Researchers grow genetically modified miniature human livers in the laboratory to emulate human liver disease progression and test therapeutics


Researchers at the University of Pittsburgh School of Medicine are the first to grow genetically modified miniature human livers in the laboratory, to emulate human liver disease progression and test therapeutics.

In a proof-of-concept paper published today in Cell Metabolism, Pitt researchers chronicle how they transformed genetically engineered human cells into functional, 3-D liver tissue that mimics non-alcoholic fatty liver disease (NAFLD) – a condition involving fat buildup in the liver, which can lead to cirrhosis or even liver failure.

With obesity rates in America climbing, NAFLD is quickly becoming the leading cause of chronic liver disease.

“This is the first time we can create genetically engineered human mini livers with a disease using stem cells in the lab,” said senior author Alejandro Soto-Gutierrez, M.D., Ph.D., associate professor of pathology at Pitt’s School of Medicine and faculty member of the McGowan Institute for Regenerative Medicine and the Pittsburgh Liver Research Center.

That’s important not only for understanding what causes the disease and how it progresses, but also for testing therapeutics. It’s common for drugs to fail in clinical trials, despite promising results in mice.

For instance, the drug Resveratrol, which acts on SIRT1 proteins commonly associated with NAFLD, was effective in mouse models, but failed in human clinical trials.

“Mice aren’t humans,” Soto-Gutierrez said. “We are born with certain mutations, polymorphisms, that will predispose us to certain diseases, but you can’t study polymorphisms in mice, so making a mini customized human liver is advantageous.”

First, the researchers genetically engineered normal human skin cells to express a chemically activated switch that could tamp down the SIRT1 gene.

Then, they reprogrammed the cells back to their stem cell state and turned them into liver cells.

After that, they seeded the genetically engineered human liver cells into rat livers stripped of their own cells, where they blossomed into functional 3-D mini livers, with blood vessels and other structural features of a normal organ.

Pitt first to grow genetically engineered mini livers to study disease and therapeutics
Photograph of rat liver, stripped of rat-specific cells and re-seeded with engineered human liver cells. Credit: UPMC

That structure is part of what distinguishes mini livers from ‘organoid’ cultures – tiny balls of cells that self-assemble to replicate simplified organ function – although the mini livers lacked the distinct zones of metabolic function that normal livers have.

Once the mini livers were mature, the researchers flipped the genetic switch to suppress the SIRT1 gene, and the bioengineered mini livers started to mimic the metabolic dysfunction observed in tissues from patients with fatty liver disease.

But just like the clinical trials, Resveratrol wasn’t effective in the lab-grown livers either.

The key, Soto-Gutierrez explained, is that Resveratrol boosts the activity of SIRT1 proteins, not SIRT1 genes. If SIRT1 gene expression is suppressed – like it is in his bioengineered livers, and perhaps also NAFLD patients – there isn’t any protein to act on, so the drug won’t work. It’s targeting the wrong step.

“That’s an insight that could only come from studying functional human tissue,” Soto-Gutierrez said.

Genetically engineered, lab-grown mini livers provide a ready and reliable test-bed for drugs at all stages of disease progression.

Photograph of the organ perfusion and culture system. Credit: UPMC

“These mini livers aren’t ready for clinical applications like transplantation anytime soon, but I imagine in the future we can make human livers where you can order what kind of function you want, or even enhance function,” Soto-Gutierrez added.

Several of the authors have patents on technology used in this paper (WO/2011/002926m, WO/2015/168254, PCT/US2018/018032, PCT/US2017/044719) as well as financial interests in Von Baer Wolff, Inc., a company aiming to produce iPS-derived human liver cells and treating liver failure by reprogramming therapy. The current study is not associated with Von Baer Wolff, Inc.

Japan’s government approved a request by scientists to conduct stem-cell experiments to create an animal-human hybrid and allow it to be brought to term.

Japan’s science ministry last week gave provisional approval to a proposal from researchers at the University of Tokyo to create animal embryos that contain human cells and transplant them into surrogate animals, Japan’s Asahi Shimbun newspaper reported.

The study would create a human pancreas in rodents using human induced pluripotent stem (iPS) cells.

These are cells that have been reprogrammed back into an embryo-like state and can be used to create virtually any other type of cell.

Final approval from the ministry is expected next month, according to the British scientific journal Nature.

According to Asahi, researchers will take fertilized eggs from rodents that have been gene-edited to remove the ability to produce a pancreas themselves.

To this, they will add human iPS cells to create hybrid animal-human embryos.

These in turn would be implanted into a host animal, in this case a rat or mouse, and allowed to grow.

According to researchers, the goal of the experiment is to create organs viable for transplant into humans.

“Finally, we are in a position to start serious studies in this field after 10 years of preparation,” said Hiromitsu Nakauchi, a researcher at the Institute of Medical Science of the University of Tokyo, according to Asahi.

“We don’t expect to create human organs immediately, but this allows us to advance our research based upon the know-how we have gained up to this point.”

Japan had previously banned such experiments.

It reversed its decision in March of this year after consulting with experts.

The government has said that experiments like these can take place as long as researchers take steps to prevent the birth of a creature that contains a mix of animal and human genetics.

Researchers will also monitor the animal-human hybrids once they are born for up to two years and will suspend the experiment if they detect that brains inside the growing animals contain more than 30% human cells, Asahi said.

While other countries have experimented on creating human-animal embryos, Japan is now the first country to support experiments that will allow the animals with human cells to come to full term.

Scientists in the US have experimented with pig-human hybrid fetuses and allowed them to develop for three to four weeks before destroying them, as required by US ethics regulations.

But according to Jun Wu, a biologist at the Salk Institute in La Jolla, California, only about 1 in 100,000 cells in the fetuses were human.

In the UK, scientists at King’s College London, Newcastle University, and Warwick University created dozens of hybrid embryos that were used to create embryonic stem cells that could potentially treat a wide range of illnesses.

Some bioethicists are disturbed by the possibility that human cells implanted into animals may behave unexpectedly, and could affect an animal’s brain or cognition.

“It is problematic, both ethically and from a safety aspect, to place human iPS cells, which are still capable of transforming into all types of cells, into the fertilized eggs of rats and mice,” said Jiro Nudeshima, a researcher specializing in the ethical implications of life science research, according to Asahi.

Nakauchi, the researcher proposing the experiment, dismissed concerns, saying that his experiments are focused on the creation of specific organs, and not the development of a new species.

“We are trying to do targeted organ generation, so the cells go only to the pancreas,” he said, according to Nature.

He added that in his previous experiments, the number of human cells inside of a sheep embryo has been extremely small.

“The number of human cells grown in the bodies of sheep is extremely small, like one in thousands or one in tens of thousands,” he said, according to Asahi.

“At that level, an animal with a human face will never be born,” Nakauchi said.

More information:Cell Metabolism (2019). DOI: 10.1016/j.cmet.2019.06.017

Journal information: Cell Metabolism
Provided by University of Pittsburgh


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