Liver Cell Types Research Articles

Morphological features of non-parenchymal liver cells of full-term stillbirths from HIV-mono-infected mothers.

Fifty-one liver tissue biopsies offull-term stillbirths from HIV-mono-infected mothers were investigated. All morphometric indices ofnon-parenchymal liver cells were calculated using theAvtandilov microscopic morphometric grid, which consisted of100 equidistant points. It was inserted into themicroscope ocular tube with atotal × 200 microscope magnification. Thenumber ofpoints that were found in thecorresponding types ofnon-parenchymal liver cells determined by immunohistochemistry was calculated. In each case, 10 random microscopic areas were selected and then all data were obtained, calculated and presented as percentages. Morphometric indices ofnon-parenchymal liver cells: Kupffer cells - 28.3±2.9% [control - 21.2 ±2.5], hepatic stellate cells - 14.1 ±1.4% [control - 9.8 ±1.2], LAL (liver-associated lymphocytes): CD4 - 29.2 ±2.2% [control - 43.3 ±4.9], CD8 - 25.3 ±1.9% [control - 23.1 ±1.7], dendritic cells - 3.9 ±0.3% [control - 2.6 ±0.3]. It was established that at thetime ofthebirth ofchildren from HIV-mono-infected mothers theKupffer cells showed signs oftheir proliferation and liver-associated CD4 lymphocytes showed signs oftheir reduction; hepatic stellate cells showed signs oftheir proliferation and hyperactivation; dendritic liver cells showed signs oftheir proliferation.

A Cytological Atlas of Human Liver Proteome from PROTEOME <sup>SKY</sup>-LIVER <sup>Hu</sup> 2.0, A Publicly Available Database

Liver plays a unique role as a metabolic center of the body, and also performs other important functions such as detoxification and immune response. The regulatory networks are formed between hepatocytes and non-parenchymal liver cells in human liver to maintain liver homeostasis. But an in-depth knowledge of the constituent proteins in different human liver cell types is still lacking. Here, we determine the healthy human liver proteome by measuring four major cell types that build classical liver lobule, hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs) by high-resolution mass spectrometry-based proteomics. Overall, we quantify a total of 8,354 proteins for four cell types and over 6,000 proteins for each cell type. Analysis of this dataset and regulatory pathway reveals the cellular labor division in human liver follows the pattern that parenchymal cells make the main components of pathways, but non-parenchymal cells trigger the pathways. The cells in human liver show some novel molecular features, HCs maintain KC and LSEC homeostasis by producing cholesterol and ketone body; HSCs participate xenobiotics metabolism as an agent deliver; KCs and LSECs mediate immune response through MHC class II-TLRs and MHC class I-TGFβ cascade, respectively; KCs play a central role in diurnal rhythms regulation through sensing diurnal IGF and temperature flux . Together, this work expands our understandings of liver physiology on the basis of cell type resolved proteomics of healthy human liver and provides a useful resource for future analyses of normal and diseased livers. Funding: This work was partially supported by National Key R&D Program of China (Nos.2018YFA0507502, 2020YFE0202200), National Natural Science Foundation of China (Nos. 82090051, 81770581 and 81570526), and Open Project Program of the State Key Laboratory of Proteomics (Academy of Military Medical Sciences, SKLP-K201801, SKLP-K201901, SKLP-O201902 and SKLP-O202001) Declaration of Interest: There is no competing interest in this study. Ethical Approval: This study was approved by the Ethics Committee of The third medical 407 center of Chinese PLA general hospital (Beijing, China).

MicroRNA-223 restricts liver fibrosis by inhibiting the TAZ-IHH-GLI2 and PDGF signaling pathways via the crosstalk of multiple liver cell types.

Background & Aims: Liver fibrosis is a common consequence of chronic liver injury and is characterized by the accumulation of extracellular matrix mainly generated from activated hepatic stellate cells (HSCs). At present, the mechanisms underlying liver fibrogenesis remain obscure and effective pharmacological therapies are lacking. Neutrophil-specific microRNA-223 (miR-223) plays an important role in controlling the development of various liver diseases; however, its role in HSC activation and liver fibrosis remains unclear.Methods: Liver fibrosis was induced by chronic carbon tetrachloride (CCl4) injection of miR-223 knockout (miR-223KO) mice and littermate wild-type controls. MiR-223 was overexpressed in cultured HSCs to determine its function and targets during HSC activation and proliferation. The expression of miR-223 and pri-miR-223 was examined in primary HSCs isolated from CCl4-treated mice and in cultured HSCs. The communication between HSCs and neutrophils was studied by performing in vitro co-culture experiments.Results: Genetic deletion of miR-223 exacerbated chronic CCl4-induced liver fibrosis. Administration of miR-223 inhibited liver fibrosis by inhibiting the transcriptional coactivator with PDZ-binding motif (TAZ)-Indian hedgehog (IHH)-GLI Family Zinc Finger 2 (GLI2) pathway via the crosstalk between hepatocytes and HSCs. Overexpression of miR-223 also directly attenuated Gli2 as well as platelet-derived growth factor receptor α/β (Pdgfra/b) expression in HSCs, thereby suppressing HSC activation and proliferation. The expression of pri-miR-223 and miR-223 was downregulated during HSC activation in vitro. Expression of pri-miR-223 was also decreased in activated HSCs in vivo in fibrotic livers but mature miR-223 expression was not reduced. Finally, in co-culture experiments, activated HSCs were able to take up miR-223-enriched extracellular vesicles from neutrophils, resulting in elevation of miR-223.Conclusion: MiR-223 restricts liver fibrosis by targeting multiple genes in hepatocytes and HSCs, providing potential therapeutic targets for the treatment of liver fibrosis.

Lateral size of graphene oxide determines differential cellular uptake and cell death pathways in Kupffer cells, LSECs, and hepatocytes.

As a representative two-dimensional (2D) nanomaterial, graphene oxide (GO) has shown high potential in many applications due to its large surface area, high flexibility, and excellent dispersibility in aqueous solutions. These properties make GO an ideal candidate for bio-imaging, drug delivery, and cancer therapy. When delivered to the body, GO has been shown to accumulate in the liver, the primary accumulation site of systemic delivery or secondary spread from other uptake sites, and induce liver toxicity. However, the contribution of the GO physicochemical properties and individual liver cell types to this toxicity is unclear due to property variations and diverse cell types in the liver. Herein, we compare the effects of GOs with small (GO-S) and large (GO-L) lateral sizes in three major cell types in liver, Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatocytes. While GOs induced cytotoxicity in KCs, they induced significantly less toxicity in LSECs and hepatocytes. For KCs, we found that GOs were phagocytosed that triggered NADPH oxidase mediated plasma membrane lipid peroxidation, which leads to PLC activation, calcium flux, mitochondrial ROS generation, and NLRP3 inflammasome activation. The subsequent caspase-1 activation induced IL-1β production and GSDMD-mediated pyroptosis. These effects were lateral size-dependent with GO-L showing stronger effects than GO-S. Amongst the liver cell types, decreased cell association and the absence of lipid peroxidation resulted in low cytotoxicity in LSECs and hepatocytes. Using additional GO samples with different lateral sizes, surface functionalities, or thickness, we further confirmed the differential cytotoxic effects in liver cells and the major role of GO lateral size in KUP5 pyroptosis by correlation studies. These findings delineated the GO effects on cellular uptake and cell death pathways in liver cells, and provide valuable information to further evaluate GO effects on the liver for biomedical applications.

Liver zonation

Liver zonation

Human Nonalcoholic Steatohepatitis on a Chip.

Nonalcoholic steatohepatitis (NASH), an advanced stage of nonalcoholic fatty liver disease (NAFLD), is a rapidly growing and global health problem compounded by the current absence of specific treatments. A major limiting factor in the development of new NASH therapies is the absence of models that capture the unique cellular structure of the liver microenvironment and recapitulate the complexities of NAFLD progression to NASH. Organ-on-a-chip platforms have emerged as a powerful approach to dynamically model diseases and test drugs. Herein, we describe a NASH-on-a-chip platform. Four main types of human primary liver cells (hepatocytes [HCs], Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells [HSCs]) were cocultured under microfluidic dynamics. Our chip-based model successfully recapitulated a functional liver cellular microenvironment with stable albumin and urea secretion for at least 2weeks. Exposing liver chips to a lipotoxic environment led to gradual development of NASH phenotypic characteristics, including intracellular lipid accumulation, hepatocellular ballooning, HSC activation, and elevation of inflammatory and profibrotic markers. Further, exposure of the chip to elafibranor, a drug under study for the therapy of NASH, inhibited the development of NASH-specific hallmarks, causing an ~8-fold decrease in intracellular lipids, a 3-fold reduction in number of ballooned HCs, a significant reduction in HSC activation, and a significant decrease in the levels of inflammatory and profibrotic markers compared with controls. Conclusion: We have successfully developed a microfluidic NASH-on-a-chip platform that recapitulates the main NASH histologic endpoints in a single chip and that can emerge as a powerful noninvasive, human-relevant, in vitro platform to study disease pathogenesis and develop novel anti-NASH drugs.

Single nucleus transcriptomics data integration recapitulates the major cell types in human liver.

The aim of this study was to explore the benefits of data integration from different platforms for single nucleus transcriptomics profiling to characterize cell populations in human liver. We generated single-nucleus RNA sequencing data from Chromium 10X Genomics and Drop-seq for a human liver sample. We utilized state of the art bioinformatics tools to undertake a rigorous quality control and to integrate the data into a common space summarizing the gene expression variation from the respective platforms, while accounting for known and unknown confounding factors. Analysis of single nuclei transcriptomes from both 10X and Drop-seq allowed identification of the major liver cell types, while the integrated set obtained enough statistical power to separate a small population of inactive hepatic stellate cells that was not characterized in either of the platforms. Integration of droplet-based single nucleus transcriptomics data enabled identification of a small cluster of inactive hepatic stellate cells that highlights the potential of our approach. We suggest single-nucleus RNA sequencing integrative approaches could be utilized to design larger and cost-effective studies.

Advances on liver cell-derived exosomes in liver diseases.

Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm, which contain several donor cell‐associated proteins as well as mRNA, miRNA, and lipids and coordinate multiple physiological and pathological functions through horizontal communication between cells. Almost all types of liver cells, such as hepatocytes and Kupffer cells, are exosome‐releasing and/or exosome‐targeted cells. Exosomes secreted by liver cells play an important role in regulating general physiological functions and also participate in the onset and development of liver diseases, including liver cancer, liver injury, liver fibrosis and viral hepatitis. Liver cell‐derived exosomes carry liver cell‐specific proteins and miRNAs, which can be used as diagnostic biomarkers and treatment targets of liver disease. This review discusses the functions of exosomes derived from different liver cells and provides novel insights based on the latest developments regarding the roles of exosomes in the diagnosis and treatment of liver diseases.

Sirt6 Alleviated Liver Fibrosis by Deacetylating Conserved Lysine 54 on Smad2 in Hepatic Stellate Cells.

Backgrounds and AimsActivation of hepatic stellate cells (HSCs) is a central driver of fibrosis. This study aimed to elucidate the role of the deacetylase sirtuin 6 (Sirt6) in HSC activation and liver fibrosis.Approach and ResultsGain‐of‐function and loss‐of‐function models were used to study the function of Sirt6 in HSC activation. Mass spectrometry was used to determine the specific acetylation site. The lecithin retinol acyltransferase–driven cyclization recombination recombinase construct (CreERT2) mouse line was created to generate HSC‐specific conditional Sirt6‐knockout mice (Sirt6△HSC). We found that Sirt6 is most abundantly expressed in HSCs as compared with other liver cell types. The expression of Sirt6 was decreased in activated HSCs and fibrotic livers of mice and humans. Sirt6 knockdown and Sirt6 overexpression increased and decreased fibrogenic gene expression, respectively, in HSCs. Mechanistically, Sirt6 inhibited the phosphorylation and nuclear localization of mothers against decapentaplegic homolog (Smad) 2. Further study demonstrated that Sirt6 could directly interact with Smad2, deacetylate Smad2, and decrease the transcription of transforming growth factor β/Smad2 signaling. Mass spectrometry revealed that Sirt6 deacetylated conserved lysine 54 on Smad2. Mutation of lysine 54 to Arginine in Smad2 abolished the regulatory effect of Sirt6. In vivo, specific ablation of Sirt6 in HSCs exacerbated hepatocyte injury and cholestasis‐induced liver fibrosis in mice. With targeted delivery of the Sirt6 agonist MDL‐800, its concentration was 9.28‐fold higher in HSCs as compared with other liver cells and alleviated hepatic fibrosis.ConclusionsSirt6 plays a key role in HSC activation and liver fibrosis by deacetylating the profibrogenic transcription factor Smad2. Sirt6 may be a potential therapeutic target for liver fibrosis.

Synthesis of some new benzoxazole derivatives and investigation of their anticancer activities.

Phortress is an anticancer prodrug, which has active metabolite (5F-203) being potent agonist of the aryl hydrocarbon receptor (AhR). The 5F-203 switches on cytochrome P450 CYP1A1 gene expression and thus exhibits anticancer activity. In this study, it is aimed to obtain new phortress analogues by bioisosteric replacement of benzothiazole core in the structure to benzoxazole ring system. Synthesis of compounds (3a-3p) were performed according to literature methods. Their structures were elucidated by IR, 1H NMR, 13C NMR, 2D-NMR and HRMS spectroscopic methods. Cytotoxicity (MTT), inhibition of DNA synthesis and flow cytometric analysis assays were applied to determine anticancer activity of the compounds on colon (HT-29), breast (MCF7), lung (A549), liver (HepG2) and brain (C6) carcinoma cell types. When compared reference agent doxorubicin, compounds 3m and 3n displayed very attractive anticancer effect against carcinogenic cell lines. Due to structural similarity to phortress, biotransformation studies for 3m and 3n were examined by LCMS-IT-TOF system and probable metabolites of these compounds were determined. Induction potential of these compounds on CYP1A1/2 enzymes was also investigated to clarify possible mechanism of action. Interaction modes between CYP1A1 enzyme and compound 3n or its some metabolites were investigated by docking studies. In conclusion, findings of these study indicate that compounds 3m and 3n possess significant anticancer activity, probably with the same mechanism of action to Phortress.

Cell-autonomous hepatocyte-specific GP130 signaling is sufficient to trigger a robust innate immune response in mice

Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.

Covering the Cover

Covering the Cover

Keeping rhythm requires communication

Circadian Rhythms In mammals, daily cycles in physiology require the synchronized activity of circadian clocks in peripheral organs such as the liver, a hub of metabolism. Guan et al. generated mice with hepatocytes that lack two transcriptional repressors known to be essential for clock function. This experimental manipulation unexpectedly disrupted rhythmic gene expression and metabolism not only in hepatocytes but also in other liver cell types. Feeding behavior also coregulated circadian rhythms in multiple liver cell types. Cell-cell communication thus appears to be important in maintaining the robustness of peripheral circadian clocks. Science , this issue p. [1388][1] [1]: /lookup/doi/10.1126/science.aba8984

An Integrative Omics Approach Reveals Involvement of BRCA1 in Hepatic Metastatic Progression of Colorectal Cancer.

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student’s t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.

Integrative analysis of scRNA-seq and GWAS data pinpoints periportal hepatocytes as the relevant liver cell types for blood lipids.

Liver, a heterogeneous tissue consisting of various cell types, is known to be relevant for blood lipid traits. By integrating summary statistics from genome-wide association studies (GWAS) of lipid traits and single-cell transcriptome data of the liver, we sought to identify specific cell types in the liver that were most relevant for blood lipid levels. We conducted differential expression analyses for 40 cell types from human and mouse livers in order to construct the cell-type specifically expressed gene sets, which we refer to as construction of the liver cell-type specifically expressed gene sets (CT-SEGS). Under the assumption that CT-SEGS represented specific functions of each cell type, we applied stratified linkage disequilibrium score regression to determine cell types that were most relevant for complex traits and diseases. We first confirmed the validity of this method (of delineating functionally relevant cell types) by identifying the immune cell types as relevant for autoimmune diseases. We further showed that lipid GWAS signals were enriched in the human and mouse periportal hepatocytes. Our results provide important information to facilitate future cellular studies of the metabolic mechanism affecting blood lipid levels.

Insights Into Extracellular Vesicles as Biomarker of NAFLD Pathogenesis.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease around the world estimated to affect up to one-third of the adult population and is expected to continue rising in the coming years. Nonalcoholic fatty liver disease is considered as the hepatic manifestation of the metabolic syndrome because it is strongly associated with obesity, insulin resistance, type 2 diabetes mellitus, and cardiovascular complications. Despite its high prevalence, factors leading to NAFLD progression from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and, ultimately hepatocellular carcinoma remain poorly understood. To date, no treatment has proven efficacy, and also no reliable method is currently available for diagnosis or staging of NAFLD beyond the highly invasive liver biopsy. Recently, extracellular vesicles (EVs) have emerged as potential candidate biomarkers for the diagnosis of NAFLD. Extracellular vesicles are circulating, cell-derived vesicles containing proteins and nucleic acids, among other components, that interact with and trigger a plethora of responses in neighbor or distant target cells. Several mechanisms implicated in NAFLD progression, such as inflammation, fibrosis, and angiogenesis, all related to metabolic syndrome–associated lipotoxicity, trigger EV production and release by liver cells. As hepatocytes represent ~80% of the liver volume, in this review we will focus on hepatocyte-derived EVs as drivers of the interactome between different liver cell types in NAFLD pathogenesis, as well as in their role as noninvasive biomarkers for NAFLD diagnosis and progression. Based on that, we will highlight the research that is currently available on EVs in this topic, the current limitations, and future directions for implementation in a clinical setting as biomarkers or targets of liver disease.

The hepatocyte clock and feeding control chronophysiology of multiple liver cell types

Most cells of the body contain molecular clocks, but the requirement of peripheral clocks for rhythmicity and their effects on physiology are not well understood. We show that deletion of core clock components REV-ERBα and REV-ERBβ in adult mouse hepatocytes disrupts diurnal rhythms of a subset of liver genes and alters the diurnal rhythm of de novo lipogenesis. Liver function is also influenced by nonhepatocytic cells, and the loss of hepatocyte REV-ERBs remodels the rhythmic transcriptomes and metabolomes of multiple cell types within the liver. Finally, alteration of food availability demonstrates the hierarchy of the cell-intrinsic hepatocyte clock mechanism and the feeding environment. Together, these studies reveal previously unsuspected roles of the hepatocyte clock in the physiological coordination of nutritional signals and cell-cell communication controlling rhythmic metabolism.

Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts.

Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor β (TGF-β) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions invitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.

Utility of Common Marmoset (Callithrix jacchus) Embryonic Stem Cells in Liver Disease Modeling, Tissue Engineering and Drug Metabolism.

The incidence of liver disease is increasing significantly worldwide and, as a result, there is a pressing need to develop new technologies and applications for end-stage liver diseases. For many of them, orthotopic liver transplantation is the only viable therapeutic option. Stem cells that are capable of differentiating into all liver cell types and could closely mimic human liver disease are extremely valuable for disease modeling, tissue regeneration and repair, and for drug metabolism studies to develop novel therapeutic treatments. Despite the extensive research efforts, positive results from rodent models have not translated meaningfully into realistic preclinical models and therapies. The common marmoset Callithrix jacchus has emerged as a viable non-human primate model to study various human diseases because of its distinct features and close physiologic, genetic and metabolic similarities to humans. C. jacchus embryonic stem cells (cjESC) and recently generated cjESC-derived hepatocyte-like cells (cjESC-HLCs) could fill the gaps in disease modeling, liver regeneration and metabolic studies. They are extremely useful for cell therapy to regenerate and repair damaged liver tissues in vivo as they could efficiently engraft into the liver parenchyma. For in vitro studies, they would be advantageous for drug design and metabolism in developing novel drugs and cell-based therapies. Specifically, they express both phase I and II metabolic enzymes that share similar substrate specificities, inhibition and induction characteristics, and drug metabolism as their human counterparts. In addition, cjESCs and cjESC-HLCs are advantageous for investigations on emerging research areas, including blastocyst complementation to generate entire livers, and bioengineering of discarded livers to regenerate whole livers for transplantation.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease

There are few invitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.