors, such as Notch-ligands, which directly influenced the expansion of blood stem cells. But the researchers didn’t know what caused it. Now, the Nature Cell Biology paper addresses the mechanism, and shows that the key to stem cell expansion is the activation state of endothelium—something usually caused by stress to the tissue nearby.

In healthy adult tissues, including bone marrow, small populations of stem cells lie dormant. The trick has been to figure out what prompts them to emerge from this hibernation-like state and start proliferating to heal damaged tissue. Now, Rafii and his colleagues have found that at least in mice, a layer of endothelial cells in the bone marrow called the sinusoidal endothelium is activated through a pathway called Akt. The Akt-activated endothelial cells subsequently turn on genes—the researchers identified nearly 250—that produce growth factors. This robustly upregulates the blood stem cells’ proliferation and differentiation into mature cells, so that they can completely regenerate the bone marrow.

To determine whether it was possible to trigger stem cell proliferation in live animals, the scientists developed a mouse model in which they could selectively activate the endothelium of adult mice. When they triggered Akt-activation only in the endothelial cells, the number of stem cells in the bone marrow increased 10-fold. When the activation was switched off, the number of stem cells decreased again.

“It’s all about cross-talk between endothelium and blood cells,” Rafii says. “Somehow when the number of blood cells decreases, endothelial cells are activated and produce the proper set of growth factors to replenish stem cells and help them regenerate the bone marrow.”

To investigate whether the endothelial-stem cell relationship was limited to bone marrow, the researchers then moved into another organ. They focused their study on the liver, which contains the same type of sinusoidal endothelial cells that spurred growth in the bone marrow. (The cells are also present in the spleen.) In work described in the Nature paper, they showed that sinusoidal endothelial cells in the liver can also be manipulated so that they prompt that organ’s facultative stem cells, hepatocytes, to expand and regenerate lost tissue.

To do this, they took advantage of the liver’s ability to regenerate. In mice, when one or more of the five lobes of the liver is removed, the remaining ones will grow until the organ has been restored to its original size and weight. Bi-Sen Ding, a post-doctoral fellow in Rafii’s lab, removed three lobes of the livers of mice that lacked key genes that help regulate activation of the sinusoidal endothelial cells. Without a fully-functional endothelium, the mice were unable to regrow any liver tissue at all.

To ensure that what they were seeing was a direct result of impaired growth-factor production, the researchers genetically engineered abnormal endothelial cells from some of their knock-out mice to produce the right hepatocyte-active growth factors, including Wnt2 and hepatocyte growth factor (HGF). Then, they transplanted those cells back into the mice that had been unable to regenerate their livers. With their endothelial-derived growth factors restored, the livers began regenerating.

“For the last decade, physician-scientists have been trying to transplant hepatocytes to regenerate the liver. But they grow for a few months then the majority die off,” Rafii says. “Based on our data, one could argue that just transplanting hepatocytes is not going to work. To regenerate long-lasting liver, we may need to transplant hepatocytes with the properly activated endothelium, which produces the right growth factors for the hepatocytes to attach, grow, and connect with other parts of the liver. Co-transplantation of primed activated endothelium with liver cells may be an important step to design future therapies to regenerate liver.”

The lab is already building evidence that activation of the endothelium may play an essential role in prompting tissue regeneration in the lungs and pancreas. Rafii predicts their research will ultimately be applicable to many organs, including the heart and brain, and possibly even have a role in preventing tumor growth.

“One of the most remarkable findings of our studies is the realization that endothelial cells within each organ are functionally different, and once activated produce unique sets of growth factors,” Rafii explains. “The challenge that lies ahead is to discover the organ-specific growth factors produced by the endothelial cells that initiate the regeneration of that particular organ. Then, these factors could be exploited therapeutically to induce selective regeneration of one organ without affecting others.”

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