Improved Organoid Research Pinpoints Genetic Variables Tied to NASH

How Organoids Created from Multiple Patient Samples Improve Our Knowledge of Genetic Susceptibility to NASH

A new method of creating miniature versions of human liver tissue using stem cells derived from multiple patient samples allows researchers at Cincinnati Children’s to pinpoint genetic variables that help protect or make someone more susceptible to non-alcoholic steatohepatitis (NASH).

Takanori Takebe, MD, Ph.D., is the director for Commercial Innovation at the Cincinnati Children’s Center for Stem Cell and Organoid Research and Medicine. He leads a team that created the process, called pooled organoid panel (POP). The panel provides a more efficient, less labor-intensive, and less expensive way to make a testing medium for en masse human organoid phenotyping.

Benefits of a Pooled Organoid Panel

POP replaces the old method of creating individual mediums and using reagents for each specific sample. The pooled panel also prevents variables from being introduced into the mix.

“We run experiments every week, but when we generate individual mediums there are differences depending on the time of induction,” Takebe says. “We wanted to establish a way to make organoids at the same time, all in the same way and in the same generation.”

The pooled approach creates 24 different donor-derived organoids at once, providing a further step to focus on certain diseases like NASH. The organoid panel lets researchers investigate the impact of metabolic status on genotype-phenotype association. 

Takebe and his team published their findings in Cell in Fall 2022.

Understanding How NASH Evolves

NASH is a form of fatty liver disease that shows fat accumulating in the liver plus signs of inflammation and liver cell damage. Nonalcoholic fatty liver disease (NAFLD) occurs when there is fat in the liver but no damage to liver tissue.

Takebe wanted to learn more about how NASH occurs and develops because 1 in 4 or 1in 5 people living in Western countries are diagnosed with the condition or are at risk for developing the condition. Today there are no specific medications approved for treatment. 

“It’s a growing burden to deal with in society,” Takebe says. “We wanted to understand how it’s evolving, how NASH differs across different patient populations, and if there are genetic or other factors.”

Using a pooled panel provides an opportunity to see how NASH forms or acts in different genetic populations and in diabetic and nondiabetic environments. Researchers:

• Induce NASH in a panel 
• Look at the lipid accumulation status in each organoid from each donor
• Refer back to each individual genome sequence

Gene Variations as Risk Factors

Previous studies identified two genetic factors that affect the amount of fat in liver organoids. They are: 

• PNPLA3 (Patatin-like phospholipase domain-containing protein 3) 
• GCKR-rs1260326 (glucokinase regulatory protein)

Takebe’s work drills down by looking at the genetic variations of the GCKR gene. 

How the allele works is a bit complicated, he says. It can promote NASH and protect the liver from it, Takebe says. He decided to study the genetic and metabolic effects of the gene variations as risk factors.

His research shows that the GCKR-TT gene variation leads to more NASH fibrosis in diabetic patients. The same factor leads to a less aggressive NASH state when diabetes is not present.

“We found a new subgrouping strategy for this that shows who is more protected and who is more susceptible,” Takebe says.

Earlier studies identified the GCKR-CC and GCKR-TT factors, but could not delineate other patient factors such as diet, lifestyle, etc. The pooled panel lets researchers capture the genetic information from the organoids using the same lipid and nutrient conditions for all the cells.

Multiple analyses found significant mitochondrial dysfunction incurred by GCKR-rs1260326. This was not reversed with metformin. 

Uncoupling oxidative mechanisms reduced the mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress. These "in-a-dish" genotype-phenotype association strategies separate the opposing roles of metabolic-associated gene variant functions and open the door to creating therapeutic treatments for diabetic NASH.

From Lab Findings to Developing Pharmacotherapies

Knowing that patients with the GCKR-TT risk factor are more at risk for NASH if they have diabetes provides guidance in developing therapies to reduce symptoms and repair mitochondria, Takebe says.

“We are looking downstream. If we know NASH is promoted in diabetic patients, then maybe we should be more proactive for nutrient modulation and other therapies,” Takebe says. “Mitochondrial modulation can be effective in treating the most aggressive patient subset.”

Takebe hopes this knowledge can help develop pharmacotherapies to prevent NASH progressions. Researchers are testing mitochondrial rejuvenators and activators in this patient subset with the goal of preventing fibrosis.

Takebe says his team is working with startup companies to apply their findings.

“The POP studies open a new perspective to address questions about human diversity,” Takebe says. “We see this as a new clinical platform to advance our understanding and practice of medicine in the future for liver and other diseases.”