A comprehensive review of advances in hepatocyte microencapsulation: selecting materials and preserving cell viability.

Liver failure represents a critical medical condition with a traditionally grim prognosis, where treatment options have been notably limited. Historically, liver transplantation has stood as the sole definitive cure, yet the stark disparity between the limited availability of liver donations and the high demand for such organs has significantly hampered its feasibility. This discrepancy has necessitated the exploration of hepatocyte transplantation as a temporary, supportive intervention. In light of this, our review delves into the burgeoning field of hepatocyte transplantation, with a focus on the latest advancements in maintaining hepatocyte function, co-microencapsulation techniques, xenogeneic hepatocyte transplantation, and the selection of materials for microencapsulation. Our examination of hepatocyte microencapsulation research highlights that, to date, most studies have been conducted in vitro or using liver failure mouse models, with a notable paucity of experiments on larger mammals. The functionality of microencapsulated hepatocytes is primarily inferred through indirect measures such as urea and albumin production and the rate of ammonia clearance. Furthermore, research on the mechanisms underlying hepatocyte co-microencapsulation remains limited, and the practicality of xenogeneic hepatocyte transplantation requires further validation. The potential of hepatocyte microencapsulation extends beyond the current scope of application, suggesting a promising horizon for liver failure treatment modalities. Innovations in encapsulation materials and techniques aim to enhance cell viability and function, indicating a need for comprehensive studies that bridge the gap between small-scale laboratory success and clinical applicability. Moreover, the integration of bioengineering and regenerative medicine offers novel pathways to refine hepatocyte transplantation, potentially overcoming the challenges of immune rejection and ensuring the long-term functionality of transplanted cells. In conclusion, while hepatocyte microencapsulation and transplantation herald a new era in liver failure therapy, significant strides must be made to translate these experimental approaches into viable clinical solutions. Future research should aim to expand the experimental models to include larger mammals, thereby providing a clearer understanding of the clinical potential of these therapies. Additionally, a deeper exploration into the mechanisms of cell survival and function within microcapsules, alongside the development of innovative encapsulation materials, will be critical in advancing the field and offering new hope to patients with liver failure.

A Novel Approach to Orthotopic Hepatocyte Transplantation Engineered With Liver Hydrogel for Fibrotic Livers, Enhancing Cell-Cell Interaction and Angiogenesis.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Kupffer cells abrogate homing and repopulation of allogeneic hepatic progenitors in injured liver site.

BACKGROUND: Allogeneic hepatocyte transplantation is an emerging approach to treat acute liver defects. However, durable engraftment of the transplanted cells remains a daunting task, as they are actively cleared by the recipient's immune system. Therefore, a detailed understanding of the innate or adaptive immune cells-derived responses against allogeneic transplanted hepatic cells is the key to rationalize cell-based therapies. METHODS: Here, we induced an acute inflammatory regenerative niche (3-96 h) on the surface of the liver by the application of cryo-injury (CI) to systematically evaluate the innate immune response against transplanted allogeneic hepatic progenitors in a sustained micro-inflammatory environment. RESULTS: The resulting data highlighted that the injured site was significantly repopulated by alternating numbers of innate immune cells, including neutrophils, monocytes and Kupffer cells (KCs), from 3 to 96 h. The transplanted allo-HPs, engrafted 6 h post-injury, were collectively eliminated by the innate immune response within 24 h of transplantation. Selective depletion of the KCs demonstrated a delayed recruitment of monocytes from day 2 to day 6. In addition, the intrasplenic engraftment of the hepatic progenitors 54 h post-transplantation was dismantled by KCs, while a time-dependent better survival and translocation of the transplanted cells into the injured site could be observed in samples devoid of KCs. CONCLUSION: Overall, this study provides evidence that KCs ablation enables a better survival and integration of allo-HPs in a sustained liver inflammatory environment, having implications for rationalizing the cell-based therapeutic interventions against liver defects.

Irreversible Electroporation of the Liver Increases the Transplant Engraftment of Hepatocytes.

INTRODUCTION: Irreversible electroporation (IRE) is a tissue ablation technology that kills cells with short electrical pulses that do not induce thermal damage, thereby preserving the extracellular matrix. Preclinical research suggests that IRE may be developed as a tool for regenerative surgery by clearing existing host cells within a solid organ and creating a supportive niche for new cell engraftment. We hypothesized that hepatocytes transplanted by injection into the portal circulation would preferentially engraft within liver parenchyma pretreated with IRE. METHODS: Transgene-positive ß-galactosidase-expressing hepatocytes were isolated from B6.129S7-Gt(ROSA)26Sor/J (ROSA26) mice and transplanted by intrasplenic injection into wild-type littermates that received liver IRE pretreatment or control sham treatment. Engraftment of donor hepatocytes in recipient livers was determined by X-gal staining. RESULTS: Significantly higher numbers of X-gal+ donor hepatocytes engrafted in the livers of IRE-treated mice as compared to sham-treated mice. X-gal+ hepatocytes persisted in IRE-treated recipients for at least 11 d post-transplant and formed clusters. Immunostaining demonstrated the presence of HNF4A/Ki67/ß-galactosidase triple-positive cells within IRE-ablation zones, indicating that transplanted hepatocytes preferentially engrafted in IRE-treated liver parenchyma and proliferated. CONCLUSIONS: IRE pretreatment of the liver increased engraftment of transplanted hepatocytes within the IRE-ablation zone. IRE treatment of the host liver may be developed clinically as a strategy to increase engraftment efficiency of primary hepatocytes and/or hepatocytes derived from stem cells in cell transplant therapies.

bFGF-releasing biodegradable nanoparticles for effectively engrafting transplanted hepatocyte sheet.

Hepatic tissue engineering has been applied for the treatment of intractable liver diseases, and hepatocyte sheets are promising for this purpose. However, hepatocyte sheets have poor survival after transplantation because of their high metabolic activity. In this study, we aimed to develop basic fibroblast growth factor (bFGF)-releasing nanoparticles to prolong the survival of hepatocyte sheets after transplantation. The nanoparticles were prepared by electrospraying a bFGF-dispersed poly(D,l-lactide-co-glycolide) emulsion. bFGF-loaded PLGA nanoparticles can be developed by optimizing the applied electrospray voltage and the oil:water ratio of the emulsion. The prepared nanoparticles exhibited prompt release at the initial duration and continuous gradual release at the subsequent duration. Hepatocyte sheet engraftment was evaluated by transplanting hepatocyte sheets containing the prepared nanoparticles into rats. The hepatocyte sheets with the prepared nanoparticles exhibited longer survival than those without the bFGF nanoparticles or solution owing to the local and continuous release of bFGF from the nanoparticles and the subsequent enhanced angiogenesis at the transplantation site. These results indicated that the prepared bFGF-releasing nanoparticles can enhance the efficiency of hepatocyte sheet transplantation. The developed bFGF-releasing nanoparticles would be useful for the transplantation of cellular tissue with post-transplantation survival challenges.

Liver cell therapies: cellular sources and grafting strategies.

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Magnetic Nanostructures and Stem Cells for Regenerative Medicine, Application in Liver Diseases.

The term "liver disease" refers to any hepatic condition that leads to tissue damage or altered hepatic function and can be induced by virus infections, autoimmunity, inherited genetic mutations, high consumption of alcohol or drugs, fat accumulation, and cancer. Some types of liver diseases are becoming more frequent worldwide. This can be related to increasing rates of obesity in developed countries, diet changes, higher alcohol intake, and even the coronavirus disease 2019 (COVID-19) pandemic was associated with increased liver disease-related deaths. Although the liver can regenerate, in cases of chronic damage or extensive fibrosis, the recovery of tissue mass is impossible, and a liver transplant is indicated. Because of reduced organ availability, it is necessary to search for alternative bioengineered solutions aiming for a cure or increased life expectancy while a transplant is not possible. Therefore, several groups were studying the possibility of stem cells transplantation as a therapeutic alternative since it is a promising strategy in regenerative medicine for treating various diseases. At the same time, nanotechnological advances can contribute to specifically targeting transplanted cells to injured sites using magnetic nanoparticles. In this review, we summarize multiple magnetic nanostructure-based strategies that are promising for treating liver diseases.

Cell therapy in end-stage liver disease: replace and remodel.

Liver disease is prevalent worldwide. When it reaches the end stage, mortality rises to 50% or more. Although liver transplantation has emerged as the most efficient treatment for end-stage liver disease, its application has been limited by the scarcity of donor livers. The lack of acceptable donor organs implies that patients are at high risk while waiting for suitable livers. In this scenario, cell therapy has emerged as a promising treatment approach. Most of the time, transplanted cells can replace host hepatocytes and remodel the hepatic microenvironment. For instance, hepatocytes derived from donor livers or stem cells colonize and proliferate in the liver, can replace host hepatocytes, and restore liver function. Other cellular therapy candidates, such as macrophages and mesenchymal stem cells, can remodel the hepatic microenvironment, thereby repairing the damaged liver. In recent years, cell therapy has transitioned from animal research to early human studies. In this review, we will discuss cell therapy in end-stage liver disease treatment, especially focusing on various cell types utilized for cell transplantation, and elucidate the processes involved. Furthermore, we will also summarize the practical obstacles of cell therapy and offer potential solutions.

Liver cell therapies: cellular sources and grafting strategies / 医学前沿

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Domino hepatocyte transplantation using explanted human livers with metabolic defects attenuates D-GalN/LPS-induced acute liver failure.

BACKGROUND: Explanted livers from patients with inherited metabolic liver diseases possess the potential to be a cell source of good-quality hepatocytes for hepatocyte transplantation (HT). This study evaluated the therapeutic effects of domino HT using hepatocytes isolated from explanted human livers for acute liver failure (ALF). METHODS: Isolated hepatocytes were evaluated for viability and function and then transplanted into D-galactosamine/lipopolysaccharide-induced ALF mice via splenic injection. The survival rate was analyzed by the Kaplan-Meier method and log-rank test. Liver function was evaluated by serum biochemical parameters, and inflammatory cytokine levels were measured by ELISA. The pathological changes in the liver tissues were assessed by hematoxylin-eosin staining. Hepatocyte apoptosis was investigated by TUNEL, and hepatocyte apoptosis-related proteins were detected by western blot. The localization of human hepatocytes in the injured mouse livers was detected by immunohistochemical analyses. RESULTS: Hepatocytes were successfully isolated from explanted livers of 10 pediatric patients with various liver-based metabolic disorders, with an average viability of 85.3% ± 13.0% and average yield of 9.2 × 106 ± 3.4 × 106 cells/g. Isolated hepatocytes had an excellent ability to secret albumin, produce urea, uptake indocyanine green, storage glycogen, and express alpha 1 antitrypsin, albumin, cytokeratin 18, and CYP3A4. Domino HT significantly reduced mortality, decreased serum levels of alanine aminotransferase and aspartate aminotransferase, and improved the pathological damage. Moreover, transplanted hepatocytes inhibited interleukin-6 and tumor necrosis factor-α levels. Domino HT also ameliorates hepatocyte apoptosis, as evidenced by decreased TUNEL positive cells. Positive staining for human albumin suggested the localization of human hepatocytes in ALF mice livers. CONCLUSION: Explanted livers from patients with inheritable metabolic disorders can serve as a viable cell source for cell-based therapies. Domino HT using hepatocytes with certain metabolic defects has the potential to be a novel therapeutic strategy for ALF.

Morphology of Acute Liver Failure During Treatment With a Combination of Drug Therapy and Transplanted Cultured Hepatocytes.

OBJECTIVES: We analyzed liver tissue morphology dynamics in experimental animals with acute liver failure during treatment with a combination of transplanted cultured embryonic hepatocytes and the hepatoprotective drug Erbisol. MATERIALS AND METHODS: We studied 30 white outbred male rats weighing 250 to 350 g, divided into 6 groups with 5 animals in each group. Acute liver damage was induced by a hepatotoxic model of acute liver failure for which carbon tetrachloride was applied (200 µg CCl4/100 g body weight with toxin-to-oil volume ratio of 1:1). To obtain cultured embryonic hepatocytes, we used an enzymatic-mechanical process that caused only minimal cell damage. This method is known to improve the output of viable cultured embryonic hepatocytes, improve the morphological-functional properties of the hepatocytes, and reduce the process time during procurement, and thereby reduce the overall time from procurement to the subsequent culturing of the obtained cells. Transplant of cultured embryonic hepatocytes was performed intrape-ritoneally at a dose of 50 million (0.5 × 108) cells. Morphological studies were performed on day 7 and day 21 of the experiment. RESULTS AND CONCLUSIONS: There were significant morphological changes in livers from animals with acute liver failure, caused by widespread necrosis and an elevated inflammatory response. Treatment with injections of cultured embryonic hepatocytes induced regeneration of the liver parenchyma cells and reduced the inflammatory response, both of which were further reduced in rats that received combined treatment of transplant with Erbisol. Combined application of cultured embryonic hepatocytes and Erbisol potentiated the effects of both treatments, which produced intensive proliferation of hepatocytes, hypertrophy and polyploidization of hepatocyte nuclei, and an early restoration of liver structure and organ mass.

Short-term inhalation of isoflurane improves the outcomes of intraportal hepatocyte transplantation.

Clinical hepatocyte transplantation (HTx) is only performed without general anesthesia, while inhalation anesthetics are usually used in animal experiments. We hypothesized that isoflurane may be a possible reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx. Syngeneic rat hepatocytes (1.0 × 107) were transplanted to analbuminemic rats with (ISO group) and without (AW group) isoflurane. The serum albumin, AST, ALT, LDH levels and several inflammatory mediators were analyzed. Immunohistochemical staining and ex vivo imaging were also performed. The serum albumin levels of the ISO group were significantly higher in comparison to the AW group (p < 0.05). The serum AST, ALT, LDH levels of the ISO group were significantly suppressed in comparison to the AW group (p < 0.0001, respectively). The serum IL-1ß, IL-10, IL-18, MCP-1, RNTES, Fractalkine and LIX levels were significantly suppressed in the ISO group. The ischemic regions of the recipient livers in the ISO group tended to be smaller than the AW group; however, the distribution of transplanted hepatocytes in the liver parenchyma was comparable between the two groups. Isoflurane may at least in part be a reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx.

Derivation of healthy hepatocyte-like cells from a female patient with ornithine transcarbamylase deficiency through X-inactivation selection.

Autologous cell replacement therapy for inherited metabolic disorders requires the correction of the underlying genetic mutation in patient's cells. An unexplored alternative for females affected from X-linked diseases is the clonal selection of cells randomly silencing the X-chromosome containing the mutant allele, without in vivo or ex vivo genome editing. In this report, we have isolated dermal fibroblasts from a female patient affected of ornithine transcarbamylase deficiency and obtained clones based on inactivation status of either maternally or paternally inherited X chromosome, followed by differentiation to hepatocytes. Hepatocyte-like cells derived from these clones display indistinct features characteristic of hepatocytes, but express either the mutant or wild type OTC allele depending on X-inactivation pattern. When clonally derived hepatocyte-like cells were transplanted into FRG® KO mice, they were able to colonize the liver and recapitulate OTC-dependent phenotype conditioned by X-chromosome inactivation pattern. This approach opens new strategies for cell therapy of X-linked metabolic diseases and experimental in vitro models for drug development for such diseases.

Transplantation of 3D adipose-derived stem cell/hepatocyte spheroids alleviates chronic hepatic damage in a rat model of thioacetamide-induced liver cirrhosis.

Cirrhosis refers to irreversible liver damage where healthy tissue is replaced by scar tissue, resulting in impaired liver function. There is no cure and current treatments only prevent further liver damage; thus, novel therapeutic options are urgently needed. Here, we report a new approach that enables the formation of self-assembled 3D spheroids of adipose-derived stem cells (ADSCs) and murine hepatocytes (AML12) via reconstituted collagen fibers. Compared with the spheroids formed in the commercially available EZSHERE dish, the collagen fiber-based ADSC/hepatocyte spheroids offer a notable benefit in structure formation and paracrine factor secretion. To test the regenerative capability of the collagen fiber-based 3D ADSC/hepatocyte spheroids, a rat model of thioacetamide (TAA)-induced liver cirrhosis was employed. The transplantation of the collagen fiber-based 3D ADSC/hepatocyte spheroids show an improvement in liver function and ameliorates pathological liver cirrhosis in TAA-treated rats. In summary, our data show collagen fiber-based self-assembled 3D ADSC/hepatocyte spheroids to possess the excellent regenerative capacity in response to TAA-induced liver injury, promising an alternative therapeutic strategy for liver cirrhosis.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

Limited Expansion of Human Hepatocytes in FAH/RAG2-Deficient Swine.

The mammalian liver's regenerative ability has led researchers to engineer animals as incubators for expansion of human hepatocytes. The expansion properties of human hepatocytes in immunodeficient mice are well known. However, little has been reported about larger animals that are more scalable and practical for clinical purposes. Therefore, we engineered immunodeficient swine to support expansion of human hepatocytes and identify barriers to their clinical application. Immunodeficient swine were engineered by knockout of the recombinase-activating gene 2 (RAG2) and fumarylacetoacetate hydrolase (FAH). Immature human hepatocytes (ihHCs) were injected into fetal swine by intrauterine cell transplantation (IUCT) at day 40 of gestation. Human albumin was measured as a marker of engraftment. Cytotoxicity against ihHCs was measured in transplanted piglets and control swine. We initially detected higher levels of human albumin in cord blood of newborn FAH/RAG2-deficient (FR) pigs compared with immunocompetent controls (196.26 ng/dL vs. 39.29 ng/dL, p = 0.008), indicating successful engraftment of ihHCs after IUCT and adaptive immunity in the fetus. Although rare hepatocytes staining positive for human albumin were observed, levels of human albumin did not rise after birth, but declined, suggesting rejection of xenografted ihHCs. Cytotoxicity against ihHCs increased after birth by 3.8% (95% CI: [2.1%-5.4%], p < 0.001) and inversely correlated with declining levels of human albumin (p = 2.1 × 10-5, R2 = 0.17). Circulating numbers of T cells and B cells were negligible in FR pigs. However, circulating natural killer (NK) cells exerted cytotoxicity against ihHCs. NK cell activity was lower in immunodeficient piglets after IUCT than in naive controls (30.4% vs. 40.1%, p = 0.011, 95% CI for difference [2.7%-16.7%]). In conclusion, ihHCs were successfully engrafted in FR swine after IUCT. NK cells were a significant barrier to expansion of hepatocytes. New approaches are needed to overcome this hurdle and allow large-scale expansion of human hepatocytes in immunodeficient swine. Impact statement There is currently a need for robust expansion of human hepatocytes. We describe an immunodeficient swine model into which we engrafted immature human hepatocytes (ihHCs). We identified the mechanism of the eventual graft rejection by the intact NK cell population, which has not been previously shown to have a significant role in xenograft rejection. By both improving engraftment and reducing NK cell-mediated cytotoxicity toward the graft through intrauterine cell transfer, we confirmed the presence of residual adaptive immunity in this model of immunodeficiency and the ability to induce hyposensitization in the NK cell population by taking advantage of the fetal microenvironment.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

Liver Regeneration and Cell Transplantation for End-Stage Liver Disease.

Liver transplantation is the only curative option for end-stage liver disease; however, the limitations of liver transplantation require further research into other alternatives. Considering that liver regeneration is prevalent in liver injury settings, regenerative medicine is suggested as a promising therapeutic strategy for end-stage liver disease. Upon the source of regenerating hepatocytes, liver regeneration could be divided into two categories: hepatocyte-driven liver regeneration (typical regeneration) and liver progenitor cell-driven liver regeneration (alternative regeneration). Due to the massive loss of hepatocytes, the alternative regeneration plays a vital role in end-stage liver disease. Advances in knowledge of liver regeneration and tissue engineering have accelerated the progress of regenerative medicine strategies for end-stage liver disease. In this article, we generally reviewed the recent findings and current knowledge of liver regeneration, mainly regarding aspects of the histological basis of regeneration, histogenesis and mechanisms of hepatocytes' regeneration. In addition, this review provides an update on the regenerative medicine strategies for end-stage liver disease. We conclude that regenerative medicine is a promising therapeutic strategy for end-stage liver disease. However, further studies are still required.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs.

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.