Liver-specific Gene Expression Research Articles

Bile acid-microbiota crosstalk in hepatitis B virus infection.

Hepatitis B virus (HBV) is a hepatotropic non-cytopathic virus characterized by liver-specific gene expression. HBV infection highjacks bile acid metabolism, notably impairing bile acid uptake via sodium taurocholate cotransporting polypeptide (NTCP), which is a functional receptor for HBV entry. Concurrently, HBV infection induces changes in bile acid synthesis and the size of the bile acid pool. Conversely, bile acid facilitates HBV replication and expression through the signaling molecule farnesoid X receptor (FXR), a nuclear receptor activated by bile acid. However, in HepaRG cells and primary hepatocytes, FXR agonists suppress HBV RNA expression and the synthesis and secretion of DNA. In the gut, the size and composition of the bile acid pool significantly influence the gut microbiota. In turn, the gut microbiota impacts bile acid metabolism and innate immunity, potentially promoting HBV clearance. Thus, the bile acid-gut microbiota axis represents a complex and evolving relationship in the context of HBV infection. This review explores the interplay between bile acid and gut microbiota in HBV infection and discusses the development of HBV entry inhibitors targeting NTCP.

ZBTB7B is a permissive regulator of hepatocellular carcinoma initiation by repressing c-Jun expression and function

Hepatocarcinogenesis is a multi-step process. However, the regulators of hepatocellular carcinoma (HCC) initiation are understudied. Adult liver-specific gene expression was globally downregulated in HCC. We hypothesize that adult liver-specific genes, especially adult liver-enriched transcription factors may exert tumor-suppressive functions in HCC. In this study, we identify ZBTB7B, an adult liver-enriched transcription factor as a permissive regulator of HCC initiation. ZBTB7B is highly expressed in hepatocytes in adult livers, compared to fetal livers. To evaluate the functions of ZBTB7B in hepatocarcinogenesis, we performed hepatocyte-specific ZBTB7B knockout in hydrodynamic oncogene transfer-induced mouse liver cancer models. Hepatocyte-specific knockout of ZBTB7B promotes activated Akt and N-Ras-induced HCC development. Moreover, ZBTB7B deficiency sensitizes hepatocytes to a single oncogene Akt-induced oncogenic transformation and HCC initiation, which is otherwise incompetent in inducing HCC. ZBTB7B deficiency accelerates HCC initiation by down-regulating adult liver-specific gene expression and priming livers to a fetal-like state. The molecular mechanism underlying ZBTB7B functions in hepatocytes was investigated by integrated transcriptomic, phosphoproteomic, and chromatin immunoprecipitation-sequencing analyses. Integrative multi-omics analyses identify c-Jun as the core signaling node in ZBTB7B-deficient liver cancer initiation. c-Jun is a direct target of ZBTB7B essential to accelerated liver cancer initiation in ZBTB7B-deficient livers. Knockdown of c-Jun expression or dominant negative c-Jun expression delays HCC development in ZBTB7B-deficient livers. In addition, ZBTB7B competes with c-Jun for chromatin binding. Ectopic ZBTB7B expression attenuates the tumor-promoting functions of c-Jun. Expression of ZBTB7B signature, composed of 140 genes co-regulated by ZBTB7B and c-Jun, is significantly downregulated in early-stage HCCs compared to adjacent normal tissues, correlates to liver-specific gene expression, and is associated with good prognosis in human HCC. Thus, ZBTB7B functions as a permissive regulator of HCC initiation by directly regulating c-Jun expression and function.

CSIG-19. CANCER STEM CELL MEDIATED F11 DRIVES HYPER-THROMBOTIC ACTIVITY IN GLIOBLASTOMA

Abstract Cancer-related thrombosis is the second leading cause of death for cancer patients and glioblastoma (GBM), the most common primary malignant brain tumor, is associated with approximately a 30% risk of venous thromboembolism, deep vein thrombosis, or pulmonary embolisms. While this remains a major clinical issue, the interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, including GBM. GBM is a tumor which contains a high degree of cellular heterogeneity and has populations of therapeutically resistant cancer stem cells (CSCs) that drive tumor growth. Histologically CSCs are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcome in many cancers and while platelets are known to promote progression of other tumors. Our previous work demonstrated that CSCs co-opt and activate platelets to promote CSC growth by intrinsically producing thrombin via a cell intrinsic functional coagulation cascade. These findings provided further evidence that CSCs readily execute a highly liver-specific gene expression program that is mechanistically linked to GBM growth. We now demonstrate thrombin and platelet expression in GBM patient specimen creates a hyper-thrombotic GBM tumor-microenvironment. Further, genetic targeting of the CSC intrinsic coagulation cascade, via knockdown of coagulation factor 11 (F11), reduces CSC growth and tumor formation while also decreasing the ability for CSCs to activate platelets, subsequently decreasing the hyper-thrombotic state seen in GBM. Our observations suggest that CSCs execute a cell intrinsic coagulation mechanism to contribute to the hyper-thrombotic tumor-microenvironment seen in GBM and may shed light onto mechanisms of cancer associated thrombosis in not only GBM, but also other malignancies.

HNF4α isoforms: the fraternal twin master regulators of liver function.

In the more than 30 years since the purification and cloning of Hepatocyte Nuclear Factor 4 (HNF4α), considerable insight into its role in liver function has been gleaned from its target genes and mouse experiments. HNF4α plays a key role in lipid and glucose metabolism and intersects with not just diabetes and circadian rhythms but also with liver cancer, although much remains to be elucidated about those interactions. Similarly, while we are beginning to elucidate the role of the isoforms expressed from its two promoters, we know little about the alternatively spliced variants in other portions of the protein and their impact on the 1000-plus HNF4α target genes. This review will address how HNF4α came to be called the master regulator of liver-specific gene expression with a focus on its role in basic metabolism, the contributions of the various isoforms and the intriguing intersection with the circadian clock.

Development of lumen-based perfusable 3D liver in vitro model using single-step bioprinting with composite bioinks.

Hepatic sinusoids are uniquely organized structures that help maintain a spectrum of hepatic functions. Although several in vitro liver models have been developed to replicate liver sinusoids, most of these platforms require complex, multi-step fabrication methods making it difficult to achieve truly three-dimensional (3D) channel geometries. In this study, a single-step bioprinting technique was demonstrated to simultaneously print a chip platform and develop a perfusable vascularized liver sinusoid in vitro model. The integrated system uses a co-axial-based bioprinting approach to develop a liver sinusoid-like model that consists of a sacrificial core compartment containing a perfusable pre-vascular structure and an alginate-collagen-based shell compartment containing hepatocytes. The lumen-based perfusable 3D liver sinusoid-on-a-chip (LSOC-P) demonstrated significantly better hepatocyte viability, proliferation, and liver-specific gene and protein expression compared to a 3D hepatocyte-based core/shell model with static media and the standard hepatocyte-based 2D sandwich culture system. A drug toxicity evaluation of hepatotoxins highlighted the comparatively higher sensitivity of the LSOC system with a close estimation of the therapeutic range of safe drug concentrations for humans. In conclusion, the current findings indicate that the combinatorial single-step co-axial bioprinting technique is a promising fabrication approach for the development of a perfusable LSOC platform for drug screening applications.

Structural insights into the HNF4 biology.

Hepatocyte Nuclear Factor 4 (HNF4) is a transcription factor (TF) belonging to the nuclear receptor (NR) family that is expressed in liver, kidney, intestine and pancreas. It is a master regulator of liver-specific gene expression, in particular those genes involved in lipid transport and glucose metabolism and is crucial for the cellular differentiation during development. Dysregulation of HNF4 is linked to human diseases, such as type I diabetes (MODY1) and hemophilia. Here, we review the structures of the isolated HNF4 DNA binding domain (DBD) and ligand binding domain (LBD) and that of the multidomain receptor and compare them with the structures of other NRs. We will further discuss the biology of the HNF4α receptors from a structural perspective, in particular the effect of pathological mutations and of functionally critical post-translational modifications on the structure-function of the receptor.

Decellularized Mouse Liver as a Small-Scale Scaffold for the Creation of a Miniaturized Human Liver

The liver is the main organ responsible for drug metabolism; hepatocyte-based culture models play a fundamental role in understanding liver physiology. While different types of two-dimensional and three-dimensional liver models have been developed, the failure to accurately mimic native liver, including extracellular matrix (ECM) composition, complex structure, and rich vascular network as well as shortage of liver donors, impede their clinical translation. Herein, we have realized a “miniature human liver” based on the infusion of primary human hepatocytes into a decellularized liver template (DC-liver) made from the right liver lobe of mouse. We performed detergent-based decellularization of the mouse liver sections via portal vein (PV) perfusion and confirmed the successful removal of cell content, and the preservation of the vascular network. Subsequently, the DC-liver templates were recellularized at varying cell densities, including 3 × 106 and 1 × 107 cells/mL, and liver specific gene expression was assessed. Overall, hepatocytes in the recellularized liver constructs (RC-liver) expressed liver-specific mRNA expression markers (Hnf4α, Alb) at a level comparable to the unseeded, freshly isolated hepatocytes and to hepatocytes seeded inside collagen gels. However, the RC-liver with the highest cell density exhibited poor cell distribution and blockage of the PV, in general, hepatocytes showed elevated levels of Cyp1a1. Further analysis hinted at hypoxic conditions inside the constructs, showing higher mRNA expression levels of hypoxia-related genes (Hif-1α, Casp3, Zo-1). Fortunately, oxygen supplementation appeared to alleviate hypoxia, which markedly reduced expression levels of Hif-1α and Cyp1α1. As a proof-of-concept, primary human hepatocytes were also seeded into the DC-liver templates, and we could confirm liver specific mRNA expression (HNF4A, ALB, CYP3A4). Altogether, the above results indicate a profound potential in the use of DC-liver tissue for the fabrication of a miniature human liver, which may have potential application prospects for regenerative medicine, tissue engineering (TE) and other related disciplines.

Raphanus sativus L. var. caudatus Extract Alleviates Impairment of Lipid and Glucose Homeostasis in Liver of High-Fat Diet-Induced Obesity and Insulin Resistance in Mice.

The present study investigated the activities of Raphanus sativus L. var. caudatus extract (RS) on abnormal lipid and glucose homeostasis in a high-fat diet (HFD)-induced obesity and insulin resistance in a mouse model. Institute of Cancer Research mice were rendered obese by 16-week HFD feeding. Obese mice were administered with 100 or 200 mg/kg/d RS orally during the last 8 weeks of diet feeding. Then, the biochemical parameters were determined. The gene and protein expressions regulating lipid and glucose homeostasis in the liver were measured. This study revealed that the state of hyperglycemia, hyperleptinemia, hyperinsulinemia, and hyperlipidemia was reduced after 8 weeks of RS treatment (100 or 200 mg/kg). Administration of RS also improved insulin sensitivity and increased serum adiponectin. The liver total cholesterol and triglyceride concentrations were decreased by both doses of RS. Notably, a decrease in the expression of liver-specific genes, including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase, was found in the RS-treated groups. Moreover, administration of RS showed a significant increase in the expression of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) proteins. These findings indicated that RS improved abnormal lipid and glucose homeostasis in the liver of obesity-associated insulin resistance mouse model, possibly through the stimulation of the AMPK/Sirt1 pathway.

STEM-01. GLIOMA STEM CELLS ACTIVATE PLATELETS BY PLASMA-INDEPENDENT THROMBIN PRODUCTION TO PROMOTE GBM TUMORIGENESIS

Abstract The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example glioblastoma (GBM) for which metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcome in many cancers. While platelets are known to promote progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Male and female NSG mice were transplanted with patient derived GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. We demonstrate that GSCs co-opt and activate platelets to promote GBM tumor progression. GSCs endogenously produce all coagulation factors of the intrinsic and extrinsic cascade generating thrombin and activating platelets in the absence of plasma. Conversely, inhibition of platelet activation and of thrombin production by GSCs abrogates platelet-mediated GSC self-renewal and growth (p< 0.0005). Similarly, inhibiting intratumoral thrombin production and function decreases tumor formation in vivo (p< 0.005). These studies challenge and revise the longstanding view that the complete coagulation cascades are found only in the liver and plasma demonstrating that cancer stem cells readily execute a highly liver-specific gene expression program that is mechanistically linked to GBM oncogenesis. We show that cancer cells can co-opt non-cancer cells to promote tumor growth, by uncovering the preferential relationship between platelets and GSCs that drive GBM malignancies and identifies a therapeutically targetable novel interaction.

High-throughput bioengineering of homogenous and functional human-induced pluripotent stem cells-derived liver organoids via micropatterning technique.

Human pluripotent stem cell-derived liver organoids are emerging as more human-relevant in vitro models for studying liver diseases and hepatotoxicity than traditional hepatocyte cultures and animal models. The generation of liver organoids is based on the Matrigel dome method. However, the organoids constructed by this method display significant heterogeneity in their morphology, size, and maturity. Additionally, the formed organoid is randomly encapsulated in the Matrigel dome, which is not convenient for in situ staining and imaging. Here, we demonstrate an approach to generate a novel type of liver organoids via micropatterning technique. This approach enables the reproducible and high-throughput formation of bioengineered fetal liver organoids with uniform morphology and deterministic size and location in a multiwell plate. The liver organoids constructed by this technique closely recapitulate some critical features of human liver development at the fetal stage, including fetal liver-specific gene and protein expression, glycogen storage, lipid accumulation, and protein secretion. Additionally, the organoids allow whole-mount in-situ staining and imaging. Overall, this new type of liver organoids is compatible with the pharmaceutical industry’s widely-used preclinical drug discovery tools and will facilitate liver drug screening and hepatotoxic assessment.

Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells.

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation.

In Vitro Differentiation of Human Amniotic Epithelial Cells into Hepatocyte-like Cells.

Human amniotic epithelial cells (hAECs) represent an interesting clinical alternative to human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells in regenerative medicine. The potential of hAECs can be enhanced ex vivo by their partial pre-differentiation. The aim of this study was to evaluate the effectiveness of 18-day differentiation of hAECs into endodermal cells, hepatic precursor cells, and cells showing functional features of hepatocytes using culture media supplemented with high (100 ng/mL) concentrations of EGF or HGF. The cells obtained after differentiation showed changes in morphology and increased expression of AFP, ALB, CYP3A4, CYP3A7, and GSTP1 genes. HGF was more effective than EGF in increasing the expression of liver-specific genes in hAECs. However, EGF stimulated the differentiation process more efficiently and yielded more hepatocyte-like cells capable of synthesizing α-fetoprotein during differentiation. Additionally, after 18 days, GST transferases, albumin, and CYP P450s, which proved their partial functionality, were expressed. In summary, HGF and EGF at a dose of 100 ng/mL can be successfully used to obtain hepatocyte-like cells between days 7 and 18 of hAEC differentiation. However, the effectiveness of this process is lower compared with hiPSC differentiation; therefore, optimization of the composition of the medium requires further research.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Proprotein convertase subtilisin/kexin type 9 regulates the production of acute-phase reactants from the liver.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) controls blood cholesterol levels by fostering the LDL receptor (LDLR) degradation in hepatocytes. Additionally, PCSK9 has been suggested to participate in immunoregulation by modulating cytokine production. We studied the immunological role of PCSK9 in Streptococcus pneumoniae bacteraemia in vivo and in a human hepatocyte cell line. CRISPR/Cas9 mutagenesis was utilized to create pcsk9 knock-out (KO) zebrafish, which were infected with S pneumoniae to assess the role of PCSK9 for the survival of the fish and in the transcriptomic response of the liver. The direct effects of PCSK9 on the expression of acute-phase reaction (APR) genes were studied in HepG2 cells. The pcsk9 KO zebrafish lines (pcsk9tpu-13 and pcsk9tpu-2,+15 ) did not show developmental defects or gross phenotypical differences. In the S pneumoniae infected zebrafish, the mortality of pcsk9 KOs was similar to the controls. A liver-specific gene expression analysis revealed that a pneumococcal challenge upregulated pcsk9, and that the pcsk9 deletion reduced the expression of APR genes, including hepcidin antimicrobial peptide (hamp) and complement component 7b (c7b). Accordingly, silencing PCSK9 in vitro in HepG2 cells using small interfering RNAs (siRNAs) decreased HAMP expression. We demonstrate that PCSK9 is not critical for zebrafish survival in a systemic pneumococcal infection. However, PCSK9 deficiency was associated with the lower expression of APR genes in zebrafish and altered the expression of innate immunity genes in a human hepatocyte cell line. Overall, our data suggest an evolutionarily conserved function for PCSK9 in APR in the liver.

<p><strong>Hepatocyte nuclear factor Foxa-2 effectively differentiates mesenchymal stem cells to functional hepatocytes</strong></p>

Overexpression of hepatocyte nuclear factor (Foxa-2), a master regulator significantly enhances the hepatic differentiation by triggering targeted liver-specific gene expression for liver development. We investigated the function of key regulators that facilitate hepatic differentiation. The functional hepatocyte was determined by observing morphological changes, expression of liver specific markers through immunocytochemical analysis, qRT-PCR, Western Blot and specific liver function assays. Our study confirmed that induction of functional hepatocyte shows typical epithelial morphology, express hepatic genes and acquire hepatocyte functions. Notably, these cells expressed the markers of mature hepatocytes, including albumin, tyrosine aminotransferase, cytokeratin, Cyp7A1 and hepatic transcription factors. Furthermore, these cells exhibited hepatic functions in vitro , including glycogen storage, albumin production, urea secretion and functional assessment of hepatocytes. We have developed a robust and efficient method to differentiate mesenchymal stem cells into induced functional hepatocytes, which exhibits characteristics of mouse hepatocytes. Apparently, induction of functional hepatocytes will pave a better cellular therapy or effective cell source within a short timeline to facilitate the development of hepatocytes for future clinical applications of regenerative medicine.

Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs

BackgroundThere are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs.MethodsThe HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development.ResultsLoss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality.ConclusionsThis work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Transcriptome Classification Reveals Molecular Subgroups in Patients with Hepatitis B Virus.

Hepatitis B virus (HBV) specifically infects hepatocytes, which can cause progressive liver fibrosis and a significantly increased risk of liver cancer. Multiple studies indicated host genetic, virological, and immunological factors could affect the HBV infection. However, the underlying mechanism involved in HBV infection remained unclear. Based on the analysis of gene expression data of 124 HBV patients (GEO accession: GSE84044), molecular subgroups of patients infected with hepatitis B virus were identified in this study, including C1, C2, and C3 groups. The age, fiber, degree of chemical and inflammation, and gene expression difference were also compared among the three sampling groups. Furthermore, the liver index was calculated using 93 liver-specific genes. The liver-specific gene expression in different molecular subgroups of HBV patients was thoroughly analyzed and then was compared with fibrosis and inflammation levels. Results showed that the C2 group was the youngest and the C3 group had the highest degree of fibrosis and inflammation. Enrichment analysis showed that metabolism-related pathways were mainly expressed in the C1 and C2 groups, and inflammation-related pathways and proteoglycans in cancer were highly expressed in the C1 and C3 groups. The liver index was higher in the C2 group than in the C1 and C3 groups, and it was the lowest in the C3 group. Macrophage M1/M2 and neutrophils were significantly different in the three groups. M1 was mainly abundant in the C3 group, and M2 and neutrophils were mainly abundant in the C2 group. This study provides novel information to understand the mechanisms of HBV infection in chronic hepatitis B (CHB) patients.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system

Examining intestine–liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine–liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

The use of design of experiments with multiple responses to determine optimal formulations for in vivo hepatic mRNA delivery

Although great advances have been made in the delivery of short RNAs by lipid nanoparticles (LNPs), the optimal formulation composition and physicochemical properties of LNPs for long RNA (including mRNA) remain unclear. In the present study, we optimized the lipid composition of liver-targeted mRNA-loaded LNPs that were prepared with pH-sensitive cationic lipids that had been previously designed for siRNA delivery through a two stepped design of experiment (DoE). Multiple responses including physicochemical properties, gene expression, and liver-specificity were analyzed in order, not only to understand the role of each formulation parameter, but also to examine parameters that would be difficult to predict. We found that particle size and the PEG-to-phospholipid (PEG/PL) ratio were additional key factors for liver-specific gene expression in addition to the other formulation factors. The optimized formulation showed a better gene expression compared to other lipid formulations from industry leaders. These findings suggest that a “DoE with multiple responses” approach can be used to predict significant parameters and permit optimized formulations to be prepared more efficiently.

Dissolved oxygen concentration regulates human hepatic organoid formation from pluripotent stem cells in a fully controlled bioreactor.

Developing technologies for scalable production of human organoids has gained increased attention for "organoid medicine" and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20-40% dissolved oxygen concentration [DO] (corresponded to 30-60 mmHg pO2 within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal-like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver-specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications.