A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR+ cells during antibody-mediated immune responses.

Human immune system (HIS) mice provide a model to study human immune responses in vivo. Currently available HIS mouse models may harbor mouse Fc Receptor (FcR)-expressing cells that exert potent effector functions following administration of human Ig. Previous studies showed that the ablation of the murine FcR gamma chain (FcR-γ) results in loss of antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vivo. We created a new FcR-γ-deficient HIS mouse model to compare host (mouse) versus graft (human) effects underlying antibody-mediated immune responses in vivo. FcR-γ-deficient HIS recipients lack expression and function of mouse activating FcRs and can be stably and robustly reconstituted with human immune cells. By screening blood B-cell depletion by rituximab Ig variants, we found that human FcγRs-mediated IgG1 effects, whereas mouse activating FcγRs were dominant in IgG4 effects. Complement played a role as an IgG1 variant (IgG1 K322A) lacking complement binding activity was largely ineffective. Finally, we provide evidence that FcγRIIIA on human NK cells could mediate complement-independent B-cell depletion by IgG1 K322A. We anticipate that our FcR-γ-deficient HIS model will help clarify mechanisms of action of exogenous administered human antibodies in vivo.

A live measles-vectored COVID-19 vaccine induces strong immunity and protection from SARS-CoV-2 challenge in mice and hamsters.

Several COVID-19 vaccines have now been deployed to tackle the SARS-CoV-2 pandemic, most of them based on messenger RNA or adenovirus vectors.The duration of protection afforded by these vaccines is unknown, as well as their capacity to protect from emerging new variants. To provide sufficient coverage for the world population, additional strategies need to be tested. The live pediatric measles vaccine (MV) is an attractive approach, given its extensive safety and efficacy history, along with its established large-scale manufacturing capacity. We develop an MV-based SARS-CoV-2 vaccine expressing the prefusion-stabilized, membrane-anchored full-length S antigen, which proves to be efficient at eliciting strong Th1-dominant T-cell responses and high neutralizing antibody titers. In both mouse and golden Syrian hamster models, these responses protect the animals from intranasal infectious challenge. Additionally, the elicited antibodies efficiently neutralize in vitro the three currently circulating variants of SARS-CoV-2.

In-vitro and in-vivo models for hepatitis B cure research.

PURPOSE OF REVIEW: Antiviral therapy for chronic hepatitis B infection is rarely curative, thus research in HBV cure strategies is a priority. Drug development and testing has been hampered by the lack of robust cell culture systems and small animal models. This review summarizes existing models for HBV cure research and focuses on recent developments since 2017 until today. RECENT FINDINGS: The field has progressed in the development of cell culture and animal models to study HBV. Although early cell culture systems relied on transfection of HBV genomes in hepatoma cell lines, novel models expressing the entry receptor for HBV are susceptible to infection. Improved culture conditions for primary human hepatocytes, the primary target of HBV, have enabled the screening and validation of novel antivirals. Mouse models grafted with partially humanized livers are suitable for testing viral entry inhibitors or direct acting antivirals, and can be reconstituted with human immune cells to analyze immunotherapies. Other immunocompetent models include mice transduced with HBV genomes or woodchucks infected with their native hepatitis virus. SUMMARY: Model systems for HBV research have helped lay the groundwork for the development and optimization of antiviral and immune-based therapeutic approaches that are now moving to clinical trials.

Regulatory T cells control toxicity in a humanized model of IL-2 therapy.

While patient selection and clinical management have reduced high-dose IL-2 (HDIL2) immunotherapy toxicities, the immune mechanisms that underlie HDIL2-induced morbidity remain unclear. Here we show that dose-dependent morbidity and mortality of IL-2 immunotherapy can be modeled in human immune system (HIS) mice. Depletion of human T cell subsets during the HDIL2 treatment reduces toxicity, pointing to the central function of T cells. Preferential expansion of effector T cells secondary to defective suppressive capacity of regulatory T (Treg) cells after HDIL2 therapy further underscores the importance of Treg in the maintenance of immune tolerance. IL-2 toxicity is induced by selective depletion or inhibition of Treg after LDIL2 therapy, and is ameliorated in HDIL2-treated HIS mice receiving the PIM-1 kinase inhibitor, Kaempferol. Modeling IL-2 pathophysiology in HIS mice offers a means to understand the functions of effector and regulatory T cells in immune-mediated toxicities associated with cancer immunotherapy.

Modeling hepatitis virus infections and treatment strategies in humanized mice.

Hepatitis viruses cause chronic liver diseases such as fibrosis, cirrhosis and hepatocellular carcinomas that are difficult to treat and constitute a global health problem. Species-specific viral tropism has limited the usefulness of small animal models to study the impact of viral hepatitis. Immunodeficient mice grafted with human hepatocytes are susceptible to hepatitis viruses B, C, D and E (HBV, HCV, HDV and HEV), developing full viral life cycles, and delivering a means to investigate virus-host interactions and antiviral treatments. These chimeric humanized mouse models have been further grafted with humanized immune systems to decipher immune responses following hepatotropic viral infections, the ensuing pathophysiology, and to test novel therapeutic strategies.

Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear. METHODS: We performed studies with BALB/c Rag2-/-Il2rg-/-SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells. RESULTS: Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. CONCLUSION: In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

A functional DC cross talk promotes human ILC homeostasis in humanized mice.

Humanized mice harboring human hematopoietic systems offer a valuable small-animal model to assess human immune responses to infection, inflammation, and cancer. Human immune system (HIS) mice develop a broad repertoire of antigen receptor bearing B and T cells that can participate in adaptive immune responses after immunization. In contrast, analysis of innate immune components, including innate lymphoid cells (ILCs) and natural killer (NK) cells, is limited in current HIS mouse models, partly because of the poor development of these rare lymphoid subsets. Here we show that novel dendritic cell (DC)-boosted BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (BRGSF) HIS mice harbor abundant NK cells and tissue-resident ILC subsets in lymphoid and nonlymphoid mucosal sites. We find that human NK cells and ILCs are phenotypically and functionally mature and provide evidence that human DC activation in BRGSF-based HIS mice can "cross talk" to human NK cells and ILCs. This novel HIS mouse model should provide the opportunity to study the immunobiology of human NK cell and ILC subsets in vivo in response to various environmental challenges.

A Transcriptomic Signature of Mouse Liver Progenitor Cells.

Liver progenitor cells (LPCs) can proliferate extensively, are able to differentiate into hepatocytes and cholangiocytes, and contribute to liver regeneration. The presence of LPCs, however, often accompanies liver disease and hepatocellular carcinoma (HCC), indicating that they may be a cancer stem cell. Understanding LPC biology and establishing a sensitive, rapid, and reliable method to detect their presence in the liver will assist diagnosis and facilitate monitoring of treatment outcomes in patients with liver pathologies. A transcriptomic meta-analysis of over 400 microarrays was undertaken to compare LPC lines against datasets of muscle and embryonic stem cell lines, embryonic and developed liver (DL), and HCC. Three gene clusters distinguishing LPCs from other liver cell types were identified. Pathways overrepresented in these clusters denote the proliferative nature of LPCs and their association with HCC. Our analysis also revealed 26 novel markers, LPC markers, including Mcm2 and Ltbp3, and eight known LPC markers, including M2pk and Ncam. These markers specified the presence of LPCs in pathological liver tissue by qPCR and correlated with LPC abundance determined using immunohistochemistry. These results showcase the value of global transcript profiling to identify pathways and markers that may be used to detect LPCs in injured or diseased liver.

Towards an HBV cure: state-of-the-art and unresolved questions--report of the ANRS workshop on HBV cure.

HBV infection is a major cause of liver cirrhosis and hepatocellular carcinoma. Although HBV infection can be efficiently prevented by vaccination, and treatments are available, to date there is no reliable cure for the >240 million individuals that are chronically infected worldwide. Current treatments can only achieve viral suppression, and lifelong therapy is needed in the majority of infected persons. In the framework of the French National Agency for Research on AIDS and Viral Hepatitis 'HBV Cure' programme, a scientific workshop was held in Paris in June 2014 to define the state-of-the-art and unanswered questions regarding HBV pathobiology, and to develop a concerted strategy towards an HBV cure. This review summarises our current understanding of HBV host-interactions leading to viral persistence, as well as the roadblocks to be overcome to ultimately address unmet medical needs in the treatment of chronic HBV infection.

Animal models to test hiPS-derived hepatocytes in the context of inherited metabolic liver diseases.

Human induced pluripotent stem (hiPS) cells are established following reprogramming of somatic cells from a wide variety of tissues. Given the scarcity of adult human hepatocytes, hiPS-derived hepatocytes would be a valuable source of cells to study differentiation programs, model patient-specific diseases, test drug toxicities, and cell transplantation therapies. Although hiPS-derived hepatocytes are extensively characterized in cell culture assays, testing these cells in animal models is necessary to fully evaluate their differentiation profile and their lack of tumorigenicity. Immunodeficient mouse models harboring liver damage are effective hosts in which xenogeneic hepatocytes can engraft, proliferate, and participate in liver regeneration, thus constituting a stringent test of hepatocyte functionality. The in vivo evaluation of disease-specific hiPS-derived hepatocytes should broaden our understanding of the cellular and molecular processes involved in inherited metabolic liver disease phenotypes. Herein, we detail our methods to test the functions of hiPS-derived hepatocytes in the context of the immunodeficient Rag2(-/-)IL2Rγc(-/-)Alb-uPAtg mouse model.

Ectopic expression of murine CD47 minimizes macrophage rejection of human hepatocyte xenografts in immunodeficient mice.

UNLABELLED: Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA (urokinase-type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. CONCLUSION: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Autonomous and extrinsic regulation of thymopoiesis in human immune system (HIS) mice.

Human Immune System (HIS) mice represent a novel biotechnology platform to dissect human haematopoiesis and immune responses. However, the limited human T-cell development that is observed in HIS mice restricts its utility for these applications. Here, we address whether reduced thymopoiesis in HIS mice reflects an autonomous defect in T-cell precursors and/or a defect in the murine thymic niche. Human thymocyte precursors seed the mouse thymus and their reciprocal interactions with murine thymic epithelial cells (TECs) led to both T-cell and TEC maturation. The human thymocyte subsets observed in HIS mice demonstrated survival, proliferative and phenotypic characteristics of their normal human counterparts, suggesting that the intrinsic developmental program of human thymocytes unfolds normally in this xenograft setting. We observed that exogenous administration of human IL-15/IL-15Rα agonistic complexes induced the survival, proliferation and absolute numbers of immature human thymocyte subsets, without any obvious effect on cell-surface phenotype or TCR Vß usage amongst the newly selected mature single-positive (SP) thymocytes. Finally, when IL-15 was administered early after stem cell transplantation, we noted accelerated thymopoiesis resulting in the more rapid appearance of peripheral naïve T cells. Our results highlight the functional capacity of murine thymic stroma cells in promoting human thymopoiesis in HIS mice but suggest that the "cross-talk" between murine thymic stroma and human haematopoietic precursors may be suboptimal. As IL-15 immunotherapy promotes early thymopoiesis, this novel approach could be used to reduce the period of T-cell immunodeficiency in the post-transplant clinical setting.

Functional CD47/signal regulatory protein alpha (SIRP(alpha)) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo.

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2(-/-)IL-2Rγc(-/-) mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4(+) or CD8(+) single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo.

IL-15 transpresentation promotes both human T-cell reconstitution and T-cell-dependent antibody responses in vivo.

Cytokine immunotherapies targeting T lymphocytes are attractive clinical interventions against viruses and tumors. In the mouse, the homeostasis of memory α/ß CD8(+) T cells and natural killer (NK) cells is significantly improved with increased IL-15 bioavailability. In contrast, the role of "transpresented" IL-15 on human T-cell development and homeostasis in vivo is unknown. We found that both CD8 and CD4 T cells in human immune system (HIS) mice are highly sensitive to transpresented IL-15 in vivo, with both naïve (CD62L(+)CD45RA(+)) and memory phenotype (CD62L(-)CD45RO(+)) subsets being significantly increased following IL-15 "boosting." The unexpected global improvement in human T-cell homeostasis involved enhanced proliferation and survival of both naïve and memory phenotype peripheral T cells, which potentiated B-cell responses by increasing the frequency of antigen-specific responses following immunization. Transpresented IL-15 did not modify T-cell activation patterns or alter the global T-cell receptor (TCR) repertoire diversity. Our results indicate an unexpected effect of IL-15 on human T cells in vivo, in particular on CD4(+) T cells. As IL-15 promotes human peripheral T-cell homeostasis and increases the frequency of neutralizing antibody responses in HIS mice, IL-15 immunotherapy could be envisaged as a unique approach to improve vaccine responses in the clinical setting.

Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development.

UNLABELLED: Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hESCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3-kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, alpha1-antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4alpha and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low-density lipoprotein uptake. After transduction with a green fluorescent protein-expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and alpha1-antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human-induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of diverse origins. CONCLUSION: We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery.

Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer.

Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.

Morphogenetic competence of HNF4 alpha-deficient mouse hepatic cells.

BACKGROUND/AIMS: To specify roles of HNF 4 alpha in mouse liver development, we have analyzed the ex vivo morphogenetic potential of HNF4 alpha-null embryonic hepatic cells. METHODS: Using mice with floxed or deficiency alleles of HNF4 alpha, hepatic cells lacking this transcription factor were explanted into primary culture and derived into cell lines. RESULTS: Contrary to behavior in vivo where HNF4 alpha-null liver cells fail to show normal polarity and epithelialization, e18.5 hepatic cells in primary culture from mutant embryos show restoration of apical expression of tight junction protein-1 and of transcripts for E-cadherin. Clones of control and HNF4 alpha-null cell lines were indistinguishable, even when differentiation of bile canalicular formation was induced. HNF4 alpha-null and control cell lines showed similar potential to colonize livers of the murine ALB-uPA/SCID model of liver regeneration, but null cells formed only bile ducts and not clusters of hepatocytes. Finally, analysis of mutant embryonic livers revealed a transcriptional signature consistent with a stress response, which could underlie the morphogenetic defects observed in vivo. CONCLUSIONS: We conclude that the lack of epithelialization characteristic of the HNF4 alpha-null embryonic liver is due, at least in part, to non-cell autonomous defects, and that null cells do not suffer intrinsic defects in polarization.

Transcriptional profiling of bipotential embryonic liver cells to identify liver progenitor cell surface markers.

The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application. As a step in this direction, we have used a bipotential liver cell line from 14 days postcoitum mouse embryonic liver to compile a list of cell surface markers expressed specifically by liver progenitor cells. These cells, known as bipotential mouse embryonic liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte-like and cholangiocyte-like cells in vitro. Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo. Microarray and Gene Ontology (GO) analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state. This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation, whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes. In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state. Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated. One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate-treated animals. We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells. Disclosure of potential conflicts of interest is found at the end of this article.