For individuals experiencing the advanced stages of hepatic dysfunction, the impairment has progressed to a point where the liver’s inherent and remarkable restorative capabilities are insufficient to achieve repair or adequate compensation. Upon crossing this critical threshold, the sole recourse available is a hepatic organ transplantation. Nevertheless, securing a liver transplant presents considerable challenges due to the pronounced disparity between the substantial demand and the restricted availability of donor organs. At any given temporal juncture, approximately 9,000 to 10,000 individuals diagnosed with liver ailments are registered on the United States national transplant waiting list. Regrettably, about one-fifth of these patients deteriorate to a state rendering them ineligible for transplantation or succumb while awaiting this life-saving intervention.
Substantial scientific endeavors are currently underway with the ultimate objective of facilitating the fabrication of complete hepatic organs suitable for implantation. However, to date, the maximal dimensions achievable for laboratory-generated hepatic constructs remain constrained and are not yet capable of conferring therapeutic advantages upon patients. Concurrently, a research consortium comprising investigators from the Wyss Institute at Harvard University, Boston University, and MIT, under the principal guidance of Christopher Chen, M.D., Ph.D., a core faculty member at the Wyss Institute, and Sangeeta Bhatia, M.D., Ph.D., an associate faculty member, has adopted an innovative approach to address this pressing issue.
“Our fundamental inquiry was whether it would be feasible to initially introduce a diminutive hepatic construct and subsequently stimulate its growth within the recipient’s organism post-engraftment. A sufficiently developed and functional auxiliary liver could promptly alleviate the metabolic strain on a compromised native liver and serve as a bridge until a definitive transplant becomes attainable,” stated Dr. Chen. “This project represents a logical progression of our longstanding collaborative efforts in the field of engineered hepatic tissue therapeutics, harmoniously integrating Dr. Bhatia’s proficiency in nanotechnologies and hepatic bioengineering with my own expertise in cellular engineering and vascularization.” Dr. Chen also holds the distinguished title of William Fairfield Warren Distinguished Professor of Biomedical Engineering and presides over the Biological Design Center at Boston University. Furthermore, he actively contributes as a leader within the Wyss Institute’s 3D Organ Engineering Initiative and serves as the principal investigator for the recently sanctioned ARPA-H PRINT-supported ImPLANT project, which is dedicated to the comprehensive engineering of hepatic organs across the Wyss Institute and its collaborating institutions. Concurrently, Dr. Bhatia occupies the esteemed positions of John J. and Dorothy Wilson Professor of Health Sciences and Technology, and Professor of Electrical Engineering and Computer Science at the Koch Institute for Integrative Cancer Research at MIT, and is also a distinguished Investigator with the Howard Hughes Medical Institute.
The research collective spearheaded by Drs. Chen and Bhatia, notably propelled by the pioneering strategy developed by Amy Stoddard, Ph.D. (MIT ’25) during her doctoral investigations and subsequent postdoctoral tenure, has ingeniously fused tissue engineering methodologies with synthetic biology principles. This integrated approach, christened “bioengineered on-demand outgrowth via synthetic biology triggering” or BOOST, was implemented to effectively orchestrate cellular behavior. By specifically re-engineering the gene expression profiles of primary hepatic cells (hepatocytes) and supportive stromal cells (fibroblasts), the researchers successfully initiated a programmed tissue expansion within small, bioengineered hepatic constructs subsequent to their implantation into murine models. The empirical findings stemming from this groundbreaking research have been disseminated in the esteemed journal, Science Advances.
Indications for Bioengineered Hepatic Growth
To successfully induce the proliferation of an implanted, diminutive hepatic construct *in situ* within the host organism, the scientific team first undertook the critical task of identifying the requisite endogenous signals that would facilitate such a process.
Recognizing that hepatic growth is inherently modulated by soluble growth factors (GFs), Dr. Stoddard conducted a systematic evaluation of a selection of candidate factors. The objective was to ascertain which factors, when introduced to cultured human primary hepatocytes (HEPs), exhibited the most pronounced growth-stimulatory effects. “Our investigation ultimately pinpointed a quartet of growth factors—HGF, TGFa, WNT2, and RSPO3—which demonstrated potent capacity to induce proliferation in sparsely distributed HEPs within a standard culture dish. However, upon evaluating their efficacy in three-dimensional hepatic tissues comprising densely packed HEPs and fibroblasts, these factors proved to be ineffective,” reported Dr. Stoddard, who benefited from the dual mentorship of Drs. Chen and Bhatia. “This observation led us to hypothesize the existence of an additional regulatory mechanism operative in human HEPs that actively impedes cellular proliferation under conditions of high cell density.”
The research group subsequently focused their attention on a notable intracellular protein known as YAP. This protein possesses the capacity to sense biomechanical stimuli and, under conditions of low cell density, translocates from the cellular cytoplasm to the nucleus, thereby promoting the transcription of genes associated with cellular proliferation. Conversely, in dense cellular environments, mechanical compression leads to the degradation of YAP within the cytoplasm, which effectively abrogates the activation of these proliferative genes and imposes a constraint on cell division. “Crucially, when we engineered HEPs to overexpress a variant of YAP resistant to degradation, which retained its nuclear localization even in high-density conditions to participate in gene regulation, we successfully circumvented this density-dependent restriction on HEP proliferation. Interestingly, our findings indicated that sustained cellular expansion in densely packed three-dimensional hepatic tissues necessitated stimulation by both YAP and specific GFs,” Dr. Stoddard elaborated, further noting, “despite nearly a century of dedicated research into liver regeneration, the preponderance of insights has been derived from studies conducted in rodent models. Our research conclusively demonstrates that the regulation of human hepatic growth exhibits a somewhat more intricate regulatory landscape.”
Translational Advancement via Synthetic Biology
In pursuit of the overarching objective to safely induce and precisely govern HEP proliferation within a living organism, with eventual applicability to human patients, the research team harnessed advanced synthetic biology tools. These tools were employed to locally orchestrate the activity of these signaling pathways within the engineered three-dimensional hepatic tissues themselves, specifically targeting HEPs and fibroblast cells. They engineered distinct fibroblast cell lines, each designed to secrete one of the four identified GFs, and developed HEPs engineered to express the constitutively active, non-degradable YAP protein. Moreover, the expression of all these engineered proteins was rendered “inducible,” meaning their production was contingent upon the presence of doxycycline (DOX), a widely utilized and benign antibiotic. Protein synthesis ceased upon the withdrawal of DOX, a standard technique employed by researchers to effect controlled expression of target proteins. Through meticulous time-course experiments, the team established that a continuous seven-day exposure to DOX resulted in robust volumetric expansion and a significant increase in cell numbers within the engineered three-dimensional hepatic tissues cultured *in vitro*. Subsequent removal of DOX effectively reversed the HEPs to a quiescent, non-proliferative state. “However, when we performed single-cell gene expression analyses comparing BOOST-engineered, DOX-induced three-dimensional hepatic tissue with non-engineered or BOOST-engineered, non-induced hepatic tissue, we observed a discernible trade-off accompanying the enhanced proliferation: elevated rates of cell division were correlated with a diminution in HEP functional capacity. While we acknowledge this as a potential inherent compromise frequently encountered across diverse biological contexts, we are optimistic about addressing this limitation in future investigations, recognizing that the liver also possesses endogenous mechanisms for regaining functionality that can be leveraged,” explained Dr. Stoddard.
Nevertheless, the definitive validation of the BOOST strategy for engineered growth within three-dimensional hepatic tissues was contingent upon demonstrating analogous expansion following implantation into live murine subjects subjected to systemic DOX administration for the identical seven-day period. Indeed, the implanted hepatic constructs exhibited a remarkable 500% increase in cellular proliferation, characterized by a doubling of the engineered HEP population alone. Furthermore, the constructs became well-vascularized, adequately supporting the metabolic demands of the augmented tissue mass. The implants were also exceptionally well-tolerated by the murine recipients, with no evidence of fibrosis resulting from infiltrating immune cells and associated inflammatory responses, nor did tumor formation occur. “These findings were particularly exhilarating for us,” remarked Dr. Stoddard. “Prior to our research, inducing hepatocyte engraftment and proliferation invariably necessitated antecedent injury to the host liver. In our study, we have successfully obviated this requirement, enabling the controlled, on-demand expansion of implanted hepatic tissue in a completely healthy host.” Future research will investigate the capacity of BOOST-engineered hepatic tissue to ameliorate host conditions in scenarios of hepatic injury.
“Our BOOST methodology establishes a foundational framework for a future where solid organ cell therapies can be administered and precisely controlled non-surgically, adapting to the evolving clinical needs of patients and their attending physicians. Beyond the treatment of hepatic diseases, the fundamental principles underlying the BOOST approach hold significant promise for application to other categories of engineered tissue therapeutics that encounter analogous constraints related to tissue scale-up. This includes, but is not limited to, engineered cardiac or pancreatic tissues intended for the management of severe systemic illnesses,” stated Dr. Bhatia.
“This collaborative undertaking, born from the distinct and complementary expertise resident within the laboratories of Drs. Chen and Bhatia, and their persistent dedication to mitigating the critical shortage of donor livers, has yielded a profoundly innovative solution for hepatic regeneration. This innovative paradigm may prove equally efficacious in confronting other challenging diseases. It serves as a compelling illustration of our operational ethos at the Wyss Institute, which is fundamentally geared towards transforming the lives of patients who frequently find themselves with limited alternative treatment options,” commented Wyss Founding Director Donald Ingber, M.D., Ph.D., who also holds the positions of Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Hansjörg Wyss Professor of Biologically Inspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
