(BOSTON) — In patients developing end-stage liver disease, the damage has become too severe for the liver's normally extraordinary regenerative capacity to repair or compensate for it. Once this "point of no return" has been reached, the only option is an organ transplant. However, getting a liver transplant is extremely difficult due to high demand and limited supply – about 9,000 to 10,000 people with liver disease are on the U.S. national transplant list at any given time, and roughly 20% of them become too sick to receive a transplant or die while waiting.
Ambitious efforts are on the way that eventually could enable the engineering of entire implantable liver organs. But thus far, the maximum size of laboratory-engineered liver constructs remains limited and cannot provide therapeutic benefits for patients yet. Now, a research team at the Wyss Institute at Harvard University , Boston University , and MIT led by Wyss Institute Core Faculty member Christopher Chen , M.D., Ph.D. and Associate Faculty member Sangeeta Bhatia , M.D., Ph.D., have approached this important problem from a different angle.
"We asked if it would be possible to first implant a small-scale liver construct and then drive it to expand in the body following its engraftment. A sufficiently grown, functional 'satellite liver' could immediately relieve the metabolic burden in a damaged liver and help bridge the time until a transplant becomes available," said Chen. "This project was a natural extension of our longtime collaboration in engineering liver tissue therapeutics, and a perfect combination of Sangeeta's expertise in nanotechnologies and liver bioengineering, and mine in cellular engineering and vascularizaton." Chen is also the William Fairfield Warren Distinguished Professor of Biomedical Engineering and Director of the Biological Design Center at Boston University . He also is a leader of the Wyss Institute's 3D Organ Engineering Initiative, and team lead of the recently awarded ARPA-H PRINT-supported ImPLANT project , which focuses on whole organ liver engineering at the Wyss and collaborating institutions. Bhatia is also the John J. and Dorothy Wilson Professor of Health Sciences and Technology and of Electrical Engineering and Computer Science at the Koch Institute for Integrative Cancer Research at MIT, and a Howard Hughes Medical Institute Investigator.
Chen's and Bhatia's team, which was spearheaded by Amy Stoddard , Ph.D. (MIT '25) who developed the strategy in her doctoral research and then as a postdoctoral fellow, integrated tissue engineering and synthetic biology tools in a genetic strategy they named "bioengineered on-demand outgrowth via synthetic biology triggering," or in short BOOST. By specifically re-wiring the gene expression of primary liver hepatocytes and supportive fibroblast cells, they were able to effectively switch on a tissue growth program in small, engineered liver constructs after their implantation into mice. Their findings are published in Science Advances.
Green signals for engineered liver growth
To be able to induce growth of an implanted small liver constructs in situ within a recipient's body, the researchers first needed to identify the relevant cues that would allow them to do so.
Since liver growth is known to be regulated by soluble growth factors (GFs), Stoddard screened a collection of candidate factors to identify those that, when added to cultured human primary hepatocyte cells (HEPs), had the strongest growth-inducing effects. "We ended up with a set of four growth factors, HGF, TGFa, WNT2 and RSPO3, that potently induced sparsely scattered HEPs to grow in the culture dish. But when we tested whether they could do the same in 3D liver tissues consisting of densely packed HEPs and fibroblasts, they turned out to be ineffective," said Stoddard, who was co-mentored by Chen and Bhatia. "This led us to hypothesize that there must be an additional mechanism at work in human HEPs that inhibits cell proliferation in high-density conditions."
The team homed in on an interesting intracellular protein known as YAP that senses mechanical signals, which was known to move from cells' cytosol to their nucleus in low-density conditions to help express genes involved in cell proliferation. However, in high-density conditions when cells are compressed, YAP is degraded in the cytosol, which prevents the activation of those target genes and restricts proliferation. "Importantly, when we over-expressed a non-degradable version of YAP in HEPs, which also reaches the nucleus in high-density conditions to partake in gene regulation, we successfully overrode this density checkpoint in HEPs. Interestingly, we found that HEPs needed to be stimulated with both YAP and GFs in order to grow in densely packed 3D liver tissues," said Stoddard, adding that "despite nearly a century of research on liver regeneration, the vast majority of insights were gained from studying rodents. We demonstrate in our study that growth regulation of human liver growth is slightly more complicated."
Translation through synthetic biology
Toward the goal of safely inducing and controlling HEP proliferation in a living organism, and eventually human patients, the researchers deployed synthetic biology tools to locally install control of these signaling pathways in HEPs and fibroblast cells within the engineered 3D liver tissues themselves. They engineered fibroblast cell lines that each secreted one of the four GFs, and HEPs that expressed the non-degradable YAP protein. And they made the expression of all proteins "inducible," meaning that they were only produced in the presence of the common and harmless antibiotic doxycycline (DOX), and not anymore once DOX is removed – a common trick used by biologists to study their proteins of interest in a controlled way. The team determined in time course experiments that a continuous seven-day treatment with DOX led 3D liver tissue composed of engineered cells to robustly expand in size and cell numbers in the culture dish, and that removal of DOX reverted HEPs back to a non-proliferating state. "However, when we compared the gene expression of single cells in BOOST-engineered, DOX-induced 3D liver tissue to that of cells in non-engineered or BOOST-engineered, non-induced 3D liver tissue, we noticed that the expansion came with a trade-off: high proliferation rates went hand in hand with a less functional HEP state. While we believe this is a natural tradeoff seen in a wide variety of biological settings, we hope to be able to address this in the future, recognizing that the liver also has native re-functionalization signals to harness," explained Stoddard.
However, the litmus test for BOOST-engineered growth in 3D liver tissues was to see whether they would similarly expand following their implantation into living mice that were systemically treated with DOX for the same seven-day duration. Indeed, the implanted tissue exhibited a striking 500% increase in proliferation with a doubling of the engineered HEPs alone and it was vascularized to accommodate the metabolic demands of the expanded tissue. They also were well tolerated by the mice, which didn't develop any signs of fibrosis due to invading immune cells and fibroblast inflammation, or tumor growth. "These results were particularly exciting to us", said Stoddard. "prior to our work, injury to the host liver has always been required to trigger hepatocyte engraftment and proliferation. Here we were able to relieve this dependence, and trigger on-demand growth of implanted liver tissue in a completely healthy host". In the future, the team will explore the capacity of BOOSTed liver tissue to rescue the host in the setting of liver injury.
"Our BOOST strategy lays the foundation for a future when solid organ cell therapies can be controlled non-surgically according to the needs of patients and their physicians. Beyond treating liver disease, the premise of BOOST could be applied to other engineered tissue therapeutics that are similarly constrained by challenges associated with tissue scale-up, such as engineered heart or pancreatic tissue to address serious diseases," said Bhatia.
"This collaborative study borne out of the unique expertise of Chris and Sangeeta's labs and their sustained efforts to meet the liver shortage challenge have led to an entirely new solution for liver that may equally valuable for confronting other diseases. This beautifully demonstrates how we work at the Wyss Institute toward changing the lives of patients who are often out of other options," said Wyss Founding Director Donald Ingber , M.D., Ph.D. who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
Other authors on the study were Vardhman Kumar, Constantine Tzouanas, Veronica Hui, Jeffrey Li, Anisha Jain, and Alanna Farrell. The study was supported by the National Institutes of Health (awards #EB00262 and #EBEB033821), the Howard Hughes Medical Institute, Wellcome Leap HOPE program, an NSF Engineering Research Center award, Paul G. Allen Frontiers Group Allen Distinguished Investigator Award, Koch Institute Support Grant (award #P30-CA14051) from the National Cancer Institute, and a Core Center Grant (award #P30-ES002109) from the National Institute of Environmental Health Sciences. Stoddard was supported by an NSF Graduate Research Fellowship (1745302 and 2141064), a Boston University Multicellular Design Program Kilachand Fellowship, and an MIT IMES fellowship.
