There are few in vitro 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 re-seeded 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 ELISA. Levels of gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) 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) vs 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 vs 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 GGT and ALP 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 non-alcoholic 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.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.

Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BRISC and BRCA1-A complex member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of the mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide; nevertheless, current therapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms in HCC biology could yield important insights for the intervention of novel therapies. Recently, various studies have reported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19; however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disrupted HCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19 yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulation of CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the 3′ untranslated region (3′ UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), and the dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107 activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves as a molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this study demonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppression as a novel approach for HCC treatment.

Chronic liver injury induces the activation of hepatic stellate cells (HSCs) into myofibroblasts, which produce excessive amounts of extracellular matrix (ECM), resulting in tissue fibrosis. If the injury persists, these fibrous scars could be permanent and disrupt liver architecture and function. Currently, effective anti-fibrotic therapies are lacking; hence, understanding molecular mechanisms that control HSC activation could hold a key to the development of new treatments. Recently, emerging studies have revealed roles of circular RNAs (circRNAs), a class of non-coding RNAs that was initially assumed to be the result of splicing errors, as new regulators in HSC activation. These circRNAs can modulate the activity of microRNAs (miRNAs) and their interacting protein partners involved in regulating fibrogenic signaling cascades. In this review, we will summarize the current knowledge of this class of non-coding RNAs for their molecular function in HSC activation and liver fibrosis progression.