Human induced pluripotent stem cell based hepatic-modeling of lipid metabolism associated TM6SF2 E167K variant.

BACKGROUND AND AIMS: TM6SF2 rs58542926 (E167K) is related to increased prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD). Conflicting mouse study results highlight the need for a human model to understand this mutation's impact. This study aims to create and characterize a reliable human in vitro model to mimic the effects of the TM6SF2-E167K mutation for future studies. APPROACH AND RESULTS: We used gene editing on human human-induced pluripotent stem cells (iPSC) from a healthy individual to create cells with the TM6SF2-E167K mutation. After hepatocyte directed differentiation, we observed decreased TM6SF2 protein expression, increased intracellular lipid droplets and total cholesterol in addition to reduced VLDL secretion. Transcriptomics revealed upregulation of genes involved in lipid, fatty acid, and cholesterol transport, flux, and oxidation. Global lipidomics showed increased lipid classes associated with ER stress, mitochondrial dysfunction, apoptosis, and lipid metabolism. Additionally, the TM6SF2-E167K mutation conferred a pro-inflammatory phenotype with signs of mitochondria and ER stress. Importantly, by facilitating protein folding within the ER of hepatocytes carrying TM6SF2-E167K mutation, VLDL secretion and ER stress markers improved. CONCLUSIONS: Our findings indicate that induced hepatocytes generated from iPSCs carrying the TM6SF2-E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to identify potential clinical targets and highlights the therapeutic potential of targeting protein misfolding to alleviate ER stress and mitigate the detrimental effects of the TM6SF2-E167K mutation on hepatic lipid metabolism.

Evaluation of Human Hepatocyte Drug Metabolism Carrying High-Risk or Protection-Associated Liver Disease Genetic Variants.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography-mass spectrometry (LC-MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.

From a Single Cell to a Whole Human Liver: Disease Modeling and Transplantation.

Although the underlying cause may vary across countries and demographic groups, liver disease is a major cause of morbidity and mortality globally. Orthotopic liver transplantation is the only definitive treatment for liver failure but is limited by the lack of donor livers. The development of drugs that prevent the progression of liver disease and the generation of alternative liver constructs for transplantation could help alleviate the burden of liver disease. Bioengineered livers containing human induced pluripotent stem cell (iPSC)-derived liver cells are being utilized to study liver disease and to identify and test potential therapeutics. Moreover, bioengineered livers containing pig hepatocytes and endothelial cells have been shown to function and survive after transplantation into pig models of liver failure, providing preclinical evidence toward future clinical applications. Finally, bioengineered livers containing human iPSC-derived liver cells have been shown to function and survive after transplantation in rodents but require considerable optimization and testing prior to clinical use. In conclusion, bioengineered livers have emerged as a suitable tool for modeling liver diseases and as a promising alternative graft for clinical transplantation. The integration of novel technologies and techniques for the assembly and analysis of bioengineered livers will undoubtedly expand future applications in basic research and clinical transplantation.

The Inside-Out of End-Stage Liver Disease: Hepatocytes are the Keystone.

Chronic liver injury results in cirrhosis and end-stage liver disease (ESLD) which represents a leading cause of death worldwide, affecting people in their most productive years of life. Medical therapy can extend life, but the only definitive treatment is liver transplantation (LT). However, LT remains limited by access to quality donor organs and suboptimal long-term outcomes. The degeneration from healthy-functioning livers to cirrhosis and ESLD involves a dynamic process of hepatocyte damage, diminished hepatic function, and adaptation. However, the mechanisms responsible for deterioration of hepatocyte function and ultimately hepatic failure in man are poorly understood. We review the current understanding of cirrhosis and ESLD as a dynamic process and outline the current mechanisms associated with the development of hepatic failure from the clinical manifestations to energy adaptations, regeneration, and regulation of nuclear transcription factors. A new generation of therapeutics could target stabilization of hepatocyte differentiation and function to avoid the need for transplantation in patients with cirrhosis and ESLD.

Expression of the type 3 InsP3 receptor is a final common event in the development of hepatocellular carcinoma.

BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.

Polymorphism in the Promoter Region of NFE2L2 Gene Is a Genetic Marker of Susceptibility to Cirrhosis Associated with Alcohol Abuse.

Alcoholic liver disease (ALD) is a highly prevalent spectrum of pathologies caused by alcohol overconsumption. Morbidity and mortality related to ALD are increasing worldwide, thereby demanding strategies for early diagnosis and detection of ALD predisposition. A potential candidate as a marker for ALD susceptibility is the transcription factor nuclear factor erythroid-related factor 2 (Nrf2), codified by the nuclear factor erythroid 2-related factor 2 gene (NFE2L2). Nrf2 regulates expression of proteins that protect against oxidative stress and inflammation caused by alcohol overconsumption. Here, we assessed genetic variants of NFE2L2 for association with ALD. Specimens from patients diagnosed with cirrhosis caused by ALD were genotyped for three NFE2L2 single nucleotide polymorphisms (SNP) (SNPs: rs35652124, rs4893819, and rs6721961). Hematoxylin & eosin and immunohistochemistry were performed to determine the inflammatory score and Nrf2 expression, respectively. SNPs rs4893819 and rs6721961 were not specifically associated with ALD, but analysis of SNP rs35652124 suggested that this polymorphism predisposes to ALD. Furthermore, SNP rs35652124 was associated with a lower level of Nrf2 expression. Moreover, liver samples from ALD patients with this polymorphism displayed more severe inflammatory activity. Together, these findings provide evidence that the SNP rs35652124 variation in the Nrf2-encoding gene NFE2L2 is a potential genetic marker for susceptibility to ALD.

Calcium signalling from the type I inositol 1,4,5-trisphosphate receptor is required at early phase of liver regeneration.

BACKGROUND & AIMS: Liver regeneration is a multistage process that unfolds gradually, with different mediators acting at different stages of regeneration. Calcium (Ca(2+) ) signalling is essential for liver regeneration. In hepatocytes, Ca(2+) signalling results from the activation of inositol 1,4,5-trisphosphate receptors (InsP3 R) of which two of the three known isoforms are expressed (InsP3 R-I and InsP3 R-II). Here, we investigated the role of the InsP3 R-I-dependent Ca(2+) signals in hepatic proliferation during liver regeneration. METHODS: Partial hepatectomy (HX) in combination with knockdown of InsP3 R-I (AdsiRNA-I) was used to evaluate the role of InsP3 R-I on liver regeneration and hepatocyte proliferation, as assessed by liver to body mass ratio, PCNA expression, immunoblots and measurements of intracellular Ca(2+) signalling. RESULTS: AdsiRNA-I efficiently infected the liver as demonstrated by the expression of ß-galactosidase throughout the liver lobules. Moreover, this construct selectively and efficiently reduced the expression of InsP3 R-I, as evaluated by immunoblots. Expression of AdsiRNA-I in liver decreased peak Ca(2+) amplitude induced by vasopressin in isolated hepatocytes 2 days after HX. Reduced InsP3 R-I expression prior to HX also delayed liver regeneration, as measured by liver to body weight ratio, and reduced hepatocyte proliferation, as evaluated by PCNA staining, at the same time point. At later stages of regeneration, control hepatocytes showed a decreased expression of InsP3 R, as well as reduced InsP3 R-mediated Ca(2+) signalling, events that did not affect liver growth. CONCLUSION: Together, these results show that InsP3 R-I-dependent Ca(2+) signalling is an early triggering pathway required for liver regeneration.

A rat model of cirrhosis with well-differentiated hepatocellular carcinoma induced by thioacetamide.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths, and commonly associated with hepatic fibrosis or cirrhosis. This study aims to establish a rat model mimicking the progression from liver fibrosis to cirrhosis and subsequently to HCC using thioacetamide (TAA). We utilized male Lewis rats, treating them with intra-peritoneal injections of TAA. These rats received bi-weekly injections of either 200 mg/kg TAA or saline (as a control) over a period of 34 weeks. The development of cirrhosis and hepatocarcinogenesis was monitored through histopathological examinations, biochemical markers, and immunohistochemical analyses. Our results demonstrated that chronic TAA administration induced cirrhosis and well-differentiated HCC, characterized by increased fibrosis, altered liver architecture, and enhanced hepatocyte proliferation. Biochemical analyses revealed significant alterations in liver function markers, including elevated alpha-fetoprotein (AFP) levels, without affecting kidney function or causing significant weight loss or mortality in rats. This TAA-induced cirrhosis and HCC rat model successfully replicates the clinical progression of human HCC, including liver function impairment and early-stage liver cancer characteristics. It presents a valuable tool for future research on the mechanisms of antitumor drugs in tumor initiation and development.

Comparison of wild-type and high-risk PNPLA3 variants in a human biomimetic liver microphysiology system for metabolic dysfunction-associated steatotic liver disease precision therapy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide health epidemic with a global occurrence of approximately 30%. The pathogenesis of MASLD is a complex, multisystem disorder driven by multiple factors, including genetics, lifestyle, and the environment. Patient heterogeneity presents challenges in developing MASLD therapeutics, creating patient cohorts for clinical trials, and optimizing therapeutic strategies for specific patient cohorts. Implementing pre-clinical experimental models for drug development creates a significant challenge as simple in vitro systems and animal models do not fully recapitulate critical steps in the pathogenesis and the complexity of MASLD progression. To address this, we implemented a precision medicine strategy that couples the use of our liver acinus microphysiology system (LAMPS) constructed with patient-derived primary cells. We investigated the MASLD-associated genetic variant patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 (I148M variant) in primary hepatocytes as it is associated with MASLD progression. We constructed the LAMPS with genotyped wild-type and variant PNPLA3 hepatocytes, together with key non-parenchymal cells, and quantified the reproducibility of the model. We altered media components to mimic blood chemistries, including insulin, glucose, free fatty acids, and immune-activating molecules to reflect normal fasting (NF), early metabolic syndrome (EMS), and late metabolic syndrome (LMS) conditions. Finally, we investigated the response to treatment with resmetirom, an approved drug for metabolic syndrome-associated steatohepatitis (MASH), the progressive form of MASLD. This study, using primary cells, serves as a benchmark for studies using "patient biomimetic twins" constructed with patient induced pluripotent stem cell (iPSC)-derived liver cells using a panel of reproducible metrics. We observed increased steatosis, immune activation, stellate cell activation, and secretion of pro-fibrotic markers in the PNPLA3 GG variant compared to the wild-type CC LAMPS, consistent with the clinical characterization of this variant. We also observed greater resmetirom efficacy in the PNPLA3 wild-type CC LAMPS compared to the GG variant in multiple MASLD metrics, including steatosis, stellate cell activation, and the secretion of pro-fibrotic markers. In conclusion, our study demonstrates the capability of the LAMPS platform for the development of MASLD precision therapeutics, enrichment of patient cohorts for clinical trials, and optimization of therapeutic strategies for patient subgroups with different clinical traits and disease stages.

Polymorphisms Associated With Metabolic Dysfunction-Associated Steatotic Liver Disease Influence the Progression of End-Stage Liver Disease.

BACKGROUND AND AIMS: Chronic liver injury that results in cirrhosis and end-stage liver disease (ESLD) causes more than 1 million deaths annually worldwide. Although the impact of genetic factors on the severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-related liver disease (ALD) has been previously studied, their contribution to the development of ESLD remains largely unexplored. METHODS: We genotyped 6 MASLD-associated polymorphisms in healthy (n = 123), metabolic dysfunction-associated steatohepatitis (MASH) (n = 145), MASLD-associated ESLD (n = 72), and ALD-associated ESLD (n = 57) cohorts and performed multinomial logistic regression to determine the combined contribution of genetic, demographic, and clinical factors to the progression of ESLD. RESULTS: Distinct sets of factors are associated with the progression to ESLD. The PNPLA3 rs738409:G and TM6SF2 rs58542926:T alleles, body mass index (BMI), age, and female sex were positively associated with progression from a healthy state to MASH. The PNPLA3 rs738409:G allele, age, male sex, and having type 2 diabetes mellitus were positively associated, while BMI was negatively associated with progression from MASH to MASLD-associated ESLD. The PNPLA3 rs738409:G and GCKR rs780094:T alleles, age, and male sex were positively associated, while BMI was negatively associated with progression from a healthy state to ALD-associated ESLD. The findings indicate that the PNPLA3 rs738409:G allele increases susceptibility to ESLD regardless of etiology, the TM6SF2 rs58542926:T allele increases susceptibility to MASH, and the GCKR rs780094:T allele increases susceptibility to ALD-associated ESLD. CONCLUSION: The PNPLA3, TM6SF2, and GCKR minor alleles influence the progression of MASLD-associated or ALD-associated ESLD. Genotyping for these variants in MASLD and ALD patients can enhance risk assessment, prompting early interventions to prevent ESLD.

Human Hepatocellular response in Cholestatic Liver Diseases.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), the most common types of cholestatic liver disease (CLD), result in enterohepatic obstruction, bile acid accumulation, and hepatotoxicity. The mechanisms by which hepatocytes respond to and cope with CLD remain largely unexplored. This study includes the characterization of hepatocytes isolated from explanted livers of patients with PBC and PSC. We examined the expression of hepatocyte-specific genes, intracellular bile acid (BA) levels, and oxidative stress in primary-human-hepatocytes (PHHs) isolated from explanted livers of patients with PBC and PSC and compared them with control normal human hepatocytes. Our findings provide valuable initial insights into the hepatocellular response to cholestasis in CLD and help support the use of PHHs as an experimental tool for these diseases.

Human Induced Pluripotent Stem Cell based Hepatic-Modeling of Lipid metabolism associated TM6SF2 E167K variant.

BACKGROUND AND AIMS: TM6SF2 rs58542926 (E167K) is associated with an increase in the prevalence of Metabolic Disfunction-Associated Steatotic Liver Disease (MASLD). Despite all the investigation related to the role of this variant in lipid metabolism, conflicting results in mouse studies underscore the importance of creating a human model for understanding the TM6SF2 mechanism. Therefore, the aim of this study is to generate a reliable human in vitro model that mimic the effects of the TM6SF2 E167K mutation and can be used for future mechanism studies. APPROACH AND RESULTS: We performed gene editing on human-induced pluripotent stem cells (iPSC) derived from a healthy individual to obtain the cells carrying the TM6SF2 E167K mutation. After hepatic differentiation, a decrease in TM6SF2 protein expression was observed in the mutated-induced hepatocyte. An increase in intracellular lipid droplets and a decrease in the efflux of cholesterol and ApoB100 were also observed. Transcriptomics analysis showed up-regulation of genes related to the transport, flux, and oxidation of lipids, fatty acids, and cholesterol in TM6SF2 E167K cells. Additionally, signs of cellular stress were observed in the ER and mitochondria. CONCLUSIONS: Our findings indicate that induced hepatocytes generated from iPSC carrying the TM6SF2 E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to help in the identification of potential clinical targets and therapies and to understand the mechanism by which the TM6SF2 E167K variant leads to vulnerability to MASLD.

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis.

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial-cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via nonintegrative and safe nucleoside-modified mRNA encapsulated into lipid nanoparticles (mRNA-LNPs) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and elimination of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This work defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals unexpected therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases.

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis.

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via non-integrative and safe nucleoside-modified mRNA encapsulated into lipid-nanoparticles (mRNA-LNP) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals novel therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases. Highlights: Complementary mouse and zebrafish models of liver injury demonstrate the therapeutic impact of VEGFA-KDR axis activation to harness BEC-driven liver regeneration.VEGFA mRNA LNPs restore two key features of the chronic liver disease in humans such as steatosis and fibrosis.Identification in human cirrhotic ESLD livers of KDR-expressing BECs adjacent to clusters of KDR+ hepatocytes suggesting their BEC origin.KDR-expressing BECs may represent facultative adult progenitor cells, a unique BEC population that has yet been uncovered.

Therapeutic treatment with phosphodiesterase-4 inhibitors alleviates kidney injury and renal fibrosis by increasing MMP-9 in a doxorubicin-induced nephrotoxicity mouse model.

Nephrotic syndrome (NS) is associated with kidney dysfunction and is an important cause of morbidity and mortality in industrialized countries. Here, we evaluated the effects of the phosphodiesterase-4 (PDE-4) inhibitors rolipram and roflumilast on a doxorubicin-induced NS model. Early-stage rolipram treatment preserved glomerular filtration barrier function, as indicated by reduced serum protein and albumin loss and the prevention of hypercholesterolemia. These effects were associated with reduced glomerular and tubular lesions and abrogated renal cell apoptosis. In addition, rolipram treatment reduced inflammation, which was characterized by a decrease in macrophage accumulation and reduced levels of CCL2 and TNF in the kidneys. Rolipram also reduced renal fibrosis, which was associated with decreased α-smooth muscle actin (α-SMA) area and increased metalloproteinase 9 (MMP9) activity in renal tissue. Late-stage rolipram or roflumilast treatment preserved glomerular filtration barrier function, as characterized by reduced serum albumin loss, decreased proteinuria, and the prevention of hypercholesterolemia. Importantly, only roflumilast treatment was associated with a reduction in glomerular and tubular lesions at this time point. In addition, both rolipram and roflumilast reduced renal tissue fibrosis and MMP9 activity in renal tissue.

Transmembrane channel activity in human hepatocytes and cholangiocytes derived from induced pluripotent stem cells.

The initial creation of human-induced pluripotent stem cells (iPSCs) set the foundation for the future of regenerative medicine. Human iPSCs can be differentiated into a variety of cell types in order to study normal and pathological molecular mechanisms. Currently, there are well-defined protocols for the differentiation, characterization, and establishment of functionality in human iPSC-derived hepatocytes (iHep) and iPSC-derived cholangiocytes (iCho). Electrophysiological study on chloride ion efflux channel activity in iHep and iCho cells has not been previously reported. We generated iHep and iCho cells and characterized them based on hepatocyte-specific and cholangiocyte-specific markers. The relevant transmembrane channels were selected: cystic fibrosis transmembrane conductance regulator, leucine rich repeat-containing 8 subunit A, and transmembrane member 16 subunit A. To measure the activity in these channels, we used whole-cell patch-clamp techniques with a standard intracellular and extracellular solution. Our iHep and iCho cells demonstrated definitive activity in the selected transmembrane channels, and this approach may become an important tool for investigating human liver biology of cholestatic diseases.

Inositol 1,4,5-trisphosphate receptor type 3 is involved in resistance to apoptosis and maintenance of human hepatocellular carcinoma.

The expression of the inositol 1,4,5-trisphosphate receptor type 3 (ITRP3) in hepatocytes is a common event in the pathogenesis of hepatocellular carcinoma (HCC), regardless of the type of underlying liver disease. However, it is not known whether ITPR3 expression in hepatocytes is involved in tumor maintenance. The aim of the present study was to determine whether there is an association between ITPR3 expression and clinical and morphological parameters using HCC samples obtained from liver explants from patients (n=53) with different etiologies of underlying chronic liver disease (CLD). ITPR3 expression, mitosis and apoptosis were analyzed in human liver samples by immunohistochemistry. Clinical and event-free survival data were combined to assess the relationship between ITPR3 and liver cancer growth in patients. RNA sequencing analysis was performed to identify apoptotic genes altered by ITPR3 expression in a liver tumor cell line. ITPR3 was highly expressed in HCC tumor cells relative to adjacent CLD tissue and healthy livers. There was an inverse correlation between ITPR3 expression and mitotic and apoptotic indices in HCC, suggesting that ITPR3 contributed to the maintenance of HCC by promoting resistance to apoptosis. This was confirmed by the upregulation of CTSB, CHOP and GADD45, genes involved in the apoptotic pathway in HCC. The expression of ITPR3 in the liver may be a promising prognostic marker of HCC.

Biofabrication of synthetic human liver tissue with advanced programmable functions.

Advances in cellular engineering, as well as gene, and cell therapy, may be used to produce human tissues with programmable genetically enhanced functions designed to model and/or treat specific diseases. Fabrication of synthetic human liver tissue with these programmable functions has not been described. By generating human iPSCs with target gene expression controlled by a guide RNA-directed CRISPR-Cas9 synergistic-activation-mediator, we produced synthetic human liver tissues with programmable functions. Such iPSCs were guide-RNA-treated to enhance expression of the clinically relevant CYP3A4 and UGT1A1 genes, and after hepatocyte-directed differentiation, cells demonstrated enhanced functions compared to those found in primary human hepatocytes. We then generated human liver tissue with these synthetic human iPSC-derived hepatocytes (iHeps) and other non-parenchymal cells demonstrating advanced programmable functions. Fabrication of synthetic human liver tissue with modifiable functional genetic programs may be a useful tool for drug discovery, investigating biology, and potentially creating bioengineered organs with specialized functions.

Is HSD17B13 Genetic Variant a Protector for Liver Dysfunction? Future Perspective as a Potential Therapeutic Target.

As diet and lifestyle have changed, fatty liver disease (FLD) has become more and more prevalent. Many genetic risk factors, such as variants of PNPLA3, TM6SF2, GCKR, and MBOAT7, have previously been uncovered via genome wide association studies (GWAS) to be associated with FLD. In 2018, a genetic variant (rs72613567, T > TA) of hydroxysteroid 17-ß dehydrogenase family 13 (HSD17B13) was first associated with a lower risk of developing alcoholic liver disease and non-alcoholic fatty liver disease (NAFLD) in minor allele carriers. Other HSD17B13 variants were also later linked with either lower inflammation scores among NAFLD patients or protection against NAFLD (rs6834314, A > G and rs9992651, G > A) respectively. HSD17B13 is a lipid droplet-associated protein, but its function is still ambiguous. Compared to the other genetic variants that increase risk for FLD, HSD17B13 variants serve a protective role, making this gene a potential therapeutic target. However, the mechanism by which these variants reduce the risk of developing FLD is still unclear. Because studies in cell lines and mouse models have produced conflicting results, human liver tissue modeling using induced pluripotent stem cells may be the best way to move forward and solve this mystery.

Alkaloid and acetogenin-rich fraction from Annona crassiflora fruit peel inhibits proliferation and migration of human liver cancer HepG2 cells.

Plant species from Annonaceae are commonly used in traditional medicine to treat various cancer types. This study aimed to investigate the antiproliferative potential of an alkaloid and acetogenin-rich fraction from the fruit peel of Annona crassiflora in HepG2 cells. A liquid-liquid fractionation was carried out on the ethanol extract of A. crassiflora fruit peel in order to obtain an alkaloid and acetogenin-rich fraction (AF-Ac). Cytotoxicity, proliferation and migration were evaluated in the HepG2 cells, as well as the proliferating cell nuclear antigen (PCNA), vinculin and epidermal growth factor receptor (EGFR) expression. In addition, intracellular Ca2+ was determined using Fluo4-AM and fluorescence microscopy. First, 9 aporphine alkaloids and 4 acetogenins that had not yet been identified in the fruit peel of A. crassiflora were found in AF-Ac. The treatment with 50 µg/mL AF-Ac reduced HepG2 cell viability, proliferation and migration (p < 0.001), which is in accordance with the reduced expression of PCNA and EGFR levels (p < 0.05). Furthermore, AF-Ac increased intracellular Ca2+ in the HepG2 cells, mobilizing intracellular calcium stores, which might be involved in the anti-migration and anti-proliferation capacities of AF-Ac. Our results support the growth-inhibitory potential of AF-Ac on HepG2 cells and suggest that this effect is triggered, at least in part, by PCNA and EGFR modulation and mobilization of intracellular Ca2+. This study showed biological activities not yet described for A. crassiflora fruit peel, which provide new possibilities for further in vivo studies to assess the antitumoral potential of A. crassiflora, especially its fruit peel.