Liver Cell Therapy Research Articles

Clinical Application of Pluripotent Stem Cells: An Alternative Cell-Based Therapy for Treating Liver Diseases?

The worldwide shortage of donor livers for organ and hepatocyte transplantation has prompted the search for alternative therapies for intractable liver diseases. Cell-based therapy is envisaged as a useful therapeutic option to recover and stabilize the lost metabolic function for acute liver failure, end-stage and congenital liver diseases, or for those patients who are not considered eligible for organ transplantation. In recent years, research to identify alternative and reliable cell sources for transplantation that can be derived by reproducible methods has been encouraged. Human pluripotent stem cells (PSCs), which comprise both embryonic and induced PSCs, may offer many advantages as an alternative to hepatocytes for liver cell therapy. Their capacity for expansion, hepatic differentiation and self-renewal make them a promising source of unlimited numbers of hepatocyte-like cells for treating and repairing damaged livers. Immunogenicity and tumorigenicity of human PSCs remain the bottleneck for successful clinical application. However, recent advances made to develop disease-corrected hepatocyte-like cells from patients' human-induced PSCs by gene editing have opened up many potential gateways for the autologous treatment of hereditary liver diseases, which may likely reduce the risk of rejection and the need for lifelong immunosuppression. Well-defined methods to reduce the expression of oncogenic genes in induced PSCs, including protocols for their complete and safe hepatic differentiation, should be established to minimize the tumorigenicity of transplanted cells. On top of this, such new strategies are currently being rigorously tested and validated in preclinical studies before they can be safely transferred to clinical practice with patients.

Human hepatic stellate cells and inflammation: A regulated cytokine network balance

Aim: Uncertainty about the safety of cell therapy continues to be a major challenge to the medical community. Inflammation and the associated immune response represent a major safety concern hampering the development of long-term clinical therapy. In vivo interactions between the cell graft and the host immune system are mediated by functional environmental sensors and stressors that play significant roles in the immunobiology of the graft. Within this context, human liver stellate cells (HSC) demonstrated marked immunological plasticity that has main importance for future liver cell therapy application. Methods: By using qPCR technique, we established the cytokine gene expression profile of HSCs and investigated the effect of an inflammatory environment on the immunobiology of HSCs. Results and discussion: HSCs present a specific immunological profile as demonstrated by the expression and modulation of major immunological cytokines. Under constitutive conditions, the cytokine pattern expressed by HSCs was characterized by the high expression of IL-6. Inflammation critically modulated the expression of major immunological cytokines. As evidenced by the induction of the expression of several inflammatory genes, HSCs acquire a pro-inflammatory profile that ultimately might have critical implications for their immunological shape. Conclusion: These new observations have to be taken into account in any future liver cell therapy application based on the use of HSCs.

Suppressing Pitx2 inhibits proliferation and promotes differentiation of iHepSCs

Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their abilities for self-renewal and bi-potential differentiation. However, the molecular mechanism regulating proliferation and differentiation of iHepSCs is poorly understood. In this study, we provide evidence that the homeodomain transcription factor, Pitx2, is essential to maintain iHepSCs stem cell characteristics. Suppressing Pitx2 expression in iHepSCs by lentivirus mediated specific shRNA markedly reduced the expression of the hepatic stem cell-associated genes (Lgr5, EpCAM, and Sox9) with concomitant inhibition of proliferation by blocking the G1/S phase transition, and these phenotypic changes were reversed upon re-expression of Pitx2. Pitx2 knockdown also resulted in up-regulation of the p53-induced Cdk inhibitor p21, and down-regulation of its downstream effector CDK2-Cyclin E kinase complex. Furthermore, we observed that iHepSCs were more efficiently induced to differentiate into both hepatocytes and cholangiocytes when Pitx2 expression was suppressed, as compared to unmanipulated iHepSCs. These findings reveal that Pitx2 expression may be leveraged to control the status of iHepSCs during expansion in vitro to provide a strategy for further application of iHepSCs in liver cell therapy.

Prospects for Adult Stem Cells in the Treatment of Liver Diseases.

Hepatocytes constitute the main bulk of the liver and perform several essential functions. After injury, the hepatocytes have a remarkable capacity to regenerate and restore functionality. However, in some cases, the endogenous hepatocytes cannot replicate or restore the function, and liver transplantation, which is not exempt of complications, is required. Stem cells offer in theory the possibility of generating unlimited supply of hepatocytes in vitro due to their capacity to self-renew and differentiate when given the right cues. Stem cells isolated from an array of tissues have been investigated for their capacity to differentiate into hepatocyte-like cells in vitro and are employed in rescue experiments in vivo. Adult stem cells have gained in attractiveness over embryonic stem cells for liver cell therapy due to their origin, multipotentiality, and the possibility of autologous transplantation. This review deals with the promise and limitations of adult stem cells in clinically restoring liver functionality.

Thalidomide promotes transplanted cell engraftment in the rat liver by modulating inflammation and endothelial integrity

For liver-directed cell therapy, efficient engraftment of transplanted cells is critical. This study delineated whether anti-inflammatory and endothelial disrupting properties of thalidomide could promote transplanted cell engraftment and proliferation in liver. We used dipeptidyl peptidase IV-deficient rats for cell transplantation studies, including gene expression analysis, morphological tissue analysis, serological assays, cell culture assays, and assays of transplanted cell engraftment and proliferation. Thalidomide-pretreatment increased engraftment and proliferation of transplanted hepatocytes due to decreased inflammation. Moreover, thalidomide exacerbated cell transplantation-induced endothelial injury. This combined anti-inflammatory and endothelial injury effect of thalidomide was superior to the anti-inflammatory effect alone of repertaxin or etanercept, which block cytokines/chemokines/receptor-dependent inflammation. In thalidomide-pretreated animals, liver repopulation accelerated, including when cells were primed with bosentan to block endothelin-1 receptors. Thalidomide improved transplanted cell engraftment and liver repopulation. Therefore, this class of drugs will advance applications of liver cell therapy in people. This work aimed to develop effective drug treatments for improving engraftment of transplanted cells because that constitutes a critical step in rebuilding liver with healthy cells. Studies in animal models of cell transplantation led to identification of an old drug, thalidomide, which blocked inflammation and altered the liver microenvironment to yield superior engraftment and proliferation of transplanted cells. This will be appropriate for liver cell therapy in people.

Assessing the therapeutic potential of lab-made hepatocytes.

Hepatocyte transplantation has potential as a bridge or even alternative to whole-organ liver transplantation. Because donor livers are scarce, realizing this potential requires the development of alternative cell sources. To be therapeutically effective, surrogate hepatocytes must replicate the complex function and ability to proliferate of primary human hepatocytes. Ideally, they are also autologous to eliminate the need for immune suppression, which can have severe side effects and may not be sufficient to prevent rejection long term. In the past decade, several methods have been developed to generate hepatocytes from other readily and safely accessible somatic cells. These lab-made hepatocytes show promise in animal models of liver diseases, supporting the feasibility of autologous liver cell therapies. Here, we review recent preclinical studies exemplifying different types of lab-made hepatocytes that can potentially be used in autologous liver cell therapies. To define the therapeutic efficacy of current lab-made hepatocytes, we compare them to primary human hepatocytes, focusing on engraftment efficiency and posttransplant proliferation and function. In addition to summarizing published results, we discuss animal models and assays effective in assessing therapeutic efficacy. This analysis underscores the therapeutic potential of current lab-made hepatocytes, but also highlights deficiencies and uncertainties that need to be addressed in future studies aimed at developing liver cell therapies with lab-made hepatocytes. (Hepatology 2016;64:287-294).

Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury

The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34(+) progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl4). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease. Hematopoietic stem/progenitor cell expansion and tissue-specific differentiation in vitro are challenges in regenerative medicine, although stem cell therapy has raised hope for the treatment of liver diseases by overcoming the scarcity of hepatocytes. This study identified and characterized a group of blood-derived progenitor cells (BDPCs) from the peripheral blood of an adult mouse. The CD34(+) progenitor-dominant BDPCs were rapidly expanded and hepatically differentiated into functional hepatocyte-like cells with our established coculture system. BDPC treatment increased animal survival and produced full regeneration in a severe liver injury mouse model caused by CCl4. BDPCs could have potential for liver cell therapies.

Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location.

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs.

Improvement of Liver Cell Therapy in Rats by Dietary Stearic Acid.

Background:Stearic acid is known as a potent anti-inflammatory lipid. This fatty acid has profound and diverse effects on liver metabolism. The aim of this study was to investigate the effect of stearic acid on markers of hepatocyte transplantation in rats with acetaminophen (APAP)-induced liver damage.Methods:Wistar rats were randomly assigned to 10-day treatment. Stearic acid was administered to the rats with APAP-induced liver damage. The isolated liver cells were infused intraperitoneally into rats. Blood samples were obtained to evaluate the changes in the serum liver enzymes, including activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and the level of serum albumin. To assess the engraftment of infused hepatocytes, rats were euthanized, and the liver DNA was used for PCR using sex-determining region Y (SRY) primers.Results:The levels of AST, ALT and ALP in the serum of rats with APAP-induced liver injury were significantly increased and returned to the levels in control group by day six. The APAP-induced decrease in albumin was significantly improved in rats through cell therapy, when compared with that in the APAP-alone treated rats. SRY PCR analysis showed the presence of the transplanted cells in the liver of transplanted rats.Conclusion:Stearic acid-rich diet in combination with cell therapy accelerates the recovering of hepatic dysfunction in a rat model of liver injury.

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy.

Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.

Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

Methods of Liver Stem Cell Therapy in Rodents as Models of Human Liver Regeneration in Hepatic Failure.

Cell therapy is a promising intervention for treating liver diseases and liver failure. Different animal models of human liver cell therapy have been developed in recent years. Rats and mice are the most commonly used liver failure models. In fact, rodent models of hepatic failure have shown significant improvement in liver function after cell infusion. With the advent of stem-cell technologies, it is now possible to re-programme adult somatic cells such as skin or hair-follicle cells from individual patients to stem-like cells and differentiate them into liver cells. Such regenerative stem cells are highly promising in the personalization of cell therapy. The present review article will summarize current approaches to liver stem cell therapy with rodent models. In addition, we discuss common cell tracking techniques and how tracking data help to direct liver cell therapy research in animal models of hepatic failure.

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells.

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.

Concise review: cell therapies for hereditary metabolic liver diseases-concepts, clinical results, and future developments.

The concept of cell-based therapies for inherited metabolic liver diseases has been introduced for now more than 40 years in animal experiments, but controlled clinical data in humans are still not available. In the era of dynamic developments in stem cell science, the "right" cell for transplantation is considered as an important key for successful treatment. Do we aim to transplant mature hepatocytes or do we consider the liver as a stem/progenitor-driven organ and replenish the diseased liver with genetically normal stem/progenitor cells? Although conflicting results from cell tracing and transplantation experiments have recently emerged about the existence and role of stem/progenitor cells in the liver, their overall contribution to parenchymal cell homeostasis and tissue repair is limited. Accordingly, engraftment and repopulation efficacies of extrahepatic and liver-derived stem/progenitor cell types are considered to be lower compared to mature hepatocytes. On the basis of these results, we will discuss the current clinical cell transplantation programs for inherited metabolic liver diseases and future developments in liver cell therapy.

Stem cell-based regenerative opportunities for the liver: State of the art and beyond

The existing mismatch between the great demand for liver transplants and the number of available donor organs highlights the urgent need for alternative therapeutic strategies in patients with acute or chronic liver failure. The rapidly growing knowledge on stem cell biology and the intrinsic repair processes of the liver has opened new avenues for using stem cells as a cell therapy platform in regenerative medicine for hepatic diseases. An impressive number of cell types have been investigated as sources of liver regeneration: adult and fetal liver hepatocytes, intrahepatic stem cell populations, annex stem cells, adult bone marrow-derived hematopoietic stem cells, endothelial progenitor cells, mesenchymal stromal cells, embryonic stem cells, and induced pluripotent stem cells. All these highly different cell types, used either as cell suspensions or, in combination with biomaterials as implantable liver tissue constructs, have generated great promise for liver regeneration. However, fundamental questions still need to be addressed and critical hurdles to be overcome before liver cell therapy emerges. In this review, we summarize the state-of-the-art in the field of stem cell-based therapies for the liver along with existing challenges and future perspectives towards a successful liver cell therapy that will ultimately deliver its demanding goals.

Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver

Engraftment of transplanted cells is critical for liver-directed cell therapy, but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells (KCs). To define whether tumor necrosis factor alpha (TNF-α) served roles in cell-transplantation-induced hepatic inflammation, we used the TNF-α antagonist, etanercept (ETN), for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas ETN before cell transplantation essentially normalized these responses. Moreover, ETN down-regulated cell-transplantation-induced intrahepatic release of secretory cytokines, such as high-mobility group box 1. These effects of ETN decreased cell-transplantation-induced activation of neutrophils, but not of KCs. Transplanted cell engraftment improved by several-fold in ETN-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with ETN before multiple cell transplantation sessions. Transplanted cell numbers did not change over time, indicating absence of cell proliferation after ETN alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after ETN pretreatment significantly accelerated liver repopulation, compared to control rats. TNF-α plays a major role in orchestrating cell-transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. Because TNF-α antagonism by ETN decreased transplanted cell clearance, improved cell engraftment, and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy.

Cryopreservation of microencapsulated human hepatocytes

To establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver. Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC). The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not. The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.

Adult Human Hepatocytes Promote CD4+ T-Cell Hyporesponsiveness Via Interleukin-10-Producing Allogeneic Dendritic Cells

The success of liver cell therapy remains closely dependent on how well the infused cells can be accepted after transplantation and is directly related to their degree of immunogenicity. In this study, we investigated the in vitro immunogenic properties of isolated human hepatocytes (hHeps) and adult-derived human liver progenitor cells (ADHLPCs), an alternative cell candidate for liver cell transplantation (LCT). The constitutive expression of immune markers was first analyzed on these liver-derived cells by flow cytometry. Human liver-derived cells were then cocultured with allogeneic human adult peripheral blood mononuclear cells (PBMCs), and the resulting activation and proliferation of PBMCs was evaluated, as well as the cytokine levels in the coculture supernatant. The effect of liver-derived cells on monocyte-derived dendritic cell (MoDC) properties was further analyzed in a secondary coculture with naive CD4(+) T-cells. We report that hHeps and ADHLPCs expressed human leukocyte antigen (HLA) class I and Fas but did not express HLA-DR, Fas ligand, and costimulatory molecules. hHeps and ADHLPCs did not induce T-cell activation or proliferation. Moreover, hHeps induced a cell contact-dependent production of interleukin (IL)-10 that was not observed with ADHLPCs. The IL-10 was produced by a myeloid DC subset characterized by an incomplete mature state. Furthermore, hHep-primed MoDCs induced an antigen-independent hyporesponsiveness of naive CD4(+) T lymphocytes that was partially reversed by blocking IL-10, whereas nonprimed MoDCs (i.e., those cultured alone) did not. hHeps and ADHLPCs present a low immunogenic phenotype in vitro. Allogeneic hHeps, but not ADHLPCs, promote a cell contact-dependent production of IL-10 by myeloid DCs, which induces naive CD4(+) T-cells antigen-independent hyporesponsiveness.

In Utero Transplantation of Human Hepatocytes Results in Sustained Immune Hyporesponsiveness in the FAH Deficient (FAH-/-) Pig.

We postulated that FAH deficiency would provide a selective advantage for robust in vivo expansion of human hepatocytes in pigs. FAH-/- pigs were produced through adeno-associated virus-mediated gene targeting. In utero transplantation of human hepatocytes was performed in FAH-/- pigs to induce fetal immune hyporesponsiveness and avoid complications of immunosuppression. Three doses of human hepatocytes were studied: 1x106 cells (Group 1, n=3 pigs), 4x106 cells (Group 2, n=3 pigs), or 0 cells (Control, n=4 pigs). Cells were injected intrahepatically by ultrasound-guidance at gestational day (GD) 40, prior to development of the fetal pig immune system. Following delivery on GD115, pigs were primed by subcutaneous inoculation of 1x107 human hepatocytes at 2 week intervals. Porcine peripheral blood mononuclear cells were collected for immune monitoring by enzyme-linked immunosorbent spot (ELISPOT) assay, which detected porcine IFNγ labeled cells. Administration of the protective drug nitisinone to FAH-/- pigs led to normal phenotype, while withdrawal of nitisinone resulted in progressive liver injury, elevated AST (205.5 ± 58.7 U/L) and hyperammonemia (629.0 ±369.1 μmol/L). Histopathological evidence of hepatocyte injury included ballooning degeneration, necrosis, and karyomegaly. Equivalent levels of immune hyporesponsiveness to human cells were observed by ELISPOT assay in pigs from Groups 1 and 2. These levels were both reduced significantly compared to control pigs at 10 weeks: 96±37 (Group 1) vs 100±41 (Group 2) vs 256±23 (Control); Group 1 and Group 2 vs Control, p<0.001. In conclusion, nitisinone withdrawal in the FAH-/- pig offers a selective environment for in vivo expansion of FAH+ donor cells. In utero transplantation of 1-4x106 human hepatocytes resulted in immune hyporesponsiveness and may allow for their robust expansion without or with reduced immunosuppression and its associated co-morbidities. DISCLOSURES:Grompe, M.: Stockholder, Yecuris Incorporated. Nyberg, S.: Stockholder, Liver Cell Therapies Incorporated.