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The safety and short-term efficacy of hepatocyte transplantation (HCTx) have been widely proven. However, issues such as reduced viability and/or function of hepatocytes, insufficient engraftment, and lack of a long-term effect have to be overcome for widespread application of HCTx. In this study, we evaluated hepatocyte spheroids (HSs), formed by self-aggregation of hepatocytes, as an alternative to hepatocytes in single-cell suspension. Hepatocytes were isolated from C57BL/6 J mice liver using a three-step collagenase perfusion technique and HSs were formed by the hanging drop method. After the spheroids formation, the HSs showed significantly higher mRNA expression of albumin, ornithine transcarbamylase, glucose-6-phosphate, alpha-1-antitrypsin, low density lipoprotein receptor, coagulation factors, and apolipoprotein E (ApoE) than 2 dimensional (2D)-cultured hepatocytes ( p < 0.05). Albumin production by HSs was significantly higher than that by 2D-cultured hepatocytes (9.5 ± 2.5 vs 3.5 ± 1.8 μg/dL, p < 0.05). The HSs, but not single hepatocytes, maintained viability and albumin mRNA expression in suspension (92.0 ± 2.8% and 1.03 ± 0.09 at 6 h). HSs (3.6 × 10 6 cells) or isolated hepatocytes (fSH, 3.6 × 10 6 cells) were transplanted into the liver of ApoE knockout (KO-/-) mice via the portal vein. Following transplantation, serum ApoE concentration (ng/mL) of HS-transplanted mice (1w: 63.1 ± 56.7, 4w: 17.0 ± 10.9) was higher than that of fSH-transplanted mice (1 w: 33.4 ± 13.0, 4w: 13.7 ± 9.6). In both groups, the mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, MCP-1, and MIP-1β) were upregulated in the liver following transplantation; however, no significant differences were observed. Pathologically, transplanted HSs were observed as flat cell clusters in contact with the portal vein wall on day 7. Additionally, ApoE positive cells were observed in the liver parenchyma distant from the portal vein on day 28. Our results indicate that HS is a promising alternative to single hepatocytes and can be applied for HCTx.

Introduction : Hepatocyte transplantation (HT) is a promising alternative to liver transplantation for the treatment of liver-based metabolic diseases and acute liver failure (ALF). However, shortage of good-quality liver tissues, early cell loss post-infusion, reduced cell engraftment and function restricts clinical application. Areas covered : A comprehensive literature search was performed to cover pre-clinical and clinical HT studies. The review discusses the latest developments to address HT limitations: cell sources from marginal/suboptimal donors to neonatal livers, differentiating pluripotent stem cells into hepatocyte-like cells, in vitro expansion, prevention of immune response to transplanted cells by encapsulation or using innate immunity-inhibiting agents, and enhancing engraftment through partial hepatectomy or irradiation. Expert opinion : To date, published data are highly encouraging specially the alginate-encapsulated hepatocyte treatment of children with ALF. Hepatocyte functions can be further improved through co-culturing with mesenchymal stromal cells. Moreover, ex-vivo genetic correction will enable the use of autologous cells in future personalized medicine.

Various liver diseases result in liver failure, and liver transplantation as a definite treatment is limited by the shortage of organs available for transplantation. The use of isolated primary hepatocytes in cell-based therapies including hepatocyte transplantation, tissue engineering liver transplantation, and bioartificial liver support systems has gained increasing interest during the past years. Human hepatocytes are the preferred source of cells. Aside from the organ shortage, the isolation of human liver cells is usually limited by obtaining a sufficient quantity of high-quality, metabolically active cells. Furthermore, livers from which hepatocytes are typically harvested are not suitable for transplantation, with the variability in quantity and quality. Porcine hepatocytes, on the other hand, have the ability to perform complex biological functions and show modifiable behavior. Primary porcine hepatocytes are currently widely used in the investigation of drug metabolism, hepatotoxicity, protein biosynthesis, and gene expression. Primary hepatocytes do not proliferate in vitro and are sensitive to freeze-thaw damage in cryopreservation and thus need to be freshly isolated for each experiment. Consequently, the methods of porcine hepatocyte isolation are being actively sought after. Our laboratories have been involved in various applications of liver cells, and we have long-lasting experiences in liver cell isolation and their application in R&D. We here summarize the present protocol of our laboratories for primary hepatocyte isolation from pig and their liver tissue engineering for xenotransplantation.

Hepatocyte transplantation (HcTx) is a promising approach for the treatment of metabolic diseases in newborns and children. The most common application route is the portal vein, which is difficult to access in the newborn. Transfemoral access to the splenic artery for HcTx has been evaluated in adults, with trials suggesting hepatocyte translocation from the spleen to the liver with a reduced risk for thromboembolic complications. Using juvenile Göttingen minipigs, we aimed to evaluate feasibility of hepatocyte transplantation by transfemoral splenic artery catheterization, while providing insight on engraftment, translocation, viability, and thromboembolic complications. Four Göttingen Minipigs weighing 5.6 kg to 12.6 kg were infused with human hepatocytes (two infusions per cycle, 1.00E08 cells per kg body weight). Immunosuppression consisted of tacrolimus and prednisolone. The animals were sacrificed directly after cell infusion ( n =2), 2 days ( n =1), or 14 days after infusion ( n =1). The splenic and portal venous blood flow was controlled via color-coded Doppler sonography. Computed tomography was performed on days 6 and 18 after the first infusion. Tissue samples were stained in search of human hepatocytes. Catheter placement was feasible in all cases without procedure-associated complications. Repetitive cell transplantations were possible without serious adverse effects associated with hepatocyte transplantation. Immunohistochemical staining has proven cell relocation to the portal venous system and liver parenchyma. However, cells were neither present in the liver nor the spleen 18 days after HcTx. Immunological analyses showed a response of the adaptive immune system to the human cells. We show that interventional cell application via the femoral artery is feasible in a juvenile large animal model of HcTx. Moreover, cells are able to pass through the spleen to relocate in the liver after splenic artery infusion. Further studies are necessary to compare this approach with umbilical or transhepatic hepatocyte administration.

A solubilized liver-specific extracellular matrix (L-ECM) substratum was obtained by decellularization of porcine liver using Triton X-100 and pepsin treatments. The L-ECM was able to immobilize hepatocyte growth factor at a high efficiency of 87%. L-ECM gelled spontaneously in a physiologically neutral environment. Primary hepatocytes embedded in the L-ECM gel showed a high albumin synthesis activity and ethoxyresorufin-O-deethylase (EROD) activity even at 3 weeks in culture. In addition, the L-ECM gel-embedded hepatocytes implanted subcutaneously into partial hepatectomized rats showed a high survival rate (18%) and formed a large liver tissue-like structure. Their efficiencies of EROD activity and large liver tissue-like structure formation were about twice those of collagen gel-embedded hepatocytes. Based on these results, we clarified the effectiveness of L-ECM gel as a substrate for hepatocyte culture and transplantation., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

BACKGROUND Hepatocyte transplantation (HCTx) has the potential for the treatment of end-stage liver disease. However, failure of engraftment and the long-term acceptance of cellular allografts remain significant challenges for its clinical application. The aim of this study was to investigate the efficacy of the immunosuppressive agents, Cyclosporine, Everolimus, and Belatacept to suppress the alloresponse of primary human hepatocytes in a mixed lymphocyte-hepatocyte culture (MLHC) and their potential hepatotoxicity in vitro. MATERIAL AND METHODS Primary human hepatocytes were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an MLHC. Proliferative alloresponses were determined by flow cytometry, and cytokine secretion was measured using Luminex-based multiplex technology. Using an MLHC, the alloresponses of primary human hepatocytes were compared in the presence and absence of Cyclosporine, Everolimus, and Belatacept. Cultured primary human hepatocytes were assessed for the production of albumin, urea, aspartate transaminase (AST) and DNA content. Metabolic activity was determined with the MTT assay. RESULTS Immune responses induced by primary human hepatocytes were effectively suppressed by Cyclosporine, Everolimus, and Belatacept. Everolimus significantly reduced the metabolic activity of primary human hepatocytes in vitro, suggesting impairment of cell viability. However, further functional analysis showed no significant differences between treated and untreated controls. CONCLUSIONS Cyclosporine, Everolimus, and Belatacept suppressed the alloresponse of primary human hepatocytes in an MLHC without significant cytotoxicity or functional cell impairment.

Stem cell-based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte-like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell-derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell-derived HLC, but not induced pluripotent stem cell-derived HLC., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Rationale : Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs) have been developed to address the shortage of primary human hepatocytes (PHHs) for therapeutic applications. However, the in vivo repopulation capacity of HLCs remains limited. This study investigated the roles of agonist antibody activating the c-Met receptor in promoting the in vivo proliferation and repopulation of engrafted PHHs and/or HLCs in mice with liver injuries due to different causes. Methods : An agonist c-Met receptor antibody (5D5) was used to treat PHHs and hiPSC-HLCs in both cell culture and hepatocyte-engrafted immunodeficient mice mimicking various inherited and acquired liver diseases. The promoting roles and potential influence on the hepatic phenotype of the 5D5 regimen in cell transplantation-based therapeutic applications were systematically evaluated. Results : In hiPSC-HLC cell cultures, 5D5 treatment significantly stimulated c-Met receptor downstream signalling pathways and accelerated cell proliferation in dose-dependent and reversible manners. In contrast, only slight but nonsignificant promotion was observed in 5D5-treated PHHs. In vivo administration of 5D5 greatly promoted the expansion of implanted hiPSC-HLCs in fumarylacetoacetate hydrolase (Fah) deficient mice, resulting in significantly increased human albumin levels and high human liver chimerism (over 40%) in the transplanted mice at week 8 after transplantation. More importantly, transplantation of hiPSC-HLCs in combination with 5D5 significantly prolonged animal survival and ameliorated liver pathological changes in mice with acute and/or chronic liver injuries caused by Fas agonistic antibody treatment, carbon tetrachloride treatment and/or tyrosinemic stress. Conclusion : Our results demonstrated that the proliferation of hiPSC-HLCs can be enhanced by antibody-mediated modulation of c-Met signalling and facilitate hiPSC-HLC-based therapeutic applications for life-threatening liver diseases., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Direct reprogramming represents an easy technique to generate induced hepatocytes (iHeps) from somatic cells. However, current protocols are accompanied by several drawbacks as iHeps are heterogenous and lack fully mature phenotypes of primary hepatocytes. Here, we established a polycistronic expression system to induce the direct reprogramming of mouse embryonic fibroblasts towards hepatocytes. The resulting iHeps are homogenous and display key properties of primary hepatocytes, such as expression of hepatocyte markers, albumin secretion, and presence of liver transaminases. iHeps also possess the capacity to repopulate decellularized liver tissue and exhibit enhanced hepatic maturation. As such, we present a novel strategy to generate homogenous and functional iHeps for applications in tissue engineering and cell therapy., (© 2018 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

Background: Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes., Methods: Pig and mouse hepatocytes were isolated, labeled with zirconium-89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography-computed tomography. Fumarylacetoacetate hydrolase-deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single-cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single-cell or spheroid hepatocytes were followed through histologic analysis and animals' ability to thrive off 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione., Results: Positron emission tomography-computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid- and single-cell-transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase-positive hepatocytes in the liver, correlating with similar weight stabilization curves., Conclusion: Ex vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase-deficient pigs can be performed using hepatocyte spheroids or single-cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Hepatocyte transplantation is a promising alternate for the treatment of hepatic diseases. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in the quantity sufficient and the quality required for biotherapy. Isolated fresh hepatocytes include damaged cells that are also early apoptotic cells, which is not ideal for hepatocyte transplantation. However, this does not reflect cell viability, although it is considered that it adversely affects cell survival after transplantation. We aimed to harvest these hepatocytes and filter the apoptotic cells using a magnetic method to provide a transplantation source., Materials and Methods: Rat hepatocytes were isolated from caudate lobes using manual enzymatic perfusion. The hepatocyte yield was 5.3 ± 0.66 × 10 9 cells/g of liver tissue, with a viability of 82.3 ± 3.5%. Two samples of hepatocytes were freshly isolated, one using the magnetic method, and the other without. The magnetic method was performed using DynaMag-15 Magnet, and Annexin V Antibody was used on the early apoptotic cells. We evaluated the viability and plate efficiency of the cells after 24 hours at 37°C. Hepatocytes were isolated using cell separation method, and 30 × 10 6 cells were mixed with 1.0 mL of Dulbecco's Modified Eagle's Medium (DMEM) and directly injected into the spleen of Lewis rats (150-250 g) using 24-gauge needles. Blood samples were collected on days 0, 3, 7, and 14, and the blood albumin level was measured using enzyme-linked immunosorbent assay (ELISA):G1, control (medium injection); G2, fresh hepatocyte transplant using the magnetic method; and G3, fresh hepatocyte transplant without the magnetic method., Results: The viability was 84.9 ± 2% for fresh hepatocytes and 80.7 ± 1.2% for hepatocytes isolated using the magnetic method. The magnetic method does not damage the cells (73.5 ± 2% vs 35.2 ± 2% after 24 hours), preserving hepatocyte. The albumin level accepted significantly increased in the magnet-treated group compared with the nonmagnet group. Simultaneously, the spleen in which these hepatocytes were transplanted could be used to observe the hepatocytes; the cells were transplanted 14 days later, and the magnet-treated group had significantly higher levels of hepatocytes than the nonmagnet group., Conclusion: We developed an effective technique for hepatocyte isolation for short-term preservation. As a result, we believe that transplantation not only improves the cell transplantation effect but also allows the cells to be stored efficiently using the magnetic method. These results demonstrate the usefulness of hepatocyte hypothermic preservation for cell transplantation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hepatic cell transplantation (HCT) continues to garner interest as an alternative to orthotopic liver transplantation and the attendant donor shortage. When compared with solid organ transplantation, advantages of cell transplantation include the potential to treat more patients with a considerably less invasive procedure, the ability to utilize organs otherwise unsuitable for transplant, and leaving the native organ in situ with the potential for regeneration. While studies date back to the early 1960s, advancement of clinical application has been slow due in part to limitations of suitable tissue supplies and reproducible robust techniques. Compared with orthotopic liver transplantation, there are fewer absolute contraindications for donor selection. And, current techniques used to harvest, isolate, store, and even transfuse cells vary little between institutions. Significant variation is seen due to a lack of consensus with maintenance therapy. Although the ideal recipient has not been clearly identified, the most significant results have been demonstrated with correction of congenital metabolic liver disorders, with a few trials examining its utility in cirrhotics and more recently acute liver failure. The most exciting new topic of discussion examines techniques to improve engraftment, with many such as ischemic preconditioning and nonselective partial embolization (microbead therapy), while not yet used in HCT study, showing promise in solid organ research. Advancements in HCT, although slow in progress, have great potential in the ability to alleviate the burden faced in solid organ transplantation and possibly become a long-term viable option, beyond that of a bridge or salvage therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes., Methods: Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry., Results: Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and β-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes., Conclusions: These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes., (Copyright © 2018 John Wiley & Sons, Ltd.)

Background: Replacement therapy for hemophilia remains a lifelong treatment. Only gene therapy can cure hemophilia at a fundamental level. The clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 (CRISPR-Cas9) system is a versatile and convenient genome editing tool which can be applied to gene therapy for hemophilia., Methods: A patient's induced pluripotent stem cells (iPSCs) were generated from their peripheral blood mononuclear cells (PBMNCs) using episomal vectors. The AAVS1-Cas9-sgRNA plasmid which targets the AAVS1 locus and the AAVS1-EF1α-F9 cDNA-puromycin donor plasmid were constructed, and they were electroporated into the iPSCs. When insertion of F9 cDNA into the AAVS1 locus was confirmed, whole genome sequencing (WGS) was carried out to detect the off-target issue. The iPSCs were then differentiated into hepatocytes, and human factor IX (hFIX) antigen and activity were measured in the culture supernatant. Finally, the hepatocytes were transplanted into non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through splenic injection., Results: The patient's iPSCs were generated from PBMNCs. Human full-length F9 cDNA was inserted into the AAVS1 locus of iPSCs of a hemophilia B patient using the CRISPR-Cas9 system. No off-target mutations were detected by WGS. The hepatocytes differentiated from the inserted iPSCs could secrete hFIX stably and had the ability to be transplanted into the NOD/SCID mice in the short term., Conclusions: PBMNCs are good somatic cell choices for generating iPSCs from hemophilia patients. The iPSC technique is a good tool for genetic therapy for human hereditary diseases. CRISPR-Cas9 is versatile, convenient, and safe to be used in iPSCs with low off-target effects. Our research offers new approaches for clinical gene therapy for hemophilia.

Background: There is no standard therapy for acute liver failure. Hepatocyte transplantation has been proposed for temporary liver function support, while the injured liver regenerates or while waiting for transplantation. We have previously shown such efficacy for microencapsulated porcine hepatocytes in mice with fulminant liver failure. We aimed to establish a large animal model for fulminant liver failure to assess the efficacy of microencapsulated porcine hepatocytes in temporary liver function support., Methods: The model was developed in baboons; for testing microencapsulated hepatocytes, the best condition was 75% hepatectomy and 60 min warm ischemia time. Fulminant liver failure was characterized by steep increases in liver biochemical parameters, severe steatosis, and massive hepatocyte necrosis during the first 10 days. Hepatocytes from miniature swine were microencapsulated in alginate-poly-l-lysine microspheres, and transplanted intraperitoneally immediately after hepatectomy and warm ischemia (80-120 mL packed hepatocytes in 200-350 mL microspheres, about 30%-50% of the baboon's native liver volume)., Results: In the control group, three of five animals were sacrificed after 6-10 days because of fulminant liver failure, and two of five animals recovered normal liver function and survived until elective euthanasia (28 days). In the treatment group of four animals, one animal developed liver failure but survived to 21 days, and three animals recovered completely with normal liver function., Conclusions: The results indicate that microencapsulated porcine hepatocytes provide temporary liver function support in baboons with fulminant liver failure. These data support development of this cell therapy product toward clinical trials in patients with acute liver failure., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo. We treated the livers of mice with NTIRE and subsequently implanted exogenous congenic hepatocytes within the zone of cell ablation. Donor hepatocytes engrafted and integrated with host liver parenchyma pre-treated with NTIRE. This new approach should have value for studying the effects of a native matrix scaffold on in vivo cell growth and may pioneer a new type of minimally-invasive regenerative surgery.

The liver has an important function in the human body and plays a crucial role in its metabolism. Orthotopic liver transplantation (OLT) is the gold standard treatment for patients presenting liver failure or end stage liver diseases, and is also applied for liver based intractable metabolic disorders. Due to organ shortage, invasive surgery and persistent mortality/morbidity, other treatments have to be explored. Amongst these, hepatocyte transplantation is an attractive alternative and has shown promising results in the treatment of miscellaneous metabolic disorders.

In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

Successful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver and the results of hepatocyte isolation from such tissue are inferior compared to normal tissue. Here we describe a modified method, combining the use of Liberase and N-acetylcysteine (NAC), for the isolation of primary human hepatocytes with high viability from normal and diseased liver.

Orthotopic liver transplantation remains the only proven cure for end-stage liver failure. Despite significant advances in the field, the clinical demand for donor organs far outweighs the supply. Hepatocyte transplantation has been proposed as an alternative approach to whole liver transplant in select diseases. Several international centers have reported experimental trials of human hepatocyte transplantation in acute liver failure and liver-based metabolic disorders. This chapter provides an introduction to hepatocyte transplantation from both a technical and clinical perspective. We will also focus on the special needs of pediatric patients, since historically the majority of clinical hepatocyte transplants have involved infants and children.

Liver transplantation has been established as a curative therapy for acute and chronic liver failure, as well as liver-based inherited metabolic diseases. Because of the complexity of organ transplantation and the worldwide shortage of donor organs, hepatocyte transplantation is being developed as a bridging therapy until donor organs become available, or for amelioration of inherited liver-based diseases. The Gunn rat is a molecular and metabolic model of Crigler-Najjar syndrome type 1, which is characterized by lifelong unconjugated hyperbilirubinemia due to the lack of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-mediated bilirubin glucuronidation. Gunn rats are convenient for evaluating the effect of hepatocyte transplantation or gene therapy, because the extent of UGT1A1 replacement can be assessed by serial determination of serum bilirubin levels, and excretion of bilirubin glucuronides in bile provide definitive evidence of the function of the transplanted hepatocytes or the effect of gene therapy. The core techniques involved in hepatocyte transplantation in Gunn rats are discussed in this chapter.

Hepatocyte transplantation is the best approach to maintain and propagate differentiated hepatocytes from different species. Host liver has to be adapted for transplanted hepatocytes productive engraftment and proliferation being required a chronic liver injury to eliminate host hepatocytes and provide a proliferative advantage to the transplanted hepatocytes. Most valuable mouse models for xenograft hepatocyte transplantation are based on genetically modified animals to cause a chronic liver damage and to limit host hepatocyte regeneration potential. We present a methodology that generates a chronic liver damage and can be applied to any host mouse strain and animal species based on the inoculation of a recombinant adenovirus to express herpes simplex thymidine kinase in host hepatocytes sensitizing them to ganciclovir treatment. This causes a prolonged liver damage that allows hepatocyte transplantation and generation of regenerative nodules in recipient mouse liver integrated by transplanted cells and host sinusoidal. Obtained chimeric animals maintain functional chimeric nodules for several weeks, ready to be used in any study.

Wilson's disease (WD) is characterized by the inability to excrete copper (Cu) from the body with progressive tissue injury, especially in liver and brain. The molecular defect in WD concerns mutations in ATP7B gene leading to loss of Cu transport from the hepatocyte to the bile canaliculus. While drugs, e.g., Cu chelators, have been available for several decades, these must be taken lifelong, which can be difficult due to issues of compliance or side effects. Many individuals may require liver transplantation, which can also be difficult due to donor organ shortages. Therefore, achieving permanent cures via cell or gene therapy are of great interest for WD. Cell therapy is feasible because transplanted hepatocytes can integrate in liver parenchyma and restore deficient functions, including transport of Cu into bile. The availability of authentic animal models that recapitulate hepatic WD, especially the Long-Evans Cinnamon (LEC) rat, has advanced cell transplantation research in WD. We describe requirements for cell therapy in animal models with several standardized methods for studies to test or refine cell therapy strategies in WD.

Cellular transplantation represents an alternative to liver transplantation for the treatment of end-stage liver disease and liver-based inborn errors of metabolism. In order for cellular transplantation to be successful, an optimal source of cells for transplantation needs to be identified and the molecular mechanisms regulating their engraftment, proliferation, and functional differentiation elucidated. Here we describe a detailed protocol for the isolation, selection, and transplantation into an injured adult rat liver of a defined population of late gestation fetal rat hepatocytes. Also described is the methodology for assessing the purity of the selected cells and the efficiency with which they repopulate the adult liver. Our approach provides an in vivo model to study the molecular pathways involved in liver repopulation.

In the context of cell transplantation in the liver parenchyma, preconditioning is essential to enhance cell engraftment and liver repopulation. The authors have developed a minimally invasive technique of temporary portal embolization using an absorbable material, called reversible portal vein embolization. We hereby describe the method for isolating hepatocytes from a donor rat before transplanting hepatocytes after reversible portal vein embolization in the recipient.

Hepatocyte transplantation is a promising approach for treatment of certain liver disorders, especially for metabolic liver disease in the juvenile. The lack of a clinically approved noninvasive imaging technique is a major limitation to the further development of hepatocyte transplantation. Cell tracking by Magnetic Resonance Imaging (MRI) is a promising approach for noninvasive monitoring of the fate of transplanted cells. Studies in large animal models are necessary to transfer this promising technique into the clinic.

Alginate encapsulation of cells is an attractive technique in which alginate becomes polymerized entrapping the cells. The structure of formed microbeads/microcapsules is semipermeable as it allows oxygen and nutrients to go in, and waste products and other materials produced by the cells to go out. Here, we describe basic protocols for alginate encapsulation of human hepatocytes and methods for assessing the microbeads produced.

Technical issues of experimental hepatocyte transplantation in pigs, i.e., selection of animals, anesthesia, route of transplantation, and segment-specific transplantation have described.

In an era of organ shortage, human fetuses donated after medically indicated abortion could be considered a potential liver donor for hepatic cell isolation. We investigated transplantation of fetal liver cells as a strategy to support liver functionality in end-stage liver disease. Here, we report our protocol of human fetal liver cells (hFLC) isolation in fetuses from 17 to 22 gestational weeks, and our clinical procedure of hFLC transplantation through the splenic artery.

Co-transplantation of hepatocytes and hepatic stellate cells has been shown to increase the engraftment of transplanted hepatocytes in the liver. Here, we describe a method for the simultaneous isolation of human primary hepatocytes and hepatic stellate cells from the same donor for co-transplantation or for use in in vitro cell culture models.

Mice with humanized chimeric liver are promising in vivo tools to evaluate the efficacy of novel compounds or vaccine induced antibodies directed against pathogens that infect the human liver. In addition they can be used to study the human-type metabolism of medicinal compounds and hepatotoxicity.

Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

Technologies for in vivo imaging of the distribution and integration of cell transplants gain significance for the use of novel cell therapy approaches in regenerative medicine. Applied to adequate animal models, they provide information on the spatio-temporal engraftment and functional performance of the cells transplanted. This chapter includes a detailed description of the in vivo tracking of transplanted hepatocytes in rat liver including the conjugation of antibodies to fluorochromes for far red imaging using a multispectral optical imager.

Primary or cryopreserved human hepatocytes (h-heps) have been used as the gold standard for in vitro metabolism and hepatotoxicity studies; however, the supply of h-heps is limited and they cannot grow in vitro. We achieved approximately 1000-fold propagation of h-heps in the liver of albumin promoter/enhancer-driven urokinase-type plasminogen activator transgenic/severe combined immunodeficiency disease (uPA/SCID) mice with genetically induced liver disease and immunodeficiency. When h-heps are transplanted into the uPA/SCID mouse liver via the spleen, the h-heps engraft in the mouse liver, resulting in its repopulation with h-heps. We have named this model "chimeric mouse with humanized liver, PXB-mouse ® ." Fresh h-heps can be isolated from the chimeric mice (PXB-cells ® ) and have been used for in vitro studies.The efficacy and safety of chemical entities for use in humans are estimated using experimental animals such as rats and mice. The drug development of many chemical entities has been halted because of metabolic differences between humans and animals during clinical studies. Therefore, chimeric mice with humanized liver have been used to predict human-type metabolism and safety conditions for h-heps. In addition, until recently there were no suitable hepatitis B or C virus (HBV or HCV) susceptible animal models aside from chimpanzees. Chimeric mice are the sole persistent infectious small animal model for HBV and HCV and they have been used to investigate the efficacy of new anti-HBV or HCV agents.In this chapter, we describe a method for producing chimeric mice with humanized liver using uPA/SCID mice.

Liver transplantation is the mainstay of treatment for end stage liver diseases, including metabolic and congenital liver diseases. The number of suitable donor organs is, however, limited, and a whole-liver transplant requires complex surgery. Cell therapy, such as intra-portal hepatocytes transplantation, has been considered as a bridging therapy to liver transplantation but has shown a mixed clinical outcome with limited success, including low level of engraftment of transplanted hepatocytes. Here, we report a novel cell delivery technique in a rat model by creating a cavity inside the liver parenchyma by non-invasive high intensity focused ultrasound histotripsy. Our in vivo experimental results together with histologic observations show that direct injection of cells inside the cavity can facilitate successful uptake, proliferation and integration of the transplanted hepatocytes in the recipient liver. We were able to restore the plasma albumin level to 50% of the normal level in Nagase analbuminemic rats (serum albumin level of the Nagase rats was initially nil) by cell therapy after high intensity focused ultrasound-mediated histotripsy. We believe that this novel technique would enable the delivery of a large number of cells into the liver to restore liver function, particularly as a treatment for metabolic liver diseases. This novel method of intra-hepatic hepatocyte transplantation might be an invaluable tool for cell therapy in the future., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Cell transplantation is a potential alternative for orthotopic liver transplantation because of the chronic donor shortage. Functional liver tissue is needed for cell transplantations. However, large functional liver tissue is difficult to construct because of the high oxygen consumption of hepatocytes. In our previous study, we developed a novel method to generate hybrid organoids. In this study, we used fetal liver cells (FLCs) to construct a hybrid organoid. Nucleus numbers, angiogenesis, and albumin production were measured in transplanted samples. Higher cell viability and larger liver tissue was found in FLC-containing samples than in hepatocyte-containing samples. Furthermore, the therapeutic efficiency of FLC-containing samples was evaluated by transplantation into Nagase analbuminemia rats. As a result, an increase in albumin concentration was found in rat blood. In summary, transplantation of a FLC-containing hybrid organoid is a potential approach for cell transplantation., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF., Methods: Intraperitoneal d-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF., Results: A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen., Conclusion: Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

Acute liver failure (ALF) is a clinical syndrome resulting from widespread damage of hepatocytes, with extremely high mortality rate. Urgent orthotopic liver transplantation was shown to be the most effective therapy for ALF but this treatment option is limited by scarcity of donor organs. Therefore, hepatocyte transplantation (Tx) has emerged as a new therapeutical measure for ALF, however, the first clinical applications proved unsatisfactory. Apparently, extensive preclinical studies are needed. Our aim was to examine if hepatocytes isolated from transgenic "firefly luciferase" Lewis rats into the recipient liver would attenuate the course of thioacetamide (TAA)-induced ALF in Lewis rats. Untreated Lewis rats after TAA administration showed a profound decrease in survival rate; no animal survived 54 h. The rats showed marked increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, in plasma level of bilirubin and ammonia (NH(3)), and in a significant decrease in plasma albumin. Hepatocyte Tx attenuated the course of TAA-induced ALF Lewis rats which was reflected by improved survival rate and reduced degree of liver injury showing as lowering of elevated plasma ALT, AST, NH(3) and bilirubin levels and increasing plasma albumin. In addition, bioluminescence imaging analyses have shown that in the TAA-damaged livers the transplanted hepatocyte were fully viable throughout the experiment. In conclusion, the results show that hepatocyte Tx into the liver can attenuate the course of TAA-induced ALF in Lewis rats. This information should be considered in attempts to develop new therapeutic approaches to the treatment of ALF.

Unlabelled: Cell transplantation-induced hepatic ischemia and recruitment of vasoconstrictors (e.g., endothelin-1; Edn1) leads to clearance of transplanted cells and poses problems for liver repopulation. Therefore, we determined whether darusentan (DAR), which potently blocks Edn1 receptor type A, could benefit cell engraftment. We transplanted primary F344 rat hepatocytes with or without DAR in dipeptidyl peptidase IV-deficient rats. Analysis of microcirculatory events included hepatic ischemia, endothelial injury, including with gene expression arrays, and activations of Kupffer cells (KCs), neutrophils, or hepatic stellate cells (HSCs). The retrorsine-partial hepatectomy model was used for liver repopulation studies. Whether DAR was directly cytoprotective was examined in cultured rat hepatocytes or CFSC-8B rat HSCs. We found that DAR induced hepatic sinusoidal vasodilation, caused more transplanted cells to be deposited in liver parenchyma, and decreased hepatic ischemia and endothelial injury. This lessened perturbations in expression of endothelial biology genes, including regulators of vessel tone, inflammation, cell adhesion, or cell damage, versus drug-untreated controls. Moreover, in DAR-treated animals, cell transplantation-induced activation of KCs, albeit not of neutrophils, decreased, and fewer HSCs expressed desmin. In DAR-treated rats, improvements in cell engraftment led to greater extent of liver repopulation, compared to drug-untreated controls. In cell-culture assays, DAR did not stimulate release of cytoprotective factors, such as vascular endothelial growth factor, from HSCs. Moreover, DAR did not protect hepatocytes from tumor necrosis factor alpha- or oxidative stress-induced toxicity. Endothelin receptor A blockade in vitro did not improve engraftment of subsequently transplanted hepatocytes., Conclusion: Systemic administration of DAR decreases hepatic ischemia-related events and thus indirectly improves cell engraftment and liver repopulation. This vascular mechanism may permit the development of combinatorial drug-based regimens to help optimize cell therapy., (© 2014 by the American Association for the Study of Liver Diseases.)

The new methodology to increase a therapeutic potential of cell transplantation was developed here by the use of three-dimensional spheroids of transplanting cells subsequent to the genetic modification with non-viral DNA vectors, polyplex nanomicelles. Particularly, spheroids in regulated size of 100-μm of primary hepatocytes transfected with luciferase gene were formed on the micropatterned culture plates coated with thermosensitive polymer, and were recovered in the form of injectable liquid suspension simply by cooling the plates. After subcutaneously transplanting these hepatocyte spheroids, efficient transgene expression was observed in host tissue for more than a month, whereas transplantation of a single-cell suspension from a monolayer culture resulted in an only transient expression. The spheroid system contributed to the preservation of innate functions of transplanted hepatocytes in the host tissue, such as albumin expression, thereby possessing high potential for expressing transgene. Intravital observation of transplanted cells showed that those from spheroid cultures had a tendency to localize in the vicinity of blood vessels, making a favorable microenvironment for preserving cell functionality. Furthermore, spheroids transfected with erythropoietin-expressing DNA showed a significantly higher hematopoietic effect than that of cell suspensions from monolayer cultures, demonstrating high potential of this genetically-modified spheroid transplantation system for therapeutic applications., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment., (Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.)

Introduction: Cell transplantation is an alternative to liver transplantation, which is hampered by short survival time and immunorejection. The goal of this study was to evaluate the therapeutic potential of hepatocytes coencapsulated with adipose-derived stem cells (ADSCs) in treating acute-on-chronic liver failure (ACLF)., Methods: Rat hepatocytes and ADSCs were isolated, and coencapsulated by alginate-poly-L-lysine-alginate microencapsulation. The morphological and functional changes of heterotypic interactions were characterized. ACLF in rats was induced by D-galactosamine administration following CCl4-induced cirrhosis. These rats were subjected to intraperitoneal transplantation of 5 × 107 coencapsulated hepatocytes with ADSCs, 5 × 107 encapsulated hepatocytes alone, or empty vehicles after 24 h, respectively. The survival rate and liver functions were assessed., Results: Hepatocyte performance levels such as albumin secretion and urea synthesis induction were all significantly enhanced in the coencapsulation group compared with the homo-encapsulated hepatocytes group (p<0.05). The results of cell cycle analysis showed that larger populations of hepatocytes with ADSC treatment were accumulated in the G2-S phase, and there were fewer in the G0-G1 phase compared to encapsulation of hepatocytes alone. Intraperitoneal transplantation of coencapsulated hepatocytes with ADSCs not only increased the survival rate, but also improved liver functions in a rat model of ACLF., Conclusions: Transplantation of coencapsulated hepatocytes and ADSCs might be a promising strategy for cell-based therapy of acute liver diseases.

1. Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology.

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P = 0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control = 6.3 ± 0.9; HOC = 3.5 ± 1.5; P = 0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.

Background: We previously generated humanized chimeric mice by transplanting h-hepatocytes into the livers of the diseased-liver transgenic mouse model with immunodeficient background. These mice with livers mostly replaced by human (h) hepatocytes have been proved to be useful for research on drug metabolism and toxicity and on intrahepatic pathogens such as hepatitis. However, their small body size prohibited collecting sufficient biological samples and made surgical manipulation difficult, which motivated us to produce humanized larger animal(s) bearing h-hepatocytes., Methods: Fischer 344 (F344) rats at 2 weeks of age were administrated with hepatotoxin retrorsine (RS) and then transplanted with syngeneic F344 rat (r)- or h-hepatocytes via the portal vein. The hosts were injected daily with FK506 immunosuppressant. The livers were harvested periodically for determining donor-cell replacement ratios and compared with those of the humanized chimeric mice, and liver-specific mRNA and protein expressions by immunohistochemistry and reverse-transcription PCR., Results: RS treatment of infant rats inhibited hepatocyte proliferation, resulting in decreased liver weight and megalocytic changes in hepatocytes. R-hepatocytes transplanted into RS-treated rats engrafted into and repopulated the liver at ratios of 16.4 ± 6.7% and 48.3 ± 29.3% at 3 and 6 weeks after transplantation, respectively. H-hepatocytes also engrafted into the rat liver and showed a repopulation ratio of 2.5 ± 1.5% at 3 weeks post-transplantation, which was comparable to the ratio in the humanized chimeric mouse model at least until 3 weeks. Propagated h-hepatocytes in the rat liver expressed hepatocyte-specific mRNA and proteins at least 3 weeks after transplantation., Conclusions: Xenogeneic hepatocytes were able to engraft rat liver and grow well therein for at least 3 weeks post-transplantation in rats when immunosuppression was combined appropriately with liver injury at comparable levels to the well-characterized humanized chimeric mouse model., (© 2013 John Wiley & Sons A/S.)

Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co-culture with non-parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering., (© 2013 John Wiley & Sons A/S.)