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Damage to the liver in patients developing end-stage liver disease has become too severe for the organ’s normally extraordinary regenerative capacity to repair or compensate for that damage. Once this point of no return has been reached the only option is an organ transplant. However, donor livers are in high demand and very limited supply.

Ambitious efforts are on the way that eventually could enable the engineering of entire implantable liver organs. However, the maximum size of laboratory-engineered liver constructs remains limited and cannot yet provide therapeutic benefits for patients. A research team at the Wyss Institute at Harvard University, Boston University, and MIT has now 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,” said Christopher Chen, MD, PhD, a Wyss Institute core faculty member and the William Fairfield Warren Distinguished professor of biomedical engineering and director of the Biological Design Center at Boston University. “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.”

Chen co-led the research together with associate faculty member Sangeeta Bhatia, MD, PhD, who is 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 is also 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.

The project, spearheaded by Amy Stoddard, PhD, (MIT ’25), who developed the approach in her doctoral research and then as a postdoctoral fellow, integrates tissue engineering and synthetic biology tools in a genetic strategy the team has named “bioengineered on-demand outgrowth via synthetic biology triggering,” or BOOST. By specifically rewiring the gene expression of primary liver hepatocytes and supportive fibroblast cells, the scientists were able to effectively switch on a tissue growth program in small, engineered liver constructs after their implantation into mice.

“Using engineered liver tissue as a proof-of-concept application, we integrated synthetic biology and tissue engineering tools to build liver tissues that can be expanded on-demand after implantation in vivo,” the team reported in their published paper in Science Advances, which is titled “Synthetic control of implanted engineered liver tissue growth.” In the paper they concluded “In this study, we define the first steps toward an unconventional approach to cell therapy scale-up: engineering a small construct and then inducing it to grow in situ … “This strategy, which we have named BOOST, could provide several key advantages, including circumventing the need for large quantities of cellular raw materials and bypassing the formidable challenge of generating a rapidly perfusable construct that can survive the engraftment period.”

The authors wrote, “Organ transplant is currently the only curative treatment for patients with end-stage organ failure, yet this therapy is inaccessible to many due to the paucity of organs available for transplant.” And while significant progress has been made in the field of engineering tissue-based cell therapies that could represent alternatives, or bridges to transplant, they acknowledge, “… scaling of these constructs to sizes of therapeutic relevance remains a barrier to clinical translation.”

In order to address current challenges associated with fabrication, Chen and colleagues looked at the problem from different angle, asking whether it would be possible to first implant a small-scale construct and then trigger it to expand in situ, after its engraftment into the host.

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. “A key first step toward this method of in situ scale-up would be the successful control of cellular growth within the engineered construct after engraftment,” they wrote. 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,” said Stoddard. “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. 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 a protein, YAP, that senses mechanical signals, and 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 overexpressed 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,” Stoddard said. “Interestingly, we found that HEPs needed to be stimulated with both YAP and GFs in order to grow in densely packed 3D liver tissues.”

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. “We set out to engineer a synthetic biology toolkit capable of locally modulating growth factor and YAP signaling within engineered liver tissue, enabling on-demand control of proliferation even after implantation,” they noted.

The team 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 doxycycline (DOX)-inducible. They 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. On DOX removal the HEPs reverted back to a non-proliferating state.

However, Stoddard noted, “… 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 trade-off 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.”

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. Experiments showed that the implanted tissue exhibited a striking 500% increase in proliferation with a doubling of the engineered HEPs alone, and was vascularized to accommodate the metabolic demands of the expanded tissue. The tissue implants were also well tolerated by the mice, with no signs of fibrosis due to invading immune cells and fibroblast inflammation, or of 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,” Bhatia noted. “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.”

In their paper the authors concluded, “… this work serves as an exciting proof-of-concept demonstration that scale-up of tissues via growth could be possible … Together, this work helps lay the foundations for a future of ‘smart’ tissue therapeutics that can be scaled to a patient’s needs and thereby offer treatment for numerous, previously incurable, diseases.”

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