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Background Numerous studies have demonstrated that NLRP3 is involved in the pathogenesis of inflammatory bowel disease (IBD). Mesenchymal stem cells (MSCs) have been highlighted as new candidates for treating IBD based on their immunomodulatory properties, including regulation of the NLRP3 inflammasome. However, the role of NLRP3 in shaping the immunoregulatory function of MSCs remains unclear. In this study, we demonstrated that NLRP3 influences the therapeutic effect of MSCs on colitis in an inflammasome-independent manner. Methods MSCs were isolated from the femurs of male C57/B6 mice or Nlrp3 KO mice, identified by osteogenic adipogenic differentiation assay and flow cytometry. In vitro, WT and Nlrp3 KO MSCs were treated with LPS, then the supernatant was collected; WT MSCs were stimulated with different concentrations of NLRP3-specific inhibitor MCC950, and the level of IL-10 was detected in the supernatant; Detect gene expression levels by RNA sequencing; Use Seahorse to detect cellular oxidative phosphorylation (OXPHOS) and glycolysis levels; The expression of glucose transporter 1 (Glut1) in MSCs was detected by Western blot; WT MSCs were stimulated with different concentrations of Glut1 inhibitor BAY876, and the level of IL-10 was detected. In vivo, DSS-induced colitis was treated with WT or Nlrp3 KO MSCs, body weight changes were monitored daily, measure colon length and collect colons for further evaluation; Then DSS-induced colitis was treated with pretreated MSCs, such as BAY876 pretreated WT MSCs or Glut1-overexpressed Nlrp3 KO MSCs (OE-Glut1), then the experimental operation is as above. Results Nlrp3 knockout did not alter MSC phenotypes, but promoted osteogenic differentiation. Further investigation indicated that deletion of Nlrp3reduced the production of the IL-10 in MSCs, leading to an impaired protective effect on DSS-induced colitis. Mechanistically, the deficiency of Nlrp3 downregulated Glut1 expression and glycolysis activation in MSCs, which reduced IL-10 production. Subsequently, overexpressing Glut1 in Nlrp3 KO MSCs recovered their therapeutic effect dampened due to Nlrp3 deletion. Conclusions These results improve the current understanding of the molecular mechanisms underlying the therapeutic effectiveness of MSCs and provide a basis for optimizing MSC-based therapeutic strategies for immune-mediated diseases.

Background & Aims Liver fibrosis is a wound healing response that arises from various aetiologies. The intermediate filament protein Nestin has been reported to participate in maintaining tissue homeostasis during wound healing responses. However, little is known about the role Nestin plays in liver fibrosis. This study investigated the function and precise regulatory network of Nestin during liver fibrosis. Methods Nestin expression was assessed via immunostaining and quantitative real-time PCR (qPCR) in fibrotic/cirrhotic samples. The induction of Nestin expression by transforming growth factor beta (TGFβ)-Smad2/3 signalling was investigated through luciferase reporter assays. The functional role of Nestin in hepatic stellate cells (HSCs) was investigated by examining the pathway activity of profibrogenic TGFβ-Smad2/3 signalling and degradation of TGFβ receptor I (TβRI) after interfering with Nestin. The in vivo effects of knocking down Nestin were examined with an adeno-associated virus vector (serotype 6, AAV6) carrying short-hairpin RNA targeting Nestin in fibrotic mouse models. Results Nestin was mainly expressed in activated HSCs and increased with the progression of liver fibrosis. The profibrogenic pathway TGFβ-Smad2/3 induced Nestin expression directly. Knocking down Nestin promoted caveolin 1-mediated TβRI degradation, resulting in TGFβ-Smad2/3 pathway impairment and reduced fibrosis marker expression in HSCs. In AAV6-treated murine fibrotic models, knocking down Nestin resulted in decreased levels of inflammatory infiltration, hepatocellular damage, and a reduced degree of fibrosis. Conclusion The expression of Nestin in HSCs was induced by TGFβ and positively correlated with the degree of liver fibrosis. Knockdown of Nestin decreased activation of the TGFβ pathway and alleviated liver fibrosis both in vitro and in vivo. Our data demonstrate a novel role of Nestin in controlling HSC activation in liver fibrosis. Lay summary Liver fibrosis has various aetiologies but represents a common process in chronic liver diseases that is associated with high morbidity and mortality. Herein, we demonstrate that the intermediate filament protein Nestin plays an essential profibrogenic role in liver fibrosis by forming a positive feedback loop with the TGFβ-Smad2/3 pathway, providing a potential therapeutic target for the treatment of liver fibrosis.

BackgroundEmerging evidence indicates that the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) axis plays a pivotal role in intrinsic antitumor immunity. Previous studies demonstrate that the conventional chemotherapy agent, teniposide, effectively promotes the therapeutic efficacy of programmed cell death protein-1 antibody (PD-1 Ab) through robust cGAS-STING activation. Unfortunately, the cGAS expression of tumor cells is reported to be severely suppressed by the hypoxic status in solid tumor. Clinically, enhancing chemotherapy-induced, DNA-activated tumor STING signaling by alleviating tumor hypoxia might be one possible direction for improving the currently poor response rates of patients with hepatocellular carcinoma (HCC) to PD-1 Ab.MethodsTeniposide was first screened out from several chemotherapy drugs according to their potency in inducing cGAS-STING signaling in human HCC cells. Teniposide-treated HCC cells were then cultured under hypoxia, normoxia or reoxygenation condition to detect change in cGAS-STING signaling. Next, oxaliplatin/teniposide chemotherapy alone or combined with hyperbaric oxygen (HBO) therapy was administered on liver orthotopic mouse tumor models, after which the tumor microenvironment (TME) was surveyed. Lastly, teniposide alone or combined with HBO was performed on multiple mouse tumor models and the subsequent anti-PD-1 therapeutic responses were observed.ResultsCompared with the first-line oxaliplatin chemotherapy, teniposide chemotherapy induced stronger cGAS-STING signaling in human HCC cells. Teniposide-induced cGAS-STING activation was significantly inhibited by hypoxia inducible factor 1α in an oxygen-deficient environment in vitro and the inhibition was rapidly removed via effective reoxygenation. HBO remarkably enhanced the cGAS-STING-dependent tumor type Ⅰ interferon and nuclear factor kappa-B signaling induced by teniposide in vivo, both of which contributed to the activation of dendritic cells and subsequent cytotoxic T cells. Combined HBO with teniposide chemotherapy improved the therapeutic effect of PD-1 Ab in multiple tumor models.ConclusionsBy combination of two therapies approved by the Food and Drug Administration, we safely stimulated an immunogenic, T cell-inflamed HCC TME, leading to further sensitization of tumors to anti-PD-1 immunotherapy. These findings might enrich therapeutic strategies for advanced HCC andwe can attempt to improve the response rates of patients with HCC to PD-1 Ab by enhancing DNA-activated STING signaling through effective tumor reoxygenation.

Type I interferon pathway is a crucial component of innate immune signaling upon pathogen infection or endogenous instability. An imbalance of type I interferon can lead to many diseases, such as autoimmune diseases and inflammatory diseases. Meanwhile, the side effects of clinical drugs on type I interferon signaling may result in impaired outcomes in clinical treatment, especially in cancer immunotherapy which is associated with type I interferon signaling. Here, we found that sorafenib, an FDA-approved drug for HCC chemotherapy, suppresses both DNA- and RNA-sensing mediated type I interferon pathway. Mechanistically, sorafenib treatment induces the autophagic degradation of MAVS, cGAS, TBK1, and IRF3, and attenuates the signaling transduction. In addition, sorafenib also inhibits the recruiting of STING or MAVS with TBK1 and IRF3. This work reveals the negative role of sorafenib in the regulation of type I interferon pathway. Sorafenib treatment is not only a potential drug for autoimmune disease and inflammation diseases, but also needs to be noticed in HCC chemotherapy.

Despite their small numbers, cancer stem cells play a central role in driving cancer cell growth, chemotherapeutic resistance, and distal metastasis. Previous studies mainly focused on how DNA or histone modification determines cell fate in cancer. However, it is still largely unknown how RNA modifications orchestrate cancer cell fate decisions. More than 170 distinct RNA modifications have been identified in the RNA world, while only a few RNA base modifications have been found in mRNA. Growing evidence indicates that three mRNA modifications, inosine, 5-methylcytosine, and N6-methyladenosine, are essential for the regulation of spatiotemporal gene expression during cancer stem cell fate transition. Furthermore, transcriptome-wide mapping has found that the aberrant deposition of mRNA modification, which can disrupt the gene regulatory network and lead to uncontrollable cancer cell growth, is widespread across different cancers. In this review, we try to summarize the recent advances of these three mRNA modifications in maintaining the stemness of cancer stem cells and discuss the underlying molecular mechanisms, which will shed light on the development of novel therapeutic approaches for eradicating cancer stem cells.

The selective macroautophagy/autophagy pathway is an important pathway of protein degradation, regulating signal transduction pathways via selective degradation of certain signaling complexes. TBK1 functions as a key protein in innate immunity or metabolic-associated fatty liver disease (MAFLD); however, the degradation of TBK1 has not been fully investigated. Recently, we have found that HNF1A functions as a novel cargo receptor to bridge TBK1 and MAP1LC3/LC3, hence promoting the degradation of TBK1 and regulating antiviral innate immunity and MAFLD.

MALAT1-associated small cytoplasmic RNA (mascRNA) is a cytoplasmic tRNA-like small RNA derived from nucleus-located long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). While MALAT1 was extensively studied and was found to function in multiple cellular processes, including tumorigenesis and tumor progression, the role of mascRNA was largely unknown. Here we show that mascRNA is upregulated in multiple cancer cell lines and hepatocellular carcinoma (HCC) clinical samples. Using HCC cells as model, we found that mascRNA and its parent lncRNA MALAT1 can both promote cell proliferation, migration, and invasion in vitro. Correspondingly, both of them can enhance the tumor growth in mice subcutaneous tumor model and can promote metastasis by tail intravenous injection of HCC cells. Furthermore, we revealed that mascRNA and MALAT1 can both activate ERK/MAPK signaling pathway, which regulates metastasis-related genes and may contribute to the aggressive phenotype of HCC cells. Our results indicate a coordination in function and mechanism of mascRNA and MALAT1 during development and progress of HCC, and provide a paradigm for deciphering tRNA-like structures and their parent transcripts in mammalian cells.

Hepatic ischemia-reperfusion injury (IRI) is the most common cause of liver damage leading to surgical failures in hepatectomy and liver transplantation. Extensive inflammatory reactions and oxidative responses are reported to be the major processes exacerbating IRI. The involvement of Yes-associated protein (YAP) in either process has been suggested, but the role and mechanism of YAP in IRI remain unclear. In this study, we constructed hepatocyte-specific YAP knockout (YAP-HKO) mice and induced a hepatic IRI model. Surprisingly, the amount of serum EVs decreased in YAP-HKO compared to WT mice during hepatic IRI. Then, we found that the activation of YAP increased EV secretion through F-actin by increasing membrane formation, while inhibiting the fusion of multivesicular body (MVB) and lysosomes in hepatocytes. Further, to explore the essential elements of YAP-induced EVs, we applied mass spectrometry and noticed CD47 was among the top targets highly expressed on hepatocyte-derived EVs. Thus, we enriched CD47+ EVs by microbeads and applied the isolated CD47+ EVs on IRI mice. We found ameliorated IRI symptoms after CD47+ EV treatment in these mice, and CD47+ EVs bound to CD172α on the surface of dendritic cells (DCs), which inhibited DC activation and the cascade of inflammatory responses. Our data showed that CD47-enriched EVs were released in a YAP-dependent manner by hepatocytes, which could inhibit DC activation and contribute to the amelioration of hepatic IRI. CD47+ EVs could be a potential strategy for treating hepatic IRI.

Lipid accumulation often leads to lipotoxic injuries to hepatocytes, which can cause nonalcoholic steatohepatitis. The association of inflammation with lipid accumulation in liver tissue has been studied for decades; however, key mechanisms have been identified only recently. In particular, it is still unknown how hepatic inflammation regulates lipid metabolism in hepatocytes. Herein, we found that PA treatment or direct stimulation of STING1 promoted, whereas STING1 deficiency impaired, MTORC1 activation, suggesting that STING1 is involved in PA-induced MTORC1 activation. Mechanistic studies revealed that STING1 interacted with several components of the MTORC1 complex and played an important role in the complex formation of MTORC1 under PA treatment. The involvement of STING1 in MTORC1 activation was dependent on SQSTM1, a key regulator of the MTORC1 pathway. In SQSTM1-deficient cells, the interaction of STING1 with the components of MTORC1 was weak. Furthermore, the impaired activity of MTORC1 via rapamycin treatment or STING1 deficiency decreased the numbers of LDs in cells. PA treatment inhibited lipophagy, which was not observed in STING1-deficient cells or rapamycin-treated cells. Restoration of MTORC1 activity via treatment with amino acids blocked lipophagy and LDs degradation. Finally, increased MTORC1 activation concomitant with STING1 activation was observed in liver tissues of nonalcoholic fatty liver disease patients, which provided clinical evidence for the involvement of STING1 in MTORC1 activation. In summary, we identified a novel regulatory loop of STING1-MTORC1 and explain how hepatic inflammation regulates lipid accumulation. Our findings may facilitate the development of new strategies for clinical treatment of hepatic steatosis. Abbreviations: AA: amino acid; ACTB: actin beta; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; DEPTOR: DEP domain containing MTOR interacting protein; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FFAs: free fatty acids; GFP: green fluorescent protein; HFD: high-fat diet; HT-DNA: herring testis DNA; IL1B: interleukin 1 beta; LAMP1: lysosomal associated membrane protein 1; LDs: lipid droplets; MAP1LC3: microtubule associated protein 1 light chain 3; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MLST8: MTOR associated protein, LST8 homolog; MT-ND1: mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 1; mtDNA: mitochondrial DNA; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NAFL: nonalcoholic fatty liver; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NPCs: non-parenchymal cells; PA: palmitic acid; PLIN2: perilipin 2; RD: regular diet; RELA: RELA proto-oncogene, NF-kB subunit; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RPTOR: regulatory associated protein of MTOR complex 1; RRAGA: Ras related GTP binding A; RRAGC: Ras related GTP binding C; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TGs: triglycerides; TREX1: three prime repair exonuclease 1.

Type I interferons (IFNs) play a central role in host defense against viral infection. Multiple posttranslational modifications including ubiquitination and deubiquitination regulate the function of diverse molecules in type I IFN signaling. Many ubiquitin ligase enzymes, such as those of the TRAF and TRIM families, have been shown to participate in the production of type I IFNs and inflammatory cytokines. However, the function of deubiquitinating enzymes (DUBs), a protein family that counteracts the action of protein ubiquitination, on the regulation of antiviral immune responses is not well understood. In this study, we used the broad-spectrum DUB inhibitor G5 to reveal their function in antiviral signaling, and then systematically analyzed mRNA expression of the DUB genes upon poly (I:C) treatment in THP-1 cells. Based on this analysis, we cloned some DUB genes whose expression changed and determined their function in antiviral signaling. Taken together, we present a comprehensive DUB gene expression analysis in THP-1 cells, and suggest the involvement of this family of proteins in the regulation of host antiviral activities.

Somatic cells can be directly reprogrammed into other cell lineages, which holds great promise for regenerative medicine. However, low efficiency and obscure mechanism hinder the application of direct reprogramming. Here, we show that overexpressing the hepatic-specific transcription factors (TFs) HNF1α, FOXA3, and GATA4 was sufficient to convert human urinary epithelial cells (hUCs) into induced hepatocyte-like cells (iHeps). The obtained iHeps were confirmed to express various hepatocyte-specific genes with multiple mature hepatocyte functions. Moreover, autophagy-related genes P62, ULK1, BECN1, VPS34, and LC3B were upregulated in the early stage of reprogramming and knockout of P62 and BECN1 in hUCs with CRISPR/Cas9 technology increased the efficiency of direct reprogramming. Collectively, we established a non-invasive approach to convert hUCs into iHeps and provided a glimpse into the role of autophagy in this process.

Liver fibrosis is a wound healing response that arises from various aetiologies. The intermediate filament protein Nestin has been reported to participate in maintaining tissue homeostasis during wound healing responses. However, little is known about the role Nestin plays in liver fibrosis. This study investigated the function and precise regulatory network of Nestin during liver fibrosis.Nestin expression was assessed via immunostaining and quantitative real-time PCR (qPCR) in fibrotic/cirrhotic samples. The induction of Nestin expression by transforming growth factor beta (TGFβ)-Smad2/3 signalling was investigated through luciferase reporter assays. The functional role of Nestin in hepatic stellate cells (HSCs) was investigated by examining the pathway activity of profibrogenic TGFβ-Smad2/3 signalling and degradation of TGFβ receptor I (TβRI) after interfering with Nestin. The in vivo effects of knocking down Nestin were examined with an adeno-associated virus vector (serotype 6, AAV6) carrying short-hairpin RNA targeting Nestin in fibrotic mouse models.Nestin was mainly expressed in activated HSCs and increased with the progression of liver fibrosis. The profibrogenic pathway TGFβ-Smad2/3 induced Nestin expression directly. Knocking down Nestin promoted caveolin 1-mediated TβRI degradation, resulting in TGFβ-Smad2/3 pathway impairment and reduced fibrosis marker expression in HSCs. In AAV6-treated murine fibrotic models, knocking down Nestin resulted in decreased levels of inflammatory infiltration, hepatocellular damage, and a reduced degree of fibrosis.The expression of Nestin in HSCs was induced by TGFβ and positively correlated with the degree of liver fibrosis. Knockdown of Nestin decreased activation of the TGFβ pathway and alleviated liver fibrosis both in vitro and in vivo. Our data demonstrate a novel role of Nestin in controlling HSC activation in liver fibrosis.Liver fibrosis has various aetiologies but represents a common process in chronic liver diseases that is associated with high morbidity and mortality. Herein, we demonstrate that the intermediate filament protein Nestin plays an essential profibrogenic role in liver fibrosis by forming a positive feedback loop with the TGFβ-Smad2/3 pathway, providing a potential therapeutic target for the treatment of liver fibrosis.

Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver damage during liver transplantation and hepatectomy, and its amelioration is an urgent unmet clinical need. Excess inflammatory response and oxidative stress contributes to the injury and Yes-associated protein (YAP) in the Hippo signaling pathway has been proved participating in it. In this study, using hepatocyte-specific YAP knockout (YAP-HKO) by crossing Yapflox/flox mice with Alb-Cre mice, we surprisingly noticed in the serum of these mice, the amount of serum extracellular vesicles (EVs) decreased after IRI. Then we found that YAP from hepatocytes regulate EVs secretion through F-actin by increasing membrane formation, as well as inhibiting the fusion of multivesicular body (MVB) and lysosomes. We further explored the essential element of YAP-induced EVs by mass spectrometry, and noticed CD47 was one of the target that highly expressed on hepatocyte-derived EVs in YAP-dependent manner. In addition, we found that CD47+ EVs can inhibit dendritic cells (DCs) activation and inflammatory responses via binding to CD172α on the surface of DCs. Finally, we isolated CD47+ EVs in IRI model and found IRI being ameliorated after treatment, suggesting that CD47+ EVs could be a potential therapeutic strategy for the treating hepatic IRI. Our data showed that CD47-enriched EVs were released in a YAP dependent manner by hepatocytes, which could inhibit DCs activation and contribute to the amelioration of hepatic IRI. Funding Statement: National Key RD the China Postdoctoral Science Foundation, Grant/Award Number: 2020M672980; the National 13th Five-Year Science and Technology Plan Major Projects of China, Grant/Award Number: 2017ZX10203205-006-001; National Natural Science Foundation of China, Grant/Award Number: 81702393, 81770648, 81670601, 81570593, 81802897, 81802897, 81900886; Key Scientific and Technological Projects of Guangdong Province, China, Grant/Award Number: 2015B020226004, 2015A030312013, 2017A030311034; Guangdong Natural Science Foundation, China, Grant/Award Number: 2017A030310373; Science and Technology Planning Project of Guangdong Province, China, Grant/Award Number:2017B030314027, 2017B020209004, 20169013, 2017A020215178; Science and Technology Planning Project of Guangzhou, China, Grant/Award Number: 2014Y2-00200, 2014Y2-00544, 201607010024, 201604020001, 201508020262, 201400000001-3 2017. Declaration of Interests: The authors have declared no conflict of interest. Ethics Approval Statement: The study protocol was approved by the Clinical Ethics Review Board of the Third Affiliated Hospital of Sun Yat- sen University. Informed consent was obtained according to the Declaration of Helsinki. All animal studies were approved and monitored by the animal experimental ethics committee of the Third Affiliated hospital of Sun-Yat Sen University.

Wogonin (5,7-dihydroxy-8-methoxyflavone) is an ingredient of the extracts from Scutellaria baicalensis, which has documented a wide spectrum of anti-inflammatory and antitumor activities, including inhibiting regulatory T cells, regulating effector T cell functions, and mediating macrophage immunity. However, the potential effect of Wogonin on B cells has not been fully understood. Here, our results showed that Wogonin inhibited IL-10 secretion in B cells. When purified B cells were activated by lipopolysaccharide (LPS) in vitro, the amount of IL-10 production in supernatant was decreased by Wogonin significantly. The protective role of B cells on dextran sulfate sodium- (DSS-) induced colitis was alleviated after exposure to Wogonin. Furthermore, administration of Wogonin on LPS-treated B cells suppressed phosphorylation of STAT3 and ERK, but not AKT. Interestingly, among those IL-10 signaling-associated transcription factors, mRNA and protein levels of Hif-1α were specifically decreased by Wogonin. Overall, our study indicates that Wogonin suppresses potentially IL-10 production in B cells via inhibition of the STAT3 and ERK signaling pathway as well as inhibition of mRNA and protein levels of the transcription factor Hif-1α. These results provide novel and potential molecular targets of Wogonin in B cells and help us further understand its mechanism of action, which could potentially improve its clinical application in the future.

α-Mangostin, a xanthone derivative from the pericarp of mangosteen, exerts both anti-proliferative and cytotoxic effects on various cancers. However, the effect of α-mangostin on hepatocellular carcinoma (HCC) cells has not yet been fully determined. Therefore, the present study investigated the anti-proliferative effect of α-mangostin on HCC cells. α-Mangostin inhibited the proliferation of HCC and malignantly transformed hepatic cells. In addition, α-mangostin induced G0/G1 phase cell cycle arrest. Furthermore, α-mangostin also reversed doxorubicin-resistance in the HCC cells induced by IL-6. Moreover, the percentage of CD133, CD44 and EpCAM positive stem-like HCC cells treated with α-mangostin was reduced. The expression of key stemness transcription factors, such as Bmi-1, c-Myc and Nanog, were also suppressed following α-mangostin treatment. In conclusion, α-mangostin has been shown to have anti-proliferative effects in HCC cells and malignantly transformed hepatic cells. This work discovered the potential effect of α-mangostin in HCC prevention and treatment.

Summary Activation of Wnt/β-catenin signaling can induce both self-renewal and differentiation in naive pluripotent embryonic stem cells (ESCs). To gain insights into the mechanism by which Wnt/β-catenin regulates ESC fate, we screened and characterized its downstream targets. Here, we show that the self-renewal-promoting effect of Wnt/β-catenin signaling is mainly mediated by two of its downstream targets, Klf2 and Tfcp2l1. Forced expression of Klf2 and Tfcp2l1 can not only induce reprogramming of primed state pluripotency into naive state ESCs, but also is sufficient to maintain the naive pluripotent state of ESCs. Conversely, downregulation of Klf2 and Tfcp2l1 impairs ESC self-renewal mediated by Wnt/β-catenin signaling. Our study therefore establishes the pivotal role of Klf2 and Tfcp2l1 in mediating ESC self-renewal promoted by Wnt/β-catenin signaling., Graphical Abstract, Highlights • Klf2 and Tfcp2l1 are downstream targets of Wnt/β-catenin • Klf2 and Tfcp2l1 overexpression maintains mESC self-renewal • Downregulation of Klf2 and Tfcp2l1 impairs mESC self-renewal mediated by 2i • KLF2 and TFCP2L1 promote reprogramming of EpiSCs to naive ESCs, Wnt/β-catenin signaling can promote self-renewal as well as differentiation of mouse embryonic stem cells (mESCs). Ying and colleagues show that Klf2 and Tfcp2l1, two downstream targets of Wnt/β-catenin, play an essential role in mediating mESC self-renewal promoted by Wnt/β-catenin signaling.

Table S1. Small molecules used for hepatic differentiation. Table S2. Primers used to amplify the transcripts during real-time quantitative PCR. Table S3. Antibodies used for detection. (DOCX 21 kb)

Background The advent of human-induced pluripotent stem cells holds great promise for producing ample individualized hepatocytes. Although previous efforts have succeeded in generating hepatocytes from human pluripotent stem cells in vitro by viral-based expression of transcription factors and/or addition of growth factors during the differentiation process, the safety issue of viral transduction and high cost of cytokines would hinder the downstream applications. Recently, the use of small molecules has emerged as a powerful tool to induce cell fate transition for their superior stability, safety, cell permeability, and cost-effectiveness. Methods In the present study, we established a novel efficient hepatocyte differentiation strategy of human pluripotent stem cells with pure small-molecule cocktails. This method induced hepatocyte differentiation in a stepwise manner, including definitive endoderm differentiation, hepatic specification, and hepatocyte maturation within only 13 days. Results The differentiated hepatic-like cells were morphologically similar to hepatocytes derived from growth factor-based methods and primary hepatocytes. These cells not only expressed specific hepatic markers at the transcriptional and protein levels, but also possessed main liver functions such as albumin production, glycogen storage, cytochrome P450 activity, and indocyanine green uptake and release. Conclusions Highly efficient and expedited hepatic differentiation from human pluripotent stem cells could be achieved by our present novel, pure, small-molecule cocktails strategy, which provides a cost-effective platform for in vitro studies of the molecular mechanisms of human liver development and holds significant potential for future clinical applications. Electronic supplementary material The online version of this article (10.1186/s13287-018-0794-4) contains supplementary material, which is available to authorized users.

// Jia-Jie Chen 1,3,4,* , Nan Cai 1,3,* , Guan-Zhong Chen 1,2,* , Chang-Chang Jia 1,3 , Dong-Bo Qiu 2,3 , Cong Du 2,3 , Wei Liu 1,2 , Yang Yang 1 , Zi-Jie Long 4 and Qi Zhang 1,2,3 1 Organ Transplantation Center, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, People’s Republic of China 2 Guangdong Provincial Key Laboratory of Liver Disease Research, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, People’s Republic of China 3 Vaccine Research Institute of Sun Yat-Sen University, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, People’s Republic of China 4 Department of Hematology, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Qi Zhang, email: // Zi-Jie Long, email: // Keywords : pimozide, hepatic cancer stem-like cells, self-renewal, STAT3 signaling, STAT3 inhibitor Received : February 11, 2015 Accepted : May 13, 2015 Published : May 27, 2015 Abstract Drug repurposing is currently an important approach for accelerating drug discovery and development for clinical use. Hepatocellular carcinoma (HCC) presents drug resistance to chemotherapy, and the prognosis is poor due to the existence of liver cancer stem-like cells. In this study, we investigated the effect of the neuroleptic agent pimozide to inhibit stem-like cell maintenance and tumorigenicity in HCC. Our results showed that pimozide functioned as an anti-cancer drug in HCC cells or stem-like cells. Pimozide inhibited cell proliferation and sphere formation capacities in HCC cells by inducing G0/G1 phase cell cycle arrest, as well as inhibited HCC cell migration. Surprisingly, pimozide inhibited the maintenance and tumorigenicity of HCC stem-like cells, particularly the side population (SP) or CD133-positive cells, as evaluated by colony formation, sphere formation and transwell migration assays. Furthermore, pimozide was found to suppress STAT3 activity in HCC cells by attenuating STAT3-dependent luciferase activity and down-regulating the transcription levels of downstream genes of STAT3 signaling. Moreover, pimozide reversed the stem-like cell tumorigenic phenotypes induced by IL-6 treatment in HCC cells. Further, the antitumor effect of pimozide was also proved in the nude mice HCC xenograft model. In short, the anti-psychotic agent pimozide may act as a novel potential anti-tumor agent in treating advanced HCC.