An international team has grown up to 20,000 vascularized liver buds at a time and reversed liver failure in 60 percent of mice that received the implants.
When an organ such as the liver becomes diseased and fails, nothing short of a transplant from another human will cure the patient. But one day, physicians may be able to cure end-stage liver disease by using bioengineered organs. An international team is moving one step closer to that goal by growing up to 20,000 vascularized liver buds at a time and reversing liver failure in 60 percent of mice that receive the implants.
Liver buds, also known as hepatic diverticula, are the part of an embryo that gives rise to the liver. When implanted in the mice, the buds behave as functioning livers.
In 2012, Takanori Takebe, now at Yokohama City University in Japan, showed he could grow liver buds by causing a mixture of stem cells and other types of cells to self-assemble to form vascularized liver buds. When transplanted in mice with liver failure, the buds linked with surrounding blood vessels and continued to grow and produce liver-specific proteins and human-specific metabolites.
The process was also general enough to use with other organs, such as kidneys and the pancreas. Yet Takebe's original technique hit several roadblocks: It could not grow the billions of cells needed to create a viable organ; the resulting liver buds were genetically unstable; and the process relied on animal byproducts that cannot be used therapeutically in humans.
Now Takebe and his team have collaborated with Cincinnati Children's Hospital Medical Center to resolve some of the problems that have held the technology back. They reported their findings in a paper published in Cell Reports in December and another in Stem Cell Reports in February.
In his latest work, Takebe's team used human-induced pluripotent stem cells, or iPSCs, to mass-produce vascularized and functional liver buds. This approach means Takebe could theoretically grow live buds from a patient's cells, eliminating the need for immunotherapy after the transplant. Their approach relied on two important elements. First, it used "reverse" screening to determine which progenitor cells would most effectively grow into liver buds. Second, it combined chemistry with engineering to develop a clinical scale platform to mass-produce liver buds.
Before even starting, the researchers ran a large set of "reverse" screen experiments to identify the progenitor cells that most reliably produce liver buds. They were hepatic endoderm cells (which express liver genes), endothelial cells (which line blood vessels), and septum mesenchyme cells (which form bone, cartilage, and fat). They then used iPSCs to produce all three types of liver progenitor cells.
They seeded a mixture of these cells into a specially molded, film-coated micro-well arrays to maximize the liver buds' development. Once in the arrays, these progenitor cells begin to self-organize into three-dimensional liver buds.
Using this process, Takebe and his colleagues grew enough hepatic cells in about two months to transplant them into 114 mice with liver failure. The buds grew into functional tissues and reversed acute liver failure. The bioengineered tissues behave much like late-stage fetal or early postnatal tissues, Takebe says.
The reference to fetal tissue is telling. Last year, Takebe and Barbara Treutlein of the Max Planck Institute for Evolutionary Anthropology described previously unknown genetic-molecular pathways that guided the growth of fetal liver. He has since applied this knowledge to the growth of human liver from iPSCs. "We need to find industry partners and develop a clinical-grade protocol that complies with good manufacturing processes," Takebe says.
In the Stem Cell Reports paper, Takebe and colleagues reported that they have overcome another barrier to productive tissue engineering: producing high volumes of genetically stable tissue.
Tissue generated directly from iPSCs can develop genetic variations or chromosome instability, causing benign tumors. But the researchers manipulated the iPSCs genetically and biochemically using three signaling genes to produce CDX2-positive posterior gut endodermal progenitor cells, or PGECs.
PGECs are programmed to form the gastrointestinal tract, but they have not yet differentiated to form specific types of cells such as liver, intestine, or stomach within the digestive tract. While other researchers have used PGECs to grow organoids, their methods used animal byproducts that regulators would not allow to be used in humans. Takebe's approach used only human iPSCs and so avoided the use of those byproducts.
The researchers transplanted the organoids created using this approach into mice with acute liver failure. Sixty percent of the transplanted mice survived at least 30 days, compared to 20 percent of the mice who did not receive the transplants. While the mass-produced, genetically stable liver buds mark a large step forward, the researchers still have challenges to overcome.
"One limitation associated with the current protocols is the lack of biliary integration," Takebe said. "As the bile duct needs to connect liver to gut, we are now interested in how biliary integration can be achieved during human development so that we can mimic the process in culture."
The researchers are working on automating some aspects of the process. While they will still do complex procedures, such as seeding liver buds, manually, they plan to automate environmental control, plate handling, and medium exchange.
Using techniques outlined in these two papers, the team hopes to start a pediatric clinical trial in the next two to five years to transplant a bioengineered liver into a patient.
Melissa Lutz Blouin is an independent technology writer.
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