Liver Cell Types Research Articles

Benign focal liver lesions: The role of magnetic resonance imaging.

Liver lesions are common findings in radiologists’ daily routine. They are a complex category of pathology that range from solitary benign lesions to primary liver cancer and liver metastases. Benign focal liver lesions can arise from different liver cell types: Epithelial (hepatocytes and biliary cells) and nonepithelial (mesenchymal cells). Liver magnetic resonance imaging (MRI) is a fundamental radiological method in these patients as it allows with its multiparametric approach optimal non-invasive tissue characterization. Furthermore, advanced liver MRI techniques such as diffusion-weighted imaging and hepatobiliary contrast agents have improved the detection of focal liver lesions and can be highly effective in differentiating pseudotumor from tumors, as well as benign from malignant lesions, and can also be used for differential diagnosis. Although histological examination can be useful in making a definitive diagnosis, MRI is an important modality in the diagnosis of liver lesions with a significant impact on patient care. This aim of this review is to provide a comprehensive overview of benign liver lesions on MRI.

Age-Related Changes in the Expression of Tenascin-C in the Liver of Albino Rat

Introduction and aim: Aging is a gradual deterioration in the function of tissues and organ systems and loss of the ability to maintain homeostasis, due to structural alteration or dysfunction. Aging impact directly on all the different types of liver cells: hepatocytes, liver sinusoidal endothelial cells (LSECs), Küpffer cells (KCs) and hepatic stellate cells (HSCs). Activation of HSCs leads to deposition of collagen in the space of Disse, decrease of fenestrations of LSECs and formation of basement membrane. These changes called pseudocapillarization. Tenascin-C is highly expressed during embryonic development, wound healing, chronic inflammation and cancer. We aimed to investigate the contribution of tenascin C (TnC) to liver pseudocapillarization by comparing expression of tenascin c and levels of fibrosis deposition in space of Disse in different age groups
 Methodology: Different ages (6, 12, 18, 24, 30 and 36 months) were chosen and sacrificed. Morphometric and serological parameters were assessed and the liver samples were processed for histological and immunohistochemical staining.
 Results: Immunohistochemical staining revealed a gradual increase in TnC deposition from first to forth group then gradual decrease occurred. Masson’s stain revealed gradual increase of fibrosis in space of Disse deposition from 2nd to 6th group.
 Conclusion: TnC appear and increase before appearance of fibrosis but when fibrosis is formed TnC is down regulated again.

GATA6 is predicted to regulate DNA methylation in an in vitro model of human hepatocyte differentiation

Hepatocytes are the dominant cell type in the human liver, with functions in metabolism, detoxification, and producing secreted proteins. Although gene regulation and master transcription factors involved in the hepatocyte differentiation have been extensively investigated, little is known about how the epigenome is regulated, particularly the dynamics of DNA methylation and the critical upstream factors. Here, by examining changes in the transcriptome and the methylome using an in vitro hepatocyte differentiation model, we show putative DNA methylation-regulating transcription factors, which are likely involved in DNA demethylation and maintenance of hypo-methylation in a differentiation stage-specific manner. Of these factors, we further reveal that GATA6 induces DNA demethylation together with chromatin activation in a binding-site-specific manner during endoderm differentiation. These results provide an insight into the spatiotemporal regulatory mechanisms exerted on the DNA methylation landscape by transcription factors and uncover an epigenetic role for transcription factors in early liver development.

Interplay Between GH-regulated, Sex-biased Liver Transcriptome and Hepatic Zonation Revealed by Single-Nucleus RNA Sequencing.

The zonation of liver metabolic processes is well-characterized; however, little is known about the cell type-specificity and zonation of sexually dimorphic gene expression or its growth hormone (GH)-dependent transcriptional regulators. We address these issues using single-nucleus RNA-sequencing of 32 000 nuclei representing 9 major liver cell types. Nuclei were extracted from livers from adult male and female mice; from males infused with GH continuously, mimicking the female plasma GH pattern; and from mice exposed to TCPOBOP, a xenobiotic agonist ligand of the nuclear receptor CAR that perturbs sex-biased gene expression. Analysis of these rich transcriptomic datasets revealed the following: 1) expression of sex-biased genes and their GH-dependent transcriptional regulators is primarily restricted to hepatocytes and is not a feature of liver nonparenchymal cells; 2) many sex-biased transcripts show sex-dependent zonation within the liver lobule; 3) gene expression is substantially feminized both in periportal and pericentral hepatocytes when male mice are infused with GH continuously; 4) sequencing nuclei increases the sensitivity for detecting thousands of nuclear-enriched long-noncoding RNAs (lncRNAs) and enables determination of their liver cell type-specificity, sex-bias and hepatocyte zonation profiles; 5) the periportal to pericentral hepatocyte cell ratio is significantly higher in male than female liver; and 6) TCPOBOP exposure disrupts both sex-specific gene expression and hepatocyte zonation within the liver lobule. These findings highlight the complex interconnections between hepatic sexual dimorphism and zonation at the single-cell level and reveal how endogenous hormones and foreign chemical exposure can alter these interactions across the liver lobule with large effects both on protein-coding genes and lncRNAs.

Generative Deep Learning of the Single Cell Dose Response of 2,3,7,8 Tetrachlorodibenzo‐p‐dioxin in Mouse Liver

Single cell RNA‐seq provides a new opportunity to study the heterogeneity of chemical perturbation within tissues. However, exploring the space of all cell type – chemical combinations is experimentally and financially unfeasible. This space is significantly expanded by the dose axis of chemical perturbation. Computational tools are needed to predict responses not only across tissues, but also across doses while capturing the nuances of cell type specific gene expression. Generative deep learning architectures such as autoencoders simplify the single cell expression space allowing cross‐tissue, ‐organ, ‐organism and ‐dose predictions using simple vector arithmetic. However, differing sensitivities and non‐linearities make cell type specific gene expression predictions following treatment at higher doses challenging. Here, improvements to vector arithmetic in the latent space achieve high dose cell type specific predictions better than other state of the art algorithms. We specifically look at a well‐studied toxicant, 2,3,7,8 Tetrachlorodibenzo‐p‐dioxin (TCDD), in the mouse liver as a case study. TCDD binds to and activates the aryl hydrocarbon receptor (AhR) to regulate gene expression associated with several physiological processes including xenobiotic metabolism, development, and circadian rhythm. Prediction of the effects of TCDD across liver cell types is non‐trivial as AhR is not only differentially expressed across cell types but is also differentially expressed within certain cell types along the porto‐central axis of the liver lobule. Our model predicts dose‐dependent gene expression across liver zones (i.e., portal to central hepatocytes), and across liver cell types (e.g., endothelial to stellate cells) using basal gene expression data. The model is also able to predict low dose TCDD gene expression using high dose data. Finally, using outputs from the model, an algorithm was developed to order cells based on their sensitivities to a particular toxicant by assigning a “pseudo‐dose” value to each cell. Given the model’s high performance on these tasks, the tool developed here will aid the exploration of the gene expression space of chemical perturbation.

Dynamic human liver proteome atlas reveals functional insights into disease pathways

Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence‐unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time course of primary cell cultures, and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serve as spectral library to characterize a human cohort of non‐alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of hepatic stellate cell activation resembling liver cirrhosis and providing functional insights. We built a web‐based dashboard application for the interactive exploration of our resource (www.liverproteome.org).

IN SITU HYBRIDIZATION AND VIRUS CHARACTERIZATION OF SKUNK ADENOVIRUS IN NORTH AMERICAN WILDLIFE REVEALS MULTISYSTEMIC INFECTIONS IN A BROAD RANGE OF HOSTS.

Skunk adenovirus-1 (SkAdV-1) has been reported infecting several North American wildlife species; however, lesions associated with disease have not yet been completely characterized, particularly in porcupines. We describe and characterize the tissue distribution and lesions associated with SkAdV-1 infection in 24 wildlife diagnostic cases submitted between 2015 and 2020, including 16 North American porcupines (Erethizon dorsatum), three striped skunks (Mephitis mephitis), and five raccoons (Procyon lotor), which constitute a new host species. The most common lesion in all species was severe necrotizing bronchopneumonia with (n=12) or without (n=10) interstitial involvement. Intranuclear inclusion bodies were common in respiratory epithelium (n=21) and less often in renal tubular (n=6) and biliary epithelium (n=1). Several cases (n=4) had secondary bacterial infections, including Bordetella bronchiseptica, Pasteurella multocida, and Streptococcus zooepidemicus. In situ hybridization in porcupine (n=6), raccoon (n=1), and skunk (n=1) revealed SkAdV-1 DNA in multiple tissue types, including lung, trachea, turbinates, liver, kidney, lymph node, and brain, and multiple cell types including epithelial, endothelial, and mesothelial cells. These findings were consistent across species. Comparison of viral genomes from a porcupine and a raccoon with that originally isolated from a skunk demonstrated DNA point mutations affecting several viral genes, including the fiber protein gene. Our findings show the spectrum of disease associated with SkAdV-1 infection in a broad host range of wildlife species.

Extracellular microparticles derived from hepatic progenitor cells deliver a death signal to hepatoma-initiating cells

The malignant transformation of normal resident hepatic stem/progenitor cells has a critical role in hepatocarcinogenesis and the recurrence of hepatocellular carcinoma (HCC). We defined such hepatic progenitor cells as hepatoma-initiating cells. An efficient strategy is required to target and kill the hepatoma-initiating cells. We isolated extracellular microparticles (MPs) derived from apoptotic hepatic progenitor cells (HPCs) and tested their ability to inhibit hepatocarcinogenesis. Extracellular MPs were isolated from HPCs, hepatocytes and liver tumor cells. Their effects on tumor growth were investigated in rat primary HCC models, in which hepatocarcinogenesis is induced by diethylnitrosamine (DEN). The extracellular MPs derived from apoptotic HPCs, apoptotic hepatocytes and apoptotic liver tumor cells were similar in morphology, diameter and zeta potential. However, they had different antitumor effects. In DEN-exposed rats, only the MPs derived from apoptotic HPCs effectively inhibit hepatocarcinogenesis. In vitro and in vivo analyses confirmed that HPCs preferentially take up MPs derived from apoptotic HPCs compared to MPs from other liver cell types. Proteomic analysis of MPs from apoptotic HPCs showed enrichment of proteins involved in cell death pathways. Thus, HPC-derived MPs contain a death signal to induce the killing of hepatoma-initiating cells. Our findings provide evidence that a death signal encapsulated in HPC-derived extracellular microparticles can efficiently clear hepatoma-initiating cells and prevent hepatocarcinogenesis.Graphical

Understanding Nanomaterial-Liver Interactions to Facilitate the Development of Safer Nanoapplications.

Nanomaterials (NMs) are widely used in commercial and medical products, such as cosmetics, vaccines, and drug carriers. Exposure to NMs via various routes such as dermal, inhalation, and ingestion has been shown to gain access to the systemic circulation, resulting in the accumulation of NMs in the liver. The unique organ structures and blood flow features facilitate the liver sequestration of NMs, which may cause adverse effects in the liver. Currently, most in vivo studies are focused on NMs accumulation at the organ level and evaluation of the gross changes in liver structure and functions, however, cell-type-specific uptake and responses, as well as the molecular mechanisms at cellular levels leading to effects at organ levels are lagging. Herein, the authors systematically review diverse interactions of NMs with the liver, specifically on major liver cell types including Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and hepatocytes as well as the detailed molecular mechanisms involved. In addition, the knowledge gained on nano-liver interactions that can facilitate the development of safer nanoproducts and nanomedicine is also reviewed.

Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells

ObjectiveMucosal-associated invariant T (MAIT) cells are the most abundant T cells in human liver. They respond to bacterial metabolites presented by major histocompatibility complex-like molecule MR1. MAIT cells exert regulatory...

Utilizing Shared Big Data to Identify Liver Cancer Dedifferentiation Markers.

Big data reanalysis has the potential to generate novel comparative analyses which aim to generate novel hypotheses and knowledge. However, this approach is underutilized in the realm of cancer research, particularly for cancer stem cells (CSCs). CSCs are a rare subset of tumor cells, which dedifferentiate from healthy adult cells, and have the potential for self-renewal and treatment resistance. This analysis utilizes two publically available single-cell RNA-seq datasets of liver cancer and adult liver cell types to demonstrate how reanalysis of big data can lead to valuable new discoveries. We identify 519 differentially expressed genes between liver CSCs and healthy liver cell types. Here we report the potential novel liver CSC dedifferentiation factor, Msh Homeobox 2, which was significantly upregulated in liver CSCs by 1.36 fold (p-value < 1E-10). These findings have the potential to further advance our knowledge of genes governing the formation of CSCs.

The Potential Role of Cellular Senescence in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) represents an increasing global health burden. Cellular senescence develops in response to cellular injury, leading not only to cell cycle arrest but also to alterations of the cellular phenotype and metabolic functions. In this review, we critically discuss the currently existing evidence for the involvement of cellular senescence in NAFLD in order to identify areas requiring further exploration. Hepatocyte senescence can be a central pathomechanism as it may foster intracellular fat accumulation, fibrosis and inflammation, also due to secretion of senescence-associated inflammatory mediators. However, in some non-parenchymal liver cell types, such as hepatic stellate cells, senescence may be beneficial by reducing the extracellular matrix deposition and thereby reducing fibrosis. Deciphering the detailed interaction between NAFLD and cellular senescence will be essential to discover novel therapeutic targets halting disease progression.

Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz.

Drug-induced liver injury (DILI) constitutes a clinical challenge due to the incomplete characterization of the mechanisms involved and potential risk factors. Efavirenz, an anti-HIV drug, induces deleterious actions in hepatocytes that could underlie induction of the NLRP3 inflammasome, an important regulator of inflammatory responses during liver injury. We assessed the potential of efavirenz to modulate the inflammatory and fibrogenic responses of major liver cell types involved in DILI. The effects of efavirenz were evaluated both in vitro and in vivo. Efavirenz triggered inflammation in hepatocytes, in a process that involved NF-κB and the NLRP3 inflammasome, and activated hepatic stellate cells (HSCs), thereby enhancing expression of inflammatory and fibrogenic markers. The NLRP3 inflammasome was not altered in efavirenz-treated macrophages, but these cells polarized towards the anti-inflammatory M2 phenotype and displayed upregulated anti-inflammatory mediators. Conversely, no evidence of damage was observed in efavirenz-treated animals, except when macrophages were depleted, which resulted in the in vivo manifestation of the deleterious effects detected in hepatocytes and HSCs. Efavirenz elicits a cell-specific activation of the NLRP3 inflammasome in hepatocytes and HSCs, but macrophages appear to counteract efavirenz-induced liver injury. Our results highlight the dynamic nature of the interaction among liver cell populations and emphasize the potential of targeting macrophage polarization as a strategy to treat NLRP3 inflammasome-induced liver injury.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endothelial Cells (LSECs) in Analyses of Hepatic Drug Distribution.

Development of the new generation of drugs (e.g., oligo- and polynucleotides administered intravascularly either as free compounds or as nano-formulations) frequently encounters major challenges such as lack of control of targeting and/or delivery. Uncontrolled or unwanted clearance by the liver is a well-known and particularly important hurdle in this respect. Hence, reliable techniques are needed to identify the type(s) of liver cells, receptors, and metabolic mechanisms that are responsible for unwanted clearance of these compounds.We describe here a method for the isolation and culture of the major cell types from mouse liver: hepatocytes (HCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs). The presently described protocol employs perfusion of the liver with a collagenase-based enzyme preparation to effectively transform the intact liver to a single cell suspension. From this initial cell suspension HCs are isolated by specified centrifugation schemes, yielding highly pure HC preparations, and KCs and LSECs are isolated by employing magnetic-activated cell sorting (MACS). The MACS protocol makes use of magnetic microbeads conjugated with specific antibodies that bind unique surface antigens on either KCs or LSECs. In this way the two cell types are specifically and separately pulled out of the initial liver cell suspension by applying a magnetic field, resulting in high purity, yield, and viability of the two cell types, allowing functional studies of the cells.If the drug compound in question is to be studied with respect to liver cell distribution of intravascularly administered drug compounds the isolated cells can be analyzed directly after isolation. Detailed studies of receptor-ligand interactions and/or dynamics of intracellular metabolism of the compound can be conducted in primary surface cultures of HCs, LSECs, and KCs established by seeding the isolated cells on specified growth substrates.

Multi-omics analyses of early liver injury reveals cell-type-specific transcriptional and epigenomic shift

BackgroundLiver fibrosis is a wound-healing response to tissue injury and inflammation hallmarked by the extracellular matrix (ECM) protein deposition in the liver parenchyma and tissue remodelling. Different cell types of the liver are known to play distinct roles in liver injury response. Hepatocytes and liver endothelial cells receive molecular signals indicating tissue injury and activate hepatic stellate cells which produce ECM proteins upon their activation. Despite the growing knowledge on the molecular mechanism underlying hepatic fibrosis in general, the cell-type-specific gene regulatory network associated with the initial response to hepatotoxic injury is still poorly characterized.ResultsIn this study, we used thioacetamide (TAA) to induce hepatic injury in adult zebrafish. We isolated three major liver cell types - hepatocytes, endothelial cells and hepatic stellate cells - and identified cell-type-specific chromatin accessibility and transcriptional changes in an early stage of liver injury. We found that TAA induced transcriptional shifts in all three cell types hallmarked by significant alterations in the expression of genes related to fatty acid and carbohydrate metabolism, as well as immune response-associated and vascular-specific genes. Interestingly, liver endothelial cells exhibit the most pronounced response to liver injury at the transcriptome and chromatin level, hallmarked by the loss of their angiogenic phenotype.ConclusionOur results uncovered cell-type-specific transcriptome and epigenome responses to early stage liver injury, which provide valuable insights into understanding the molecular mechanism implicated in the early response of the liver to pro-fibrotic signals.

Intracellular Exposure Dose-Associated Susceptibility of Steatotic Hepatocytes to Metallic Nanoparticles.

Non-alcoholic fatty liver disease (NAFLD), mainly characterized by the accumulation of excess fat in hepatocytes, is the most prevalent liver disorder afflicting ~25% of adults worldwide. In vivo studies have shown that adult rodents with NAFLD were more sensitive to metallic nanoparticles (MNPs) than healthy MNPs. However, due to the complex interactions between various cell types in a fatty liver, it has become a major challenge to reveal the toxic effects of MNPs to specific types of liver cells such as steatotic hepatocytes. In this study, we reported the susceptibility of steatotic hepatocytes in cytotoxicity and the induction of oxidative stress to direct exposures to MNPs with different components (silver, ZrO2, and TiO2 NPs) and sizes (20–30 nm and 125 nm) in an oleic acid (OA) -induced steatotic HepG2 (sHepG2) cell model. Furthermore, the inhibitory potential of MNPs against the process of fatty acid oxidation (FAO) were obvious in sHepG2 cells, even at extremely low doses of 2 or 4 μg/mL, which was not observed in non-steatotic HepG2 (nHepG2) cells. Further experiments on the differential cell uptake of MNPs in nHepG2 and sHepG2 cells demonstrated that the susceptibility of steatotic hepatocytes to MNP exposures was in association with the higher cellular accumulation of MNPs. Overall, our study demonstrated that it is necessary and urgent to take the intracellular exposure dose into consideration when assessing the potential toxicity of environmentally exposed MNPs.

Regulatory effects of microRNAs on hepatic stellate cell activation and liver fibrogenesis

Hepatic fibrogenesis (HF) is the common consequence of various chronic liver diseases (CLD) induced by a variety of pathogenic factors. The mechanism of HF involves the interactions within different types of liver cells, cytokines, chemokines, cell mediators and multiple signaling pathways in a way of networks. As a result, excessive production and deposition of extracellular matrix (ECM) mainly composed of type I and type III fibril forming collagen destroys the original morphology, structure and function of the liver. The activation of hepatic stellate cells (HSCs), the major scar forming cells in liver, plays a crucial role in hepatic fibrogenesis. MicroRNAs are a group of short, single stranded, non-coding RNAs that can inhibit mRNA expression at the transcriptional and post transcriptional levels. They can be loaded and transferred as cargos by exosomes, to regulate the function of nearby and distant receptive cells. The expressions of many microRNAs such as miR-21, miR-29, miR-708, miR-101, miR-455, miR-146, miR-193 change significantly in activated HSCs, which regulate the activation, fibrogenic function, proliferation, apoptosis and autophagy of HSCs via affecting target genes expression and signaling pathway molecules. They are important substances and regulatory mechanism that mediate the initiation and progression of HF.

Extracellular vesicle release and uptake by the liver under normo- and hyperlipidemia

Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20–30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with 99mTc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with 99mTc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using 99mTc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution.

Wnt ligands 3a and 5a regulate proliferation and migration in human fetal liver progenitor cells.

Since human fetal liver progenitor cells (hFLPC) can differentiate into multiple liver cell types in vitro and in vivo, hFLPC may be a suitable source for cell therapy and regeneration strategies. Imperative for effective clinical applications of hFLPC is the enhanced knowledge of growth factors that mediate and improve migration and proliferation. The canonical wingless/int-1 (Wnt) signal transduction pathway is known to play a key role in proliferation and migration of stem cells. So, we investigated a role for Wnt3a and Wnt5a ligands in regulating the proliferation and migration of hFLPC. We used alamarBlue assay and transwell migration assay and examined proliferation and migration of hFLPC to Wnt3a and Wnt5a. In addition, the target genes of Wnt signal transduction pathway was identified using microarray analysis and validated by quantitative real-time polymerase chain reaction (qPCR). We found that Wnt3a or Wnt5a independently significantly increased migration and proliferation in a dose-dependent manner which was significantly inhibited by Wnt inhibitors Wnt-C59 or KN-62. Addition of Wnt3a to hFLPC resulted in increased mRNA expression of the known Wnt target genes Axin-2, DKK2, while Wnt5a increased CXCR7, all of which are closely associated with an enhanced proliferation capacity of stem cells. Thus, we report that Wnt3a and Wnt5a may play an important role in the proliferation and migration of hFLPC by possibly regulating key target genes-involved in these processes. Incorporating recombinant human Wnt3a and Wnt5a in regenerative strategies using liver stem/progenitor cells might improve the process of liver regeneration.