Allogeneic Hepatocytes Research Articles

Liver Ultrasound Histotripsy: Novel Analysis of the Histotripsy Site Cell Constituents with Implications for Histotripsy Application in Cell Transplantation and Cancer Therapy.

Introduction: Allogenic hepatocyte transplantation is an attractive alternative to whole-organ transplantation, particularly for the treatment of metabolic disorders and acute liver failure. However, the shortage of human donor organs for cell isolation, the low cell yield from decellularisation regimes, and low engraftment rates from portal administration of donor cells have restricted its clinical application. Using ultrasound histotripsy to provide a nidus in the liver for direct cell transplantation offers a new approach to overcoming key limitations in current cell therapy. We have analysed the liver cavity constituents to assess their potential as a site for cell delivery and implantation. Methods: Using human organ retrieval techniques, pig livers were collected from the abattoir and transported in ice-cold storage to the laboratory. Following 2 h of cold storage, the livers were flushed with organ preservation solution and placed on an organ perfusion circuit to maintain viability. Organs were perfused with Soltran™ organ preservation solution via the portal vein at a temperature of 24-30 °C. The perfusion circuit was oxygenated through equilibration with room air. Perfused livers (n=5) were subjected to ultrasound histotripsy, producing a total of 130 lesions. Lesions were generated by applying 50 pulses at 1 Hz pulse repetition frequency and 1% duty cycle using a single element 2 MHz bowl-shaped transducer (Sonic Concepts, H-148). Following histotripsy, a focal liver lesion was produced, which had a liquid centre. The fluid from each lesion was aspirated and cultured in medium (RPMI) at 37 °C in an incubator. Cell cultures were analysed at 1 and 7 days for cell viability and a live-dead assay was performed. The histotripsy sites were excised following aspiration and H&E staining was used to characterise the liver lesions. Cell morphology was determined by histology. Results: Histotripsy created a subcapsular lesion (~5 mm below the liver capsule; size ranging from 3 to 5 mm), which contained a suspension of cells. On average, 61×104 cells per mL were isolated. Hepatocytes were present in the aspirate, were viable at 24 h post isolation and remained viable in culture for up to 1 week, as determined by phalloidin/DAPI cell viability stains. Cultures up to 21 days revealed metabolically active live hepatocyte. Live-dead assays confirmed hepatocyte viability at 1 week (Day 1: 12% to Day 7: 45% live cells; p < 0.0001), which retained metabolic activity and morphology, confirmed on assay and microscopy. Cell Titre-GloTM showed a peak metabolic activity at 1 week (average luminescence 24.6 RLU; p < 0.0001) post-culture compared with the control (culture medium alone), reduced to 1/3 of peak level (7.85 RLU) by day 21. Conclusions: Histotripsy of the liver allows isolation and culture of hepatocytes with a high rate of viability after 1 week in culture. Reproducing these findings using human livers may lead to wide clinical applications in cell therapy.

Alterations in The Functional and Structural Properties of Erythrocytes Due to Alcohol Intoxication; Correction of Disorders

Alcohol abuse is among the leading reasons for disability and accounts for at least 5% of the global health burden. This study aims to define changes in the functional and structural properties of erythrocytes in chronic ethanol intoxication and to design methods for correction of the identified disorders. The experiments has been conducted on 93 healthy sexually mature and newborn Wistar rats. Acute and chronic alcohol intoxication has been designed by forced intragastric administration of 20% ethanol solution at a dose of 3 ml/kg (2.92 g/kg) on a daily basis for 5, 30 and 60 days. From the newborn animals we obtained allogeneic hepatocytes and their cultural fluid, from which proteins were isolated. The proteins of allogeneic hepatocyte and the combined administration of immunomodulatory, hepatoprotective and antioxidant agents were used to correct disorders in chronic intoxication. The membranes of circulating erythrocytes were obtained by the method of Dodge; electrophoretic separation of membrane proteins was carried out according to the method of Laemli. The lipid content in the erythrocyte membrane was determined by liquid thin-layer chromatography. It has been elucidated that in presence of chronic alcohol intoxication there is a change in the content of the membrane proteins in circulating erythrocytes, which are responsible for flexibility and shaping, intrinsic cellular metabolism, stabilization and structure formation, and a disruption in the lipids representativeness and ratio that form the basis for the lipid framework of the plasmalemma and play the major role in arrangement of protein macromolecules and the normal metabolism of red blood cells as well. When analyzing the corrective activity of the proteins of allogeneic hepatocytes and the combination Glutoxim + Mexidol + Heptral, we have established their high efficiency towards the disorders in the functional and structural properties of circulating erythrocytes caused by chronic alcohol intoxication.

Invariant NKT Cells Promote the Development of Highly Cytotoxic Multipotent CXCR3+CCR4+CD8+ T Cells That Mediate Rapid Hepatocyte Allograft Rejection.

Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8+ T cells, with or without CD4+ T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type) and iNKT-deficient Jα18 knockout mice (cohorts CD4 depleted) were transplanted with allogeneic hepatocytes. Recipients were evaluated for alloprimed CD8+ T cell subset composition, allocytotoxicity, and hepatocyte rejection. We found that CD8-mediated allocytotoxicity was significantly decreased in iNKT-deficient recipients and was restored by adoptive transfer of iNKT cells. In the absence of both iNKT cells and CD4+ T cells, CD8-mediated allocytotoxicity and hepatocyte rejection was abrogated. iNKT cells enhance the proportion of a novel subset of multipotent, alloprimed CXCR3+CCR4+CD8+ cytolytic T cells that develop after hepatocyte transplant and are abundant in the liver. Alloprimed CXCR3+CCR4+CD8+ T cells express cytotoxic effector molecules (perforin/granzyme and Fas ligand) and are distinguished from alloprimed CXCR3+CCR4-CD8+ T cells by a higher proportion of cells expressing TNF-α and IFN-γ. Furthermore, alloprimed CXCR3+CCR4+CD8+ T cells mediate higher allocytotoxicity and more rapid allograft rejection. Our data demonstrate the important role of iNKT cells in promoting the development of highly cytotoxic, multipotent CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of allogeneic hepatocytes engrafted in the liver. Targeting iNKT cells may be an efficacious therapy to prevent rejection of intrahepatic cellular transplants.

Proteins of allogeneic hepatocytes and pharmacological preparations for the correction of immunometabolic disorders in experimental liver pathology

Introduction: The relationship of numerous metabolic shifts, disorders of hepatocytes functional activity resulting from hypoxia and toxic liver damage with the function of the immune system has not been sufficiently studied so far, nor have the most effective methods of pharmacological correction been established.&#x0D; Materials and Methods: The studies were carried out on 603 mature male Wistar rats and 45 mice. Acute toxic liver damage (ATLD) was modeled by intramuscular introduction of carbon tetrachloride; acute liver ischemia (ALI) was caused by clamping the hepatoduodenal ligament for 20 minutes; chronic alcohol intoxication (CAI) was modeled by forced intragastric administration of 20% ethanol solution for 60 days. Isolation of xenogeneic (murine) and allogeneic (rat) hepatocytes from newborn mice and rats was carried out according to the method of Berry and Friend (1969); culture fluid of hepatocytes and its protein fractions were prepared according to our developed techniques. The obtained biological material was intraperitoneally introduced into the rats with ATLD, ALI, and CAI.&#x0D; Results and Discussion: In all the models of the liver damage, there developed morphological and biochemical signs of the liver damage, impaired congenital and adaptive immunity, oxidative stress, increased lipid peroxidation processes.&#x0D; Conclusion: The introduction of allogeneic hepatocytes, culture fluid obtained on their basis,and proteins isolated from it with MW less than 130 kDa to the recipients with toxic and ischemic liver damage more effectively corrects the pathologic changes in the liver in comparison with xenogeneic hepatocytes, their culture fluid and pharmacological preparations (combinations of Essentiale N and Hypoxenum or Heptral and Mexicor).

Invariant NKT cells promote expansion of novel CXCR3+CCR4+CD8+ T cells in the liver following allogeneic hepatocyte transplant

Abstract Hepatocyte transplant (HcTx) is a treatment for metabolic diseases and a bridge to liver transplant but is limited by immunogenicity. Our prior work identified the critical role of CD4-dependent and CD4-independent CD8+ T cells in HcTx rejection. Herein, we evaluated the influence of invariant natural killer T cells (iNKTs), abundant in the liver, upon CD8+ T cell responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type; WT) or iNKT deficient Jα18 KO (H-2b) received FVB/N (H-2q) HcTx. Cohorts were depleted of CD4+ T cells and/or reconstituted with iNKTs. Recipients were evaluated for cytotoxicity, CD8+ T cell phenotype, and HcTx rejection. By comparing CD4-sufficient WT and Jα18 KO recipients, we found that the presence of iNKTs significantly enhanced in vivo CD8-mediated cytotoxicity (83±3% vs 23±3%; p&amp;lt;0.001) but did not affect survival (10 days vs 14 days; p=ns). However, in CD4-deficient recipients, iNKTs were critical, since without CD4+ T cells and iNKTs, CD8-mediated in vivo cytotoxicity was completely abrogated (31±3% vs 2±1%; p&amp;lt;0.001) and HcTx survival was significantly prolonged (14 days vs 56 days; p&amp;lt;0.001). iNKTs enhanced the quantity of a novel CD8+ T cell subset, CD44+IFN-γ+CXCR3+CCR4+CD8+ T cells, in the liver (2-fold) in all HcTx recipients (p&amp;lt;0.03 for all comparisons). CXCR3+CCR4+CD8+ T cells, when compared to CXCR3+CCR4−CD8+ T cells, express higher IFN-γ (32±3% vs 9±1%; p&amp;lt;0.001) and TNF-α (37±3% vs 6±1%; p&amp;lt;0.001). They also exhibit greater in vivo cytotoxicity (13±1% vs 5±1%; p&amp;lt;0.001) and mediate rapid HcTx rejection in RAG1 KO recipients (5 days vs 16 days; p=0.02). We show that iNKTs enhance the expansion of highly cytotoxic CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of HcTx.

Tumor Necrosis Factor Directs Allograft-Related Innate Responses and Its Neutralization Improves Hepatocyte Engraftment in Rats

The innate immune system plays a critical role in allograft rejection. Alloresponses involve numerous cytokines, chemokines, and receptors that cause tissue injury during rejection. To dissect these inflammatory mechanisms, we developed cell transplantation models in dipeptidylpeptidase-deficient F344 rats using mycophenolate mofetil and tacrolimus for partial lymphocyte-directed immunosuppression. Syngeneic hepatocytes engrafted in liver, whereas allogeneic hepatocytes were rejected but engrafted after immunosuppression. These transplants induced mRNAs for >40 to 50 cytokines, chemokines, and receptors. In allografts, innate cell type-related regulatory networks extended to granulocytes, monocytes, and macrophages. Activation of Tnfa and its receptors or major chemokine receptor-ligand subsets persisted in the long term. An examination of the contribution of Tnfa in allograft response revealed that it was prospectively antagonized by etanercept or thalidomide, which resolved cytokine, chemokine, and receptor cascades. In bioinformatics analysis of upstream regulator networks, the Cxcl8 pathway exhibited dominance despite immunosuppression. Significantly, Tnfa antagonism silenced the Cxcl8 pathway and decreased neutrophil and Kupffer cell recruitment, resulting in multifold greater engraftment of allogeneic hepatocytes and substantially increased liver repopulation in retrorsine/partial hepatectomy model. We conclude that Tnfa is a major driver for persistent innate immune responses after allogeneic cells. Neutralizing Tnfa should help in avoiding rejection and associated tissue injury in the allograft setting.

Эффективность белков аллогенных гепатоцитов в коррекции иммунометаболических нарушений при остром ишемическом поражении печени

Objective. The aim of the study was to establish the effectiveness of the culture fluid of allogeneic hepatocytes (CFAH) and proteins isolated from them in correcting metabolic and immune disorders in acute ischemic liver damage. Material and methods. The study has been conducted on 105 healthy mature Wistar rats. Acute ischemic liver damage (AILD) was induced by cross clamping of the hepatoduodenal ligament for 20 minutes. Isolated allogeneic hepatocytes from newborn animals were used to prepare a culture fluid from which proteins were isolated. The culture fluid of allogeneic hepatocytes (CFAH) and their proteins obtained were administered with the start of modeling acute liver ischemia five times (with a 24-hour interval) intraperitoneally to rats with AILD at the rate of 5 mg/kg. Results. Acute liver ischemia causes the development of biochemical syndromes of the liver damage (cytolysis, intra- and extrahepatic cholestasis, toxic damage, insufficiency of synthetic processes) activates the processes of lipid peroxidation, disturbs intra-erythrocyte metabolism, and causes the development of oxidative stress and suppression of forming the adaptive and innate immunity. Introduction of CFAH, undivided CFAH proteins or CFAH proteins with molecular mass (MM) less than 130 kDa to animals with AILD normalizes or corrects the studied biochemical and immunological parameters. CFAH proteins with MM more than 130 kDa do not have such effects. Conclusion. During the analysis of CFAH or CFAH proteins corrective activity on the dynamics of biochemical markers of AILD, formation of humoral and cellular immunity, impaired functional and metabolic activity of peripheral blood neutrophils, intraerythrocyte metabolism, activation of free radical oxidation, the following sequence has been established (according to increasing the effectiveness): the lack of this activity in CFAH proteins with MM more than 130 kDa; the presence of normalizing and correcting activity of: CFAH - CFAH undivided proteins - CFAH proteins less than 130 kDa.

Critical Role of Macrophage FcγR Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection.

Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known. We hypothesized that macrophage-mediated destruction of allogeneic hepatocytes occurs by cell-cell interactions requiring FcγRs. To examine this, alloantibody-dependent hepatocyte rejection in CD8-depleted wild-type (WT) and Fcγ-chain knockout (KO; lacking all functional FcγR) transplant recipients was evaluated. Alloantibody-mediated hepatocellular allograft rejection was abrogated in recipients lacking FcγR compared with WT recipients. We also investigated anti-FcγRI mAb, anti-FcγRIII mAb, and inhibitors of intracellular signaling (to block phagocytosis, cytokines, and reactive oxygen species [ROS]) in an in vitro alloantibody-dependent, macrophage-mediated hepatocytoxicity assay. Results showed that in vitro alloantibody-dependent, macrophage-mediated hepatocytotoxicity was critically dependent on FcγRs and ROS. The adoptive transfer of WT macrophages into CD8-depleted FcγR-deficient recipients was sufficient to induce alloantibody-mediated rejection, whereas adoptive transfer of macrophages from Fcγ-chain KO mice or ROS-deficient (p47 KO) macrophages was not. These results provide the first evidence, to our knowledge, that alloantibody-dependent hepatocellular allograft rejection is mediated by host macrophages through FcγR signaling and ROS cytotoxic effector mechanisms. These results support the investigation of novel immunotherapeutic strategies targeting macrophages, FcγRs, and/or downstream molecules, including ROS, to inhibit humoral immune damage of transplanted hepatocytes and perhaps other cell and solid organ transplants.

Human hepatocyte transplantation for liver disease: current status and future perspectives.

Liver transplantation is the accepted treatment for patients with acute liver failure and liver-based metabolic disorders. However, donor organ shortage and lifelong need for immunosuppression are the main limitations to liver transplantation. In addition, loss of the native liver as a target organ for future gene therapy for metabolic disorders limits the futuristic treatment options, resulting in the need for alternative therapeutic strategies. A potential alternative to liver transplantation is allogeneic hepatocyte transplantation. Over the last two decades, hepatocyte transplantation has made the transition from bench to bedside. Standardized techniques have been established for isolation, culture, and cryopreservation of human hepatocytes. Clinical hepatocyte transplantation safety and short-term efficacy have been proven; however, some major hurdles-mainly concerning shortage of donor organs, low cell engraftment, and lack of a long-lasting effect-need to be overcome to widen its clinical applications. Current research is aimed at addressing these problems, with the ultimate goal of increasing hepatocyte transplantation efficacy in clinical applications.

КЛЕТОЧНЫЕ ТЕХНОЛОГИИ В КОРРЕКЦИИ НАРУШЕНИЙ МЕТАБОЛИЗМА ЭРИТРОЦИТОВ ПРИ ОСТРОМ ТОКСИЧЕСКОМ ПОРАЖЕНИИ ПЕЧЕНИ

Цель. Установление эффективности использования аллогенных гепатоцитов и мексидола в коррекции нарушений содержания белков и липидов мембран и показателей метаболизма эритроцитов в условиях острого токсического поражения печени.Материалы и методы. Исследования проведены на 65 крысах-самцах Вистар. Острое токсическое поражение печени моделировали путем внутримышечного введения четыреххлористого углерода. Выделение аллогенных гепатоцитов от животных через 5–6 дней после рождения производилась по методике M.N. Berry, D.S. Friend. Мексидол вводили одновременно с токсикантом, пятикратно, через 24 часа, внутрибрюшинно в дозе 50 мг/кг отдельно или вместе с аллогенными гепатоцитами.Результаты. В статье изложены данные о нарушениях содержания белков и липидов мембран и показателей метаболизма эритроцитов у экспериментальных животных в условиях острого токсического поражения печени (ОТПП), вызванного введением четыреххлористого углерода. В работе определена эффективность использования аллогенных гепатоцитов и мексидола в коррекции нарушенных показателей липидного спектра мембран и метаболизма эритроцитов у животных при ОТПП.Заключение. Эффект мексидола при остром токсическом поражении печени обусловлен нормализацией липидного обмена в клеточной мембране и, соответственно, белкового спектра мембран.Область применения результатов. Полученные результаты могут быть использованы при разработке эффективных способов коррекции метаболических нарушений при различных видах патологии печени.

Применение аллогенных гепатоцитов для коррекции метаболических нарушений при хронической алкогольной интоксикации

According to the experiment 60-day introduction of ethanol leads to the development of the main biochemical syndromes of liver damage: intracellular cytolysis and cholestasis, toxic hepatopathy of immuno-inflammatory type and failure of the synthetic processes, oxidizing stress, imbalance of functional and metabolic activity of neutrophils and erythrocytes of the circulating blood. Against this background the introduction of xenogenic hepatocytes enables to normalize 20.7% of the changed metabolic indicators and corrects 62.1% but not to the normal values. The use of allogeneic hepatocytes normalizes and corrects 44.8% and 55.2% of parameters, but the use of cultural liquid of the allogeneic hepatocytes was the most effective.

In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats

Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver.

The Protective Effect of Transplanting Liver Cells Into the Mesentery on the Rescue of Acute Liver Failure After Massive Hepatectomy.

Postoperative liver failure is one of the most critical complications following extensive hepatectomy. Although transplantation of allogeneic hepatocytes is an attractive therapy for posthepatectomy liver failure, transplanting cells via the portal veins typically causes portal vein embolization. The embolization by transplanted cells would be lethal in patients who have undergone massive hepatectomy. Thus, transplant surgeons need to select extrahepatic sites as transplant sites to prevent portal vein embolization. We aimed to investigate the mechanism of how liver cells transplanted into the mesentery protect recipient rats from acute liver failure after massive hepatectomy. We induced posthepatectomy liver failure by 90% hepatectomy in rats. Liver cells harvested from rat livers were transplanted into the mesenteries of hepatectomized rats. Twenty percent of the harvested cells, which consisted of hepatocytes and nonparenchymal cells, were transplanted into each recipient. The survival rate improved significantly in the liver cell transplantation group compared to the control group 7 days after hepatectomy (69 vs. 7%). Histological findings of the transplantation site, in vivo imaging system study findings, quantitative polymerase chain reaction assays of the transplanted cells, and serum albumin measurements of transplanted Nagase analbuminemic rats showed rapid deterioration of viable transplanted cells. Although viable transplanted cells deteriorated in the transplanted site, histological findings and an adenosine-5'-triphosphate (ATP) assay showed that the transplanted cells had a protective effect on the remaining livers. These results indicated that the paracrine effects of transplanted liver cells had therapeutic effects. The same protective effects were observed in the hepatocyte transplantation group, but not in the liver nonparenchymal cell transplantation group. Therefore, this effect on the remnant liver was mainly due to the hepatocytes among the transplanted liver cells. We demonstrated that transplanted liver cells protect the remnant liver from severe damage after massive hepatectomy.

IMMUNE AND METABOLIC DISTURBANCES IN EXPERIMENTAL ACUTE TOXIC HEPATITIS: CORRECTION BY XENOGENIC AND ALLOGENIC HEPATOCYTES

For correspondence: Konoplya Alexander Ivanovich. Address: 3,K. Marx St.,Kursk, 305041,Russian Federation. Tel.: job. (4712) 58-81-76; mob. (910) 317-87-88. E-mail: konoplya51@mail.ru.Aim. To study the corrective effects of allogeneic and xenogeneic hepatocytes on metabolic disturbances in acute liver toxicity.Material and methods. Investigations were carried out on 75 adult male Wistar rats weighing 120–160 g, 15 rats and 25 mice on the 5–6th days after birth. Acute toxic hepatitis (ATH) was modeled by intramuscular injection of carbon tetrachloride at a dose of 3 ml / kg as a 50% solution in olive oil, five times at 24-hour intervals. Isolating xenogeneic (mouse) and allogeneic hepatocytes was performed by method of Berry M.N., Friend D.S. The cell suspension was prepared daily and administered at a concentration of 2 × 106 /kg in recipients with ATH intraperitoneally, five times at 24-hour intervals, simultaneously with the first injection of hepatotropic poison.Results. Intoxication by carbon tetrachloride causes development of the biochemical syndromes of liver damage, activation of the functional metabolic activity of peripheral blood neutrophils and free-radical oxidation, breaks intraerythrocytic metabolism. The introduction of allogeneic hepatocytes in recipients with toxic hepatopathy is more efficiently compared with xenogeneic hepatocytes, it corrects local and systemic metabolic disturbances arising due to the impact of hepatotropic poison. Conclusion. Transplantation of xenogenic hepatocytes, and, to a greater extent, of allogenic hepatocytes in ATH conditions is an effective means to restore the functional metabolic activity of hepatocytes, neutrophils and erythrocytes.

Cell-based therapies of liver diseases: age-related challenges.

The scope of this review is to revise recent advances of the cell-based therapies of liver diseases with an emphasis on cell donor’s and patient’s age. Regenerative medicine with cell-based technologies as its integral part is focused on the structural and functional restoration of tissues impaired by sickness or aging. Unlike drug-based medicine directed primarily at alleviation of symptoms, regenerative medicine offers a more holistic approach to disease and senescence management aimed to achieve restoration of homeostasis. Hepatocyte transplantation and organ engineering are very probable forthcoming options of liver disease treatment in people of different ages and vigorous research and technological innovations in this area are in progress. Accordingly, availability of sufficient amounts of functional human hepatocytes is crucial. Direct isolation of autologous hepatocytes from liver biopsy is problematic due to related discomfort and difficulties with further expansion of cells, particularly those derived from aging people. Allogeneic primary human hepatocytes meeting quality standards are also in short supply. Alternatively, autologous hepatocytes can be produced by reprogramming of differentiated cells through the stage of induced pluripotent stem cells. In addition, fibroblasts and mesenchymal stromal cells can be directly induced to undergo advanced stage hepatogenic differentiation. Reprogramming of cells derived from elderly people is accompanied by the reversal of age-associated changes at the cellular level manifesting itself by telomere elongation and the U-turn of DNA methylation. Cell reprogramming can provide high quality rejuvenated hepatocytes for cell therapy and liver tissue engineering. Further technological advancements and establishment of national and global registries of induced pluripotent stem cell lines homozygous for HLA haplotypes can allow industry-style production of livers for immunosuppression-free transplantation.

Ectopic Liver Tissue Formation in Rats with Induced Liver Fibrosis.

IntroductionThe possible alternative approach to whole-organ transplantation is a cell-based therapy, which can also be used as a “bridge” to liver transplantation. However, morphological and functional changes in the liver of patients suffering from chronic liver fibrosis and cirrhosis restrict the effectiveness of direct cell transplantation. Therefore, extra hepatic sites for cell transplantation, including the spleen, pancreas, peritoneal cavity, and subrenal capsule, could be a useful therapeutic approach for compensation of liver functions. However, a method of transplantation of hepatocytes into ectopic sites is needed to improve hepatocyte engraftment. Previously published data has demonstrated that mouse lymph nodes can support the engraftment and proliferation of hepatocytes as ES and rescue Fah mice from lethal liver failure. Thus, the aim of the study was to evaluate the engraftment of i.p. injected allogeneic hepatocytes into extra hepatic sites in albino rats with chemically induced liver fibrosis (LF).Materials and methodsAlbino rats were randomly divided into 4 groups: (1) intact group (n = 18); (2) rats with induced LF (n = 18); (3) rats with induced LF and transplanted with hepatocytes (n = 18); (4) as a control, rats were treated with cyclosporine A only (n = 18). In order to prevent an immune response, groups 2 and 3 were subjected to immunosuppression by cyclosporine A (25 mg/kg per day). LF was induced using N-nitrosodimethylamine (NDMA), i.p., 10 mg/kg, three times a week for 4 weeks and confirmed by histological analysis of the liver samples. Hepatocytes transplantation (HT) was performed two days after NDMA exposure cessation by i.p. injection of 5×106 freshly isolated allogeneic hepatocytes. Liver function was assessed by quantifying blood biochemical parameters (ALT, AST, GGT, total protein, bilirubin, and albumin) at 1 week, 1 month, and 2 months after hepatocytes transplantation (HT). To confirm a hepatocytes’ engraftment, we conducted immunohistochemical staining against HepPar1.ResultsWe observed a 30% mortality rate among rats with LF within 1 week after NDMA exposure cessation, while 100% of animals with HT survived. ALT, AST, and GGT activities and bilirubin levels were markedly elevated in blood samples of LF rats compared to the control animals. However, HT significantly improved ALT, AST, and GGT activity as well as bilirubin levels. We also observed decreased levels of total protein and albumin in the blood serum of rats with LF, while HT normalized these parameters. At the same time, we have not detected any statistical differences of the studied parameters in the group 4, which was treated with Cyclosporine A only, compared with the intact animals. HepPar1 immunohistochemical staining of the different tissue sections demonstrated the presence of engrafted hepatocytes, mainly within enlarged Peyer’s patches (aggregated lymphoid nodules in the lowest portion of the small intestine).ConclusionThe results of our study provide evidence that HT improves animal survival and liver functions. One potential reason for these results is that ectopic hepatic mass inside the Peyer’s patches can rescue rats from liver failure.

Allogenic hepatocyte transplantation in immunomodulated Lewis rats with acute liver insufficiency following hepatectomy.

Orthotropic Liver transplantation (OLT) is a conventional management for end-stage acute or chronic liver insufficiency, but the shortage of donor organs continues to be the restrictive factor throughout the world. Hepatocyte transplantation (HCTx) might be the promising treatment for several liver diseases and can be used as a "bridge" to OLT. Hepatocytes transplantation can protect and even save human lives, its' applicability remains limited by the large deficiency of liver organs and hepatocytes (HC), and cellular loss after engraftment. Host elimination of grafted cells is called Early Graft Dysfunction. This study was developed for an efficient protocol of HCT. Several conditions have been met in order to achieve a high yield of harvested viable HC, overcome the detached-cell apoptosis, attenuation of innate immune reaction against transplanted cells and a receptive cell environment. HC were isolated from Lewis rats (n = 8) weighing 250 gm, by the 2 step collagen a seper fusion technique, and bone marrow cells (BMCs) were obtained from the rats tibia and femur by centrifugation in a buffer solution. The mean viability of harvested HC and BMCs were 90% and 95% respectively. To minimize the rejection of HC, Lewis rat recipients (n = 14) weighing 250 gm, were irradiated with 6 Gy and received 0.1 mg of anti-aisle GMI antiserum intravenously as immunosuppressive drug. The isolated HC were intra-splenically transplanted and 10(7) bone marrow cells were injected in a penile vein into the recipients on the third day. Simultaneously, 70% hepatectomy and ligation of common bile duct were done. Thirty days later; the grafted spleen had areas with external appearance of a normal liver in ten out of 14 surviving rats (71%). Hematoxlin and eosin (H & E) staining of sections from these fragments showed sinusoids and portal areas, an evidence of successful hepatocyte engraftment and bile canaliculea formation. Large number of HC clusters of 15 to 20 cells and 2 to 4 distended small bile canaliculea were seen per 50 HC. The intrasplenic route for transplanting freshly isolated HC in an immune-compromised animal model was found to give good results regarding cell engraftment and tissue formation.

Usage of Adenovirus Expressing Thymidine Kinase Mediated Hepatocellular Damage for Enabling Mouse Liver Repopulation with Allogenic or Xenogenic Hepatocytes

It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2-/- γc-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

Human Pluripotent Stem Cells for Modelling Human Liver Diseases and Cell Therapy

The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly. However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes. The presence of a niche of “facultative” progenitor and stem cells in the normal liver has recently been confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery.

Placental stem cell correction of murine intermediate maple syrup urine disease

There is improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model after allogenic hepatocyte transplantation, confirming that a small number of enzyme-proficient liver-engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAECs) share stem cell characteristics without the latter's safety and ethical concerns and differentiate to hepatocyte-like cells. Eight direct hepatic hAEC transplantations were performed in iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at 35 and 100 days. Treatment at the neonatal stage is clinically relevant for MSUD and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplantations compared with untreated iMSUD mice, which were severely cachectic and died ≤28 days after birth. Branched chain α-keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. The branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in serum and brain, as were other large neutral amino acids. Placental-derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver-based metabolic diseases.