Liver Repopulation Research Articles

Hematopoietic stem cells and liver regeneration: differentially acting hematopoietic stem cell mobilization agents reverse induced chronic liver injury.

Bone marrow (BM) could serve as a source of cells facilitating liver repopulation in case of hepatic damage. Currently available hematopoietic stem cell (HSC) mobilizing agents, were comparatively tested for healing potential in liver fibrosis. Carbon tetrachloride (CCl4)-injured mice previously reconstituted with Green Fluorescent Protein BM were mobilized with Granulocyte-Colony Stimulating Factor (G-CSF), Plerixafor or G-CSF+Plerixafor. Hepatic fibrosis, stellate cell activation and oval stem cell frequency were measured by Gomori and by immunohistochemistry for a-Smooth Muscle Actin and Cytokeratin-19, respectively. Angiogenesis was evaluated by ELISA and immunohistochemistry. Quantitative real-time PCR was used to determine the mRNA levels of liver Peroxisome Proliferator-Activated Receptor gamma (PPAR-γ), Interleukin-6 (IL-6) and Tumor Necrosis-alpha (TNFα). BM-derived cells were tracked by double immunofluorescence. The spontaneous migration of mobilized HSCs towards injured liver and its cytokine secretion profile was determined in transwell culture systems. Either single-agent mobilization or the combination of agents significantly ameliorated hepatic damage by decreasing fibrosis and restoring the abnormal vascular network in the liver of mobilized mice compared to CCl4-only mice. The degree of fibrosis reduction was similar among all mobilized mice despite that G-CSF+Plerixafor yielded significantly higher numbers of circulating HSCs over other agents. The liver homing potential of variously mobilized HSCs differed among the agents. An extended G-CSF treatment provided the highest anti-fibrotic effect over all tested modalities, induced by the proliferation of hepatic stem cells and decreased hepatic inflammation. Plerixafor-mobilized HSCs, despite their reduced liver homing potential, reversed fibrosis mainly by increasing hepatic PPAR-γ and VEGF expression. In all groups, BM-derived mature hepatocytes as well as liver-committed BM stem cells were detected only at low frequencies, further supporting the concept that alternative mechanisms rather than direct HSC effects regulate liver recovery. Overall, our data suggest that G-CSF, Plerixafor and G-CSF+Plerixafor act differentially during the wound healing process, ultimately providing a potent anti-fibrotic effect.

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation.

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.

644 A Genetic Screen to Identify and Rank Regulators of Liver Repopulation Following Toxic Injury

644 A Genetic Screen to Identify and Rank Regulators of Liver Repopulation Following Toxic Injury

The Development of Humanized Liver With Rag1 Knockout Rats

BackgroundThe animal model with humanized liver is useful for testing drug metabolism and toxicity in preclinical studies. A mouse model has been reported in which the liver was repopulated more than 90% with human hepatocytes; however, in the rat, the target is far from being reached. In this study, we attempt to develop a humanized liver model with an immunodeficient rat. MethodsRag1 knockout rats were treated with neonatal thymectomy. At 3 and 4 weeks of age, they were injected with hepatotoxin retrorsine; 2 weeks after, the animals were subjected to 70% partial hepatectomy and transplanted with immature human hepatocytes via portal vein. The recipients were also treated with anti-asialo GM1 antibody weekly from the day before transplantation and were injected with FK506 every 3 days after transplantation. ResultsIn Rag1 knockout rats, B lymphocytes were deleted almost completely in peripheral blood. However, T and natural killer (NK) lymphocytes were kept present. When they were treated additionally with neonatal thymectomy for T-lymphocyte deletion and suppressed neutralized NK lymphocytes with anti-asialo GM1, B, T, and NK cells in lymphocytes were reduced to very low levels of 0.75%, 1.58%, and 0.26%, respectively. After transplanting human donor hepatocytes into retrorsine-treated recipient livers, at week 3 the human cell-derived hepatic colonies were expanded in the recipient liver and the liver repopulation rate with human hepatocytes reached approximately 17%. The human hepatocyte-specific genes, albumin, CYP3A4, CYP2C18, and CYP2C9, also could be detected in the recipient rat. ConclusionIt is possible to generate a chimera animal with humanized liver in a novel severely immunodeficient rat model.

Cell therapy to remove excess copper in Wilson's disease.

To achieve permanent correction of Wilson's disease by a cell therapy approach, replacement of diseased hepatocytes with healthy hepatocytes is desirable. There is a physiological requirement for hepatic ATP7B-dependent copper (Cu) transport in bile, which is deficient in Wilson's disease, producing progressive Cu accumulation in the liver or brain with organ damage. The ability to repopulate the liver with healthy hepatocytes raises the possibility of cell therapy in Wilson's disease. Therapeutic principles included reconstitution of bile canalicular network as well as proliferation in transplanted hepatocytes, despite toxic amounts of Cu in the liver. Nonetheless, cell therapy studies in animal models elicited major differences in the mechanisms driving liver repopulation with transplanted hepatocytes in Wilson's disease versus nondiseased settings. Recently, noninvasive imaging was developed to demonstrate Cu removal from the liver, including after cell therapy in Wilson's disease. Such developments will help advance cell/gene therapy approaches, particularly by offering roadmaps for clinical trials in people with Wilson's disease.

Cell turnover in the repopulated rat liver: distinct lineages for hepatocytes and the biliary epithelium

The dynamics of cell renewal in the normal adult liver remains an unresolved issue. We investigate the possible contribution of a common biliary precursor cell pool to hepatocyte turnover in the chimeric long-term repopulated rat liver. The retrorsine (RS)-based model of massive liver repopulation was used. Animals not expressing the CD26 marker (CD26-) were injected with RS, followed by transplantation of 2 million syngeneic hepatocytes isolated from a normal CD26-expressing donor. Extensive (80-90 %) replacement of resident parenchymal cells was observed at 1 year post-transplantation and persisted at 2 years, as expected. A panel of specific markers, including cytokeratin 7, OV6, EpCAM, claudin 7 and α-fetoprotein, was employed to locate the in situ putative progenitor and/or biliary epithelial cells in the stably repopulated liver. No overlap was observed between any of these markers and the CD26 tag identifying transplanted cells. Exposure to RS was not inhibitory to the putative progenitor and/or biliary epithelial cells, nor did we observe any evidence of cell fusion between these cells and the transplanted cell population. Given the long-term (>2 years) stability of the donor cell phenotype in this model of liver repopulation, the present findings suggest that hepatocyte turnover in the repopulated liver is fuelled by a cell lineage distinct from that of the biliary epithelium and relies largely on the differentiated parenchymal cell population. These results support the solid biological foundation of liver repopulation strategies based on the transplantation of isolated hepatocytes.

Lack of CD47 on Donor Hepatocytes Promotes Innate Immune Cell Activation and Graft Loss: A Potential Barrier to Hepatocyte Xenotransplantation

We have previously shown that interspecies incompatibility of CD47 plays an important role in triggering rejection of xenogeneic hematopoietic cells by macrophages. However, whether CD47 incompatibility also induces rejection of nonhematopoietic cellular xenografts remains unknown. Herein, we have addressed this question in a mouse model of hepatocyte transplantation in which CD47(-/-) hepatocytes were used to resemble xenografts for CD47 incompatibility. We show that intrasplenic transplantation of CD47(-/-), but not wild-type (WT) hepatocytes, into partially hepatectomized syngeneic WT mice resulted in a rapid increase in Mac-1(+) cells with an activation phenotype (i.e., Mac-1(+)CD14(+) and Mac-1(+)CD16/32(high)), compared to nontransplant controls. In addition, CD47(-/-) hepatocytes were more severely damaged than WT hepatocytes as indicated by the greater AST and ALT serum levels in these mice. Furthermore, long-term donor hepatocyte survival and liver repopulation were observed in mice receiving WT hepatocytes, whereas CD47(-/-) hepatocytes were completely rejected within 2 weeks. These results suggest that CD47 on donor hepatocytes prevents recipient myeloid innate immune cell activation, hence aiding in graft survival after hepatocyte transplantation. Thus, CD47 incompatibility is likely to present an additional barrier to hepatocyte xenotransplantation.

Extrahepatic stem cells mobilized from the bone marrow by the supplementation of branched‐chain amino acids ameliorate liver regeneration in an animal model

In recent years, bone marrow (BM)-derived stem cell repopulation of injured organs has been increasingly observed; however, the extent to which it occurs and its clinical relevance remain unclear. Here, we investigated on the potential of extrahepatic stem cells to become hepatocytes using the treatment of the oral supplementation of branched-chain amino acids (BCAA). In the first, Sprague-Dawley (SD) rats were administered BCAA to promote liver regeneration; in the second, syngenic liver transplantations using wild-type SD rats that do not express green fluorescent protein (GFP) as syngenic donors and GFP-transgenic SD rats as recipients to confirm that an extrahepatic source of cells (GFP(+)) could repopulate the transplanted (GFP(-)) liver were performed. Treatment of the oral supplementation of BCAA for 2-3 weeks before transplantation to promote liver regeneration resulted in greater than 7 days graft volume, with extensive spotty conversion of a small wild-type graft to the recipient GFP(+) genotype. The treatment by oral supplementation of BCAA resulted in higher levels of CD34+SDF+c-kit+ stem cells in the blood and liver after liver transplantation. Liver repopulation could be achieved with hepatocytes that bone marrow-derived from stem cells proliferated. We have identified extrahepatic stem cell migration from the BM to the injured liver as a mechanism underlying liver regeneration that supports hepatocyte proliferation in diseased liver. Our results suggested that BCAA is able to mobilize a population of BM-derived cells that contribute to hepatic regeneration.

Liver regeneration and energetic changes in rats following hepatic radiation therapy and hepatocyte transplantation by ³¹P MRSI.

Radiation-induced liver damage (RILD) is a poorly understood and potentially devastating complication of hepatic radiation therapy (RT) for liver cancers. Previous work has demonstrated that hepatocyte transplantation (HT) can ameliorate RILD in rats. We hypothesized that RT inhibits generation of cellular ATP and suppresses hepatic regeneration. To study the metabolic changes that occur in RILD with and without HT, (31)P MRSI data were acquired in rats treated with partial hepatectomy (PH) alone, PH with hepatic irradiation (PHRT) or PHRT with HT (PHRT+HT). Both [γ -ATP] and ATP/Pi (31)P MRSI signal ratio initially decreased and subsequently returned to baseline levels within 2 weeks after PH, which is consistent with other published data. Persistently reduced [γ-ATP] and ATP/Pi (31)P MRSI signal ratio were observed in rats up to 20 weeks after PHRT. However, progressive increases in [γ -ATP] were observed over time in the group of rats receiving PHRT+HT. Normal [γ -ATP] was observed 20 weeks after PHRT+HT (vs. PH alone), although, ATP/Pi levels did not return to normal after PHRT +HT. Ex vivo histological studies were performed to confirm liver repopulation with transplanted hepatocytes and the amelioration of pathologic changes of RILD. These findings suggest that (31)P MRSI can be used to monitor the progress of RILD and its amelioration using transplanted hepatocytes to simultaneously restore metabolic function while replacing host hepatocytes damaged by RT.

Current status of human hepatocyte transplantation and its potential for Wilson's disease

Wilson's disease (WD) is a genetic disorder of liver copper excretion leading to its accumulation in various vital organs like the liver, brain, and kidneys. Drugs such as penicillamine, trientine, and zinc salts are the mainstay of treatment, with good outcomes; but nonresponders or a lack of compliance to the drug treatment can result in disease progression and acute liver failure (ALF). Current treatment for WD with ALF is an emergency liver transplantation and lifelong immunosuppression. Human hepatocyte transplantation (HTx) is increasingly used as treatment for liver-based metabolic defects. HTx may benefit WD patients with ALF, either as transient support until chelation treatment shows its effect or as a definitive cure through liver repopulation by healthy donor cells, as shown in animal models of WD. Although clinical trials of HTx have already proven safety and efficacy in different ALF etiologies, it remains to be demonstrated similarly in cases of WD.

Shortcut Route for Generation of Functional Hepatocyte Cells from Human Skin Allogenically for Autologous Treatment of Chronic Liver Diseases

Zhu Sa, Rezvani Mb, Harbell Jc, Mattis ANb,d,e, Wolfe ARf, Benet LZf, Willenbring Hb,c,d, Ding Sa,g. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature. 2014;508(7494):93–97.

Mouse liver repopulation with hepatocytes generated from human fibroblasts

Human induced pluripotent stem cells (iPSCs) promise to revolutionize research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modeling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1–3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. As a solution to this problem, we report generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs, but shortcut reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells (iMPC-EPCs) and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this, we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to aHeps. Unfractionated iMPC-Heps did not form tumors, most likely because they never entered a pluripotent state. To our knowledge, this is the first demonstration of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Endothelin-1 receptor A blocker darusentan decreases hepatic changes and improves liver repopulation after cell transplantation in rats

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. 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.

Preconditioning of the liver for efficient repopulation by primary hepatocyte transplants.

The therapeutic potential of liver cell transplantation has been demonstrated in multiple clinical studies to correct hereditary metabolic or chronic liver diseases. However, there are several outstanding issues, which need to be investigated: most notably donor cell engraftment and the subsequent selective expansion of transplanted cells. This protocol describes the preconditioning of the liver in a dipeptidyl peptidase-IV (DPPIV(-))-deficient rat model of efficient repopulation utilizing a selective external beam irradiation technique combined with regional transient portal ischemia (RTPI). Irradiation of the host liver impairs endogenous cell division, and the subsequent RTPI constitutes a strongly proliferative stimulus. Transplanted cells benefit from this stimulus, whereas endogenous cells have no ability to respond, due to a reduction in the mitotic capacity of the host liver. As described here, an effective preparative regime for liver repopulation is external beam liver irradiation in the form of a single dose of 25 Gy applied to the whole organ followed (4 days later) by RTPI of the right liver lobes lasting 90 min. After 1 h of reperfusion, the donor hepatocytes may be transplanted directly into the spleen as implantation site for further redistribution into the portal system and liver. This preparative regime certainly has the potential to be implemented in the clinic, since neither toxins nor highly potent carcinogens are used.

Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes

Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV(+) F344 rats were transplanted into DPPIV(-) rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. After chronic TAA administration, DPPIV(-) F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1(+), AFP(+), CD133(+), Sox-9(+), FoxJ1(+)) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet-derived growth factor receptor β, desmin, vimentin, tissue inhibitor of metalloproteinase-1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells. This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects.

Human Liver Endothelial Cells, But Not Macrovascular or Microvascular Endothelial Cells, Engraft in the Mouse Liver

Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro- and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy.

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.

Adult Human Liver Mesenchymal Stem/Progenitor Cells Participate in Mouse Liver Regeneration after Hepatectomy

The advances in stem cell science have promoted research on their use in liver regenerative medicine. Beyond the demonstration of their ability to display metabolic functions in vitro, candidate cells should demonstrate achievable in situ differentiation and ability to participate to liver repopulation. In this work, we studied the in vivo behavior of adult liver mesenchymal stem/progenitor cells (ADHLSCs) after transplantation into immunodeficient mice. The kinetics of engraftment and in situ hepatogenic differentiation were analyzed. Response of transplanted ADHLSCs to regenerative stimulus was also evaluated. Nondifferentiated ADHLSCs were intrasplenically transplanted into SCID mice. Efficiency of transplantation was evaluated at the level of engraftment and in situ differentiation using immunohistochemistry, in situ hybridization, and RT-PCR. After bromodeoxyuridine (BrdU) implantation, proliferation of transplanted ADHLSCs in response to 20% hepatectomy was assessed using immunohistochemistry. We demonstrated that ADHLSC engraftment in the SCID mouse liver was low but remained stable up to 60 days posttransplantation, when albumin (ALB) immunopositive ADHLSCs were still detected and organized as clusters. Coexpression of ornithine transcarbamylase (OTC) demonstrated ADHLSC in situ differentiation mostly near the hepatic portal vein. After 20% hepatectomy on 1 month transplanted mice, the percentage of BrdU and human ALB immunopositive ADHLSCs increased from 3 to 28 days post-BrdU implantation to reach 31.3 ± 5.4% of the total analyzed human cells. In the current study, we demonstrate that transplanted ADHLSCs are able to differentiate in the non preconditioned SCID mouse liver mainly in the periportal area. In response to partial hepatectomy, integrated ADHLSCs proliferate and participate to recipient mouse liver regeneration.

Serum Apolipoprotein E as a Marker to Monitor Graft Function After Hepatocyte Transplantation in a Clinically Relevant Mouse Model

IntroductionHepatocyte transplantation, a promising treatment for patients with acute hepatic failure or metabolic liver diseases, requires improvement in engraftment as well as long-term function of the liver cells. We established a hepatocyte transplantation model in apolipoprotein E (ApoE) knockout mice, evaluating serum ApoE and lipoprotein profiles as markers of engraftment of transplanted wild-type hepatocytes. Herein we have described a method to monitor the function of transplanted hepatocytes at low levels of engraftment, corresponding to those reported in clinical cases. We also investigated whether pretreatment with anakinra, an anti-interleukin-1 antagonist, methylprednisolone, or a combination of the two agents improved engraftment. MethodsApoE (−/−) mice were transplanted with hepatocytes isolated from wild-type C57/bl6 mice. A total of 6 × 106 hepatocytes were transplanted by 3 separate intrasplenic injections. Animals were treated before transplantation and daily thereafter for 7 days with anakinra, methylprednisolone, or a combination of both. Graft function was monitored by lipoprotein analysis and quantification of ApoE by enzyme-linked immunosorbent assay. Expression of hepatic ApoE mRNA was quantitated by reverse-transcriptase polymerase chain reaction. ResultsTreatment with anakinra with or without methylprednisolone did not significantly increase serum or hepatic mRNA ApoE expression. The low level of hepatocyte engraftment did not normalize lipoprotein profiles, but produced a significant decline in very low-density lipoprotein and total cholesterol. Repeated transplantations significantly enhanced liver repopulation; serum ApoE levels increased with each infusion, correlating well with hepatic mRNA expression. ConclusionsThe model of serum ApoE, a sensitive marker of engraftment and transplanted hepatocyte function, allowed us to study hepatocyte transplantation in a clinically relevant manner, that is, without pretreatments such as retrorsine or carbon tetrachloride.

470. Human Parthenogenetic Stem Cell-Derived Hepatocytes for Liver Repopulation: Cell Therapy Demonstrated in a Pilot Study with Gunn Rats

470. Human Parthenogenetic Stem Cell-Derived Hepatocytes for Liver Repopulation: Cell Therapy Demonstrated in a Pilot Study with Gunn Rats