Photograph of rat liver, stripped of rat-specific cells and re-seeded with engineered human liver cells.[UPMC]

Scientists at the University of Pittsburgh School of Medicine claim to be the first to have successfully grown genetically modified miniature 3D human livers in the laboratory, which mimic the progression of non-alcoholic fatty liver disease (NAFLD) and can be used to test candidate drugs. The team’s proof-of-concept studies using the mini livers are published in Cell Metabolism, and indicate how a human gene that is involved in key metabolic processes regulates fat accumulation in the liver. The studies may also explain why one drug candidate against fatty liver disease that seemed promising in mouse models, wasn’t effective in clinical trials.

“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, MD, PhD, associate professor of pathology at the University of Pittsburgh School of Medicine, and faculty member of the McGowan Institute for Regenerative Medicine and the Pittsburgh Liver Research Center. Soto-Gutierrez and colleagues reported on their achievements in a paper titled, “Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.”

NAFLD is a complex disorder that can increase the risk of diabetes mellitus, cardiovascular and kidney diseases, and potentially lead to liver failure, the authors explained. Already affecting more than 100 million people in the United States, the incidence of NAFLD is rapidly on the rise in parallel with the global epidemic of obesity and diabetes. Liver failure resulting from progressive NAFLD-related damage and liver cirrhosis is now the second leading indication for liver transplantation. “However, a shortage of donor livers, the high cost of the procedure, and the requirement for patients to remain on lifelong immunosuppression limit the use of transplantation.”

Environmental, lifestyle, and genetic factors may all play a role in the development and progression of NAFLD, but, the researchers noted, the underlying mechanisms aren’t well understood, “… tools for early detection and knowledge of the molecular mechanisms responsible for NAFLD and its progression remain elusive.” There are clues from studies in mouse models and on liver samples from NAFLD patients, which have collectively implicated deacetylase sirtuin-1 (SIRT1). “There is increasing evidence, based on rodent studies, that SIRT1 plays a significant role in liver metabolism, and it is implicated in the development of NAFLD,” the investigators wrote. “Several studies have demonstrated a decrease in liver SIRT1 expression on liver biopsies from patients with NAFLD and plasma levels have been found to be lower in patients with severe liver steatosis.”

Given the apparent role for SIRT1 in NAFLD a SIRT1 agonist, resveratrol, has been tested in both animals and humans. Unfortunately, clinical trials have been “inconclusive,” the investigators noted. They suggest that this failure may be partly because animal models don’t exactly recapitulate human NAFLD. “These results could be explained by interspecies differences between the disease in man and the disease in current animal models of NAFLD, which do not accurately replicate the full spectrum of the disease, from insulin resistance and associated metabolic abnormalities to progression to NASH, cirrhosis, and hepatocellular carcinomas.” As Soto-Gutierrez pointed out, “Mice aren’t humans. We are born with certain mutations, polymorphisms, that will predispose us to certain diseases, but you can’t study polymorphisms in mice.”

Ideally, scientists would be able to study the disease in human tissue, but human hepatocytes aren’t widely available, culturing the cells is tricky, and hepatocytes derived from different donors would have very different genetic make-up. Taking a different approach, the University of Pittsburgh team, working with collaborators in the U.S., China, Japan, and Germany, developed a process for making mini customized human livers. They first engineered human fibroblasts to express a chemically activated switch that could disable SIRT1 production. They then deprogrammed these engineered fibroblasts back into induced pluripotent stem cells (iPSCs), and triggered the iPSCs to redifferentiate into liver cells, which they called iHeps. Initial tests with the iHeps showed that switching off mRNA expression of SIRT1 speeded fat accumulation. When exposed to fatty acids, these cells accumulated more triglycerides than did control cells.

Photograph of the organ perfusion and culture system. [UPMC]
Next, the team seeded the genetically engineered human liver cells—together with human mesenchymal cells, fibroblasts, and macrophages—into rat livers that had been denuded of their own cells. The constructs grew into functional 3D mini livers with blood vessels and other features of normal liver structures. Although the resulting mini livers didn’t have the defined metabolically functional zones of a full human liver, they were nevertheless structured distinctly from “organoids”, which are more simple balls of cells that self-assemble to replicated simplified organ function.

The researchers next turned off the SIRT1 gene in the mature mini livers, which then started to exhibit the hallmarks of metabolic dysfunction typical of liver tissue from patients with fatty liver disease. “… the human iPSC-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype, and shared a similar lipid and metabolic profiling to human fatty livers.” Interestingly, resveratrol wasn’t effective in these human mini livers, just as it had been ineffective in clinical trials, although it had worked in mouse models of NAFLD.

Soto-Gutierrez explained that the reason for this may be that resveratrol increases the activity of the SIRT1 protein, not the SIRT1 gene. So, suppressing the SIRT1 gene in the bioengineered livers, which is potentially what happens in NAFLD patients, means that there is no protein produced for the resveratrol to act on, so the drug doesn’t work. “That’s an insight that could only come from studying functional human tissue,” Soto-Gutierrez said.

The investigators acknowledge that the mini liver technology does have its limitations, but suggest that the approach could be used to interrogate other pathways, molecules, and diseases, and allow scientists to create genetically engineered, lab-grown mini livers as a test-bed for drugs at different stages of disease progression. “These studies outline a strategy for custom-engineering liver tissue for the purpose of interrogating disease pathogenesis for the study of NAFLD in man,” they wrote. “Biofabrication of genetically edited human liver tissue may become an important tool for investigating human liver biology and disease.”

As Soto-Gutierrez concluded, “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.”

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