STANFORD, Calif.—While the regenerative promise of stem cells continues to be a huge draw for research in that field, moral quandaries and the risk of tumor generation remain as stumbling blocks. But a team of scientists at Stanford University School of Medicine has discovered a new method that can bypass both issues: turning fat into liver cells.
For this experiment, the researchers used adipose stem cells from human liposuction aspirates, which then developed into human, liver-like cells and were transplanted into mouse models of liver damage. While a healthy liver is capable of regenerating to a certain extent, the organ isn’t capable of overcoming damage from issues such as acute liver poisoning, chronic alcoholism or viral hepatitis. A method of addressing liver damage other than transplantation—which is risky and often requires patients to take immunosuppressant drugs for the rest of their lives—could be a huge boon, as approximately 25,000 hospitalizations each year are the result of acute liver failure from acetaminophen, with some 6,300 transplants performed each year in the United States alone.
A method for converting adipose stem cells to liver-like cells—also known as induced hepatocytes, or i-Heps—was developed in 2006 by a team of Japanese researchers, but it required chemical stimulation and took more than a month.
Gary Peltz, M.D., Ph.D., professor of anesthesia and senior author of the study, and colleagues used a technique known as spherical culture, adapted by Dan Xu, Ph.D., a postdoctoral scholar and lead author of the study. This method cultures adipose stem cells in a liquid suspension where they form spheroids. With this approach, the team was able to covert the adipose cells to liver cells in only nine days, with an efficiency of 37 percent. As a result of working to improve the process, the team has, since the publication of the study, reached yields of more than 50 percent within seven to eight days.
The liver cells were then injected into immune-deficient laboratory mice capable of accepting human grafts. The mice also carry an extra gene that converts gancyclovir, an antiviral compound, into a potent toxin, so that when the mice are treated with gancyclovir, liver cells die off quickly, simulating acute liver damage in humans.
The team injected five million i-Heps into the mice’s livers, and four weeks later, samples of the mice’s blood presented with the protein human serum albumin, which is only produced by human liver cells. Substantial levels of the protein were found in the blood—nearly tripling in the following four weeks—and corresponded with the repopulation of nearly 10 percent to 20 percent of the mice’s destroyed livers by new human liver tissue. In addition, the new liver tissue was undertaking standard waste-filtration responsibilities, with the transplanted cells having integrated into the liver, expressed surface markers of mature human hepatocytes and produced the multi-cell structures necessary for human bile duct formation. In addition, the spherically cultured i-Heps more closely resembled normal human hepatocytes than did i-Heps generated from iPS cells.
One of many benefits of this approach is that it does not require a large amount of adipose cells to work with, says Peltz.
“The liver is the largest organ in your body, so that’s the challenge—the liver has about 200 billion cells, and you may need somewhere around 100 billion cells to replace it, maybe a little less, but somewhere on that order,” he explains. “We think we can generate a sufficient amount of cells from one liter of liposuction material. One liter is not a huge amount. It is possible we could do it from even less, because we’re working out more efficient methods for doing it, but that’s an easily attainable amount of material.”
Even better news is that two months after the spherically cultured i-Heps were injected into the mice, there was no sign of tumor formation. This is a significant change over i-Heps generated from iPS cells, which developed multiple tumors within three weeks.
“At present, we see a very low risk for malignant potential for forming tumors, and that was what was shown in the paper: while the iPS cells would readily form tumors after they were transplanted, our cells do not,” says Peltz. “They have a normal karyotpye, meaning their chromosomes look normal, and so far, they seem to not have the malignant potential associated with the pluripotent state of the iPS cells; however we’re in the midst of studies now to further test that and make sure that everything is safe. But it looks very, very promising from a risk of tumor, which is the major risk with organ regeneration.”
This approach has potential beyond hepatocytes, according to Peltz, who says they are thinking of working with other cell types to tackle other kinds of diseases. Other work in their lab includes modeling human genetic liver diseases in mice to try and reproduce the disease in mouse tissue, and investigating whether these mouse models with human livers are capable of predicting human drug toxicities to various pharmacological compounds.
Stanford’s Office of Technology Licensing has filed a patent on the use of the spherical culture method, and as they move this research forward, the team is optimizing the process and working to scale things up for human studies. Demonstrating the safety to be in line with U.S. Food and Drug Administration mandates is one of their next orders of business, says Peltz, who estimated that this method could be ready for clinical trials in two to three years.