Regeneration Defects in Yap and Taz Mutant Mouse Livers Are Caused by Bile Duct Disruption and Cholestasis.

BACKGROUND AND AIMS: The Hippo pathway and its downstream effectors YAP and TAZ (YAP/TAZ) are heralded as important regulators of organ growth and regeneration. However, different studies provided contradictory conclusions about their role during regeneration of different organs, ranging from promoting proliferation to inhibiting it. Here we resolve the function of YAP/TAZ during regeneration of the liver, where Hippo's role in growth control has been studied most intensely. METHODS: We evaluated liver regeneration after carbon tetrachloride toxic liver injury in mice with conditional deletion of Yap/Taz in hepatocytes and/or biliary epithelial cells, and measured the behavior of different cell types during regeneration by histology, RNA sequencing, and flow cytometry. RESULTS: We found that YAP/TAZ were activated in hepatocytes in response to carbon tetrachloride toxic injury. However, their targeted deletion in adult hepatocytes did not noticeably impair liver regeneration. In contrast, Yap/Taz deletion in adult bile ducts caused severe defects and delay in liver regeneration. Mechanistically, we showed that Yap/Taz mutant bile ducts degenerated, causing cholestasis, which stalled the recruitment of phagocytic macrophages and the removal of cellular corpses from injury sites. Elevated bile acids activated pregnane X receptor, which was sufficient to recapitulate the phenotype observed in mutant mice. CONCLUSIONS: Our data show that YAP/TAZ are practically dispensable in hepatocytes for liver development and regeneration. Rather, YAP/TAZ play an indirect role in liver regeneration by preserving bile duct integrity and securing immune cell recruitment and function.

Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury.

BACKGROUND & AIM: Chronic liver diseases are characterized by expansion of the small immature cholangiocytes - a mechanism named ductular reaction (DR) - which have the capacity to differentiate into hepatocytes. We investigated the kinetics of this differentiation, as well as analyzing several important features of the newly formed hepatocytes, such as functional maturity, clonal expansion and resistance to stress in mice with long-term liver damage. METHODS: We tracked cholangiocytes using osteopontin-iCreERT2 and hepatocytes with AAV8-TBG-Cre. Mice received carbon tetrachloride (CCl4) for >24 weeks to induce chronic liver injury. Livers were collected for the analysis of reporter proteins, cell proliferation and death, DNA damage, and nuclear ploidy; hepatocytes were also isolated for RNA sequencing. RESULTS: During liver injury we observed a transient DR and the differentiation of DR cells into hepatocytes as clones that expanded to occupy 12% of the liver parenchyma by week 8. By lineage tracing, we confirmed that these new hepatocytes derived from cholangiocytes but not from native hepatocytes. They had all the features of mature functional hepatocytes. In contrast to the exhausted native hepatocytes, these newly formed hepatocytes had higher proliferative capability, less apoptosis, a lower proportion of highly polyploid nuclei and were better at eliminating DNA damage. CONCLUSIONS: In chronic liver injury, DR cells differentiate into stress-resistant hepatocytes that repopulate the liver. The process might account for the observed parenchymal reconstitution in livers of patients with advanced-stage hepatitis and could be a target for regenerative purposes. LAY SUMMARY: During chronic liver disease, while native hepatocytes are exhausted and genetically unstable, a subset of cholangiocytes clonally expand to differentiate into young, functional and robust hepatocytes. This cholangiocyte cell population is a promising target for regenerative therapies in patients with chronic liver insufficiency.

How Search Engine Data Enhance the Understanding of Determinants of Suicide in India and Inform Prevention: Observational Study.

BACKGROUND: India is home to 20% of the world's suicide deaths. Although statistics regarding suicide in India are distressingly high, data and cultural issues likely contribute to a widespread underreporting of the problem. Social stigma and only recent decriminalization of suicide are among the factors hampering official agencies' collection and reporting of suicide rates. OBJECTIVE: As the product of a data collaborative, this paper leverages private-sector search engine data toward gaining a fuller, more accurate picture of the suicide issue among young people in India. By combining official statistics on suicide with data generated through search queries, this paper seeks to: add an additional layer of information to more accurately represent the magnitude of the problem, determine whether search query data can serve as an effective proxy for factors contributing to suicide that are not represented in traditional datasets, and consider how data collaboratives built on search query data could inform future suicide prevention efforts in India and beyond. METHODS: We combined official statistics on demographic information with data generated through search queries from Bing to gain insight into suicide rates per state in India as reported by the National Crimes Record Bureau of India. We extracted English language queries on "suicide," "depression," "hanging," "pesticide," and "poison". We also collected data on demographic information at the state level in India, including urbanization, growth rate, sex ratio, internet penetration, and population. We modeled the suicide rate per state as a function of the queries on each of the 5 topics considered as linear independent variables. A second model was built by integrating the demographic information as additional linear independent variables. RESULTS: Results of the first model fit (R2) when modeling the suicide rates from the fraction of queries in each of the 5 topics, as well as the fraction of all suicide methods, show a correlation of about 0.5. This increases significantly with the removal of 3 outliers and improves slightly when 5 outliers are removed. Results for the second model fit using both query and demographic data show that for all categories, if no outliers are removed, demographic data can model suicide rates better than query data. However, when 3 outliers are removed, query data about pesticides or poisons improves the model over using demographic data. CONCLUSIONS: In this work, we used search data and demographics to model suicide rates. In this way, search data serve as a proxy for unmeasured (hidden) factors corresponding to suicide rates. Moreover, our procedure for outlier rejection serves to single out states where the suicide rates have substantially different correlations with demographic factors and query rates.

Prospects in non-invasive assessment of liver fibrosis: Liquid biopsy as the future gold standard?

Liver fibrosis is the result of persistent liver injury, and is characterized by sustained scar formation and disruption of the normal liver architecture. The extent of fibrosis is considered as an important prognostic factor for the patient outcome, as an absence of (early) treatment can lead to the development of liver cirrhosis and hepatocellular carcinoma. Till date, the most sensitive and specific way for the diagnosis and staging of liver fibrosis remains liver biopsy, an invasive diagnostic tool, which is associated with high costs and discomfort for the patient. Over time, non-invasive scoring systems have been developed, of which the measurements of serum markers and liver stiffness are validated for use in the clinic. These tools lack however the sensitivity and specificity to detect small changes in the progression or regression of both early and late stages of fibrosis. Novel non-invasive diagnostic markers with the potential to overcome these limitations have been developed, but often lack validation in large patient cohorts. In this review, we will summarize novel trends in non-invasive markers of liver fibrosis development and will discuss their (dis-)advantages for use in the clinic.

Brief Report: The Deletion of the Phosphatase Regulator NIPP1 Causes Progenitor Cell Expansion in the Adult Liver.

The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8(-/-) livers developed a ductular reaction, that is, bile-duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene-expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver-injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8(-/-) livers displayed an increased sensitivity to diet-supplemented 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which also causes bile-duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256-2262.

Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche.

BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in human hepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.

The Hippo pathway effector YAP controls mouse hepatic stellate cell activation.

BACKGROUND & AIMS: Hepatic stellate cell activation is a wound-healing response to liver injury. However, continued activation of stellate cells during chronic liver damage causes excessive matrix deposition and the formation of pathological scar tissue leading to fibrosis and ultimately cirrhosis. The importance of sustained stellate cell activation for this pathological process is well recognized, and several signalling pathways that can promote stellate cell activation have been identified, such as the TGFß-, PDGF-, and LPS-dependent pathways. However, the mechanisms that trigger and drive the early steps in activation are not well understood. METHODS AND RESULTS: We identified the Hippo pathway and its effector YAP as a key pathway that controls stellate cell activation. YAP is a transcriptional co-activator and we found that it drives the earliest changes in gene expression during stellate cell activation. Activation of stellate cells in vivo by CCl4 administration to mice or activation in vitro caused rapid activation of YAP as revealed by its nuclear translocation and by the induction of YAP target genes. YAP was also activated in stellate cells of human fibrotic livers as evidenced by its nuclear localization. Importantly, knockdown of YAP expression or pharmacological inhibition of YAP prevented hepatic stellate cell activation in vitro and pharmacological inhibition of YAP impeded fibrogenesis in mice. CONCLUSIONS: YAP activation is a critical driver of hepatic stellate cell activation and inhibition of YAP presents a novel approach for the treatment of liver fibrosis.

Spatially Self-Organized Three-Dimensional Neural Concentroid as a Novel Reductionist Humanized Model to Study Neurovascular Development.

Although human pluripotent stem cell (PSC)-derived brain organoids have enabled researchers to gain insight into human brain development and disease, these organoids contain solely ectodermal cells and are not vascularized as occurs during brain development. Here it is created less complex and more homogenous large neural constructs starting from PSC-derived neuroprogenitor cells (NPC), by fusing small NPC spheroids into so-called concentroids. Such concentroids consisted of a pro-angiogenic core, containing neuronal and outer radial glia cells, surrounded by an astroglia-dense outer layer. Incorporating PSC-derived endothelial cells (EC) around and/or in the concentroids promoted vascularization, accompanied by differential outgrowth and differentiation of neuronal and astroglia cells, as well as the development of ectodermal-derived pericyte-like mural cells co-localizing with EC networks. Single nucleus transcriptomic analysis revealed an enhanced neural cell subtype maturation and diversity in EC-containing concentroids, which better resemble the fetal human brain compared to classical organoids or NPC-only concentroids. This PSC-derived "vascularized" concentroid brain model will facilitate the study of neurovascular/blood-brain barrier development, neural cell migration, and the development of effective in vitro vascularization strategies of brain mimics.

Human iPSC-derived liver co-culture spheroids to model liver fibrosis.

The lack of adequate humanin vitromodels that recapitulate the cellular composition and response of the human liver to injury hampers the development of anti-fibrotic drugs. The goal of this study was to develop a human spheroid culture model to study liver fibrosis by using induced pluripotent stem cell (iPSC)-derived liver cells. iPSCs were independently differentiated towards hepatoblasts (iHepatoblasts), hepatic stellate cells (iHSCs), endothelial cells (iECs) and macrophages (iMΦ), before assembly into free floating spheroids by culturing cells in 96-well U-bottom plates and orbital shaking for up to 21 days to allow further maturation. Through transcriptome analysis, we show further maturation of iECs and iMΦ, the differentiation of the iHepatoblasts towards hepatocyte-like cells (iHeps) and the inactivation of the iHSCs by the end of the 3D culture. Moreover, these cultures display a similar expression of cell-specific marker genes (CYP3A4, PDGFRß, CD31andCD68) and sensitivity to hepatotoxicity as spheroids made using freshly isolated primary human liver cells. Furthermore, we show the functionality of the iHeps and the iHSCs by mimicking liver fibrosis through iHep-induced iHSC activation, using acetaminophen. In conclusion, we have established a reproducible human iPSC-derived liver culture model that can be used to mimic fibrosisin vitroas a replacement of primary human liver derived 3D models. The model can be used to investigate pathways involved in fibrosis development and to identify new targets for chronic liver disease therapy.

Conditional deletion of CEACAM1 causes hepatic stellate cell activation.

Objectives: Hepatic CEACAM1 expression declines with advanced hepatic fibrosis stage in patients with MASH. Global and hepatocyte-specific deletions of Ceacam1 impair insulin clearance to cause hepatic insulin resistance and steatosis. They also cause hepatic inflammation and fibrosis, a condition characterized by excessive collagen production from activated hepatic stellate cells (HSCs). Given the positive effect of PPARγ on CEACAM1 transcriptoin and on HSCs quiescence, the current studies investigated whether CEACAM1 loss from HSCs causes their activation. Methods: We examined whether lentiviral shRNA-mediated CEACAM1 donwregulation (KD-LX2) activates cultured human LX2 stellate cells. We also generated LratCre+Cc1 fl/fl mutants with conditional Ceacam1 deletion in HSCs and characterized their MASH phenotype. Media transfer experiments were employed to examine whether media from mutant human and murine HSCs activate their wild-type counterparts. Results: LratCre+Cc1 fl/fl mutants displayed hepatic inflammation and fibrosis but without insulin resistance or hepatic steatosis. Their HSCs, like KD-LX2 cells, underwent myofibroblastic transformation and their media activated wild-type HDCs. This was inhibited by nicotinic acid treatment which stemmed the release of IL-6 and fatty acids, both of which activate the epidermal growth factor receptor (EGFR) tyrosine kinase. Gefitinib inhibition of EGFR and its downstream NF-κB/IL-6/STAT3 inflammatory and MAPK-proliferation pathways also blunted HSCs activation in the absence of CEACAM1. Conclusions: Loss of CEACAM1 in HSCs provoked their myofibroblastic transformation in the absence of insulin resistance and hepatic steatosis. This response is mediated by autocrine HSCs activation of the EGFR pathway that amplifies inflammation and proliferation.

Orphan receptor GPR176 in hepatic stellate cells exerts a profibrotic role in chronic liver disease.

Background & Aims: Chronic liver disease (CLD) remains a global health issue associated with a significant disease burden. Liver fibrosis, a hallmark of CLD, is characterised by the activation of hepatic stellate cells (HSCs) that gain profibrotic characteristics including increased production of extracellular matrix protein. Currently, no antifibrotic therapies are available clinically, in part because of the lack of HSC-specific drug targets. Here, we aimed to identify HSC-specific membrane proteins that can serve as targets for antifibrotic drug development. Methods: Small interfering RNA-mediated knockdown of GPR176 was used to assess the in vitro function of GPR176 in HSCs and in precision cut liver slices (PCLS). The in vivo role of GPR176 was assessed using the carbon tetrachloride (CCl4) and common bile duct ligation (BDL) models in wild-type and GPR176 knockout mice. GPR176 in human CLD was assessed by immunohistochemistry of diseased human livers and RNA expression analysis in human primary HSCs and transcriptomic data sets. Results: We identified Gpr176, an orphan G-protein coupled receptor, as an HSC-enriched activation associated gene. In vitro, Gpr176 is strongly induced upon culture-induced and hepatocyte-damage-induced activation of primary HSCs. Knockdown of GPR176 in primary mouse HSCs or PCLS cultures resulted in reduced fibrogenic characteristics. Absence of GPR176 did not influence liver homeostasis, but Gpr176-/- mice developed less severe fibrosis in CCl4 and BDL fibrosis models. In humans, GPR176 expression was correlated with in vitro HSC activation and with fibrosis stage in patients with CLD. Conclusions: GPR176 is a functional protein during liver fibrosis and reducing its activity attenuates fibrogenesis. These results highlight the potential of GPR176 as an HSC-specific antifibrotic candidate to treat CLD. Impact and implications: The lack of effective antifibrotic drugs is partly attributed to the insufficient knowledge about the mechanisms involved in the development of liver fibrosis. We demonstrate that the G-protein coupled receptor GPR176 contributes to fibrosis development. Since GPR176 is specifically expressed on the membrane of activated hepatic stellate cells and is linked with fibrosis progression in humans, it opens new avenues for the development of targeted interventions.

Novel prediction models for genotoxicity based on biomarker genes in human HepaRG™ cells.

Transcriptomics-based biomarkers are promising new approach methodologies (NAMs) to identify molecular events underlying the genotoxic mode of action of chemicals. Previously, we developed the GENOMARK biomarker, consisting of 84 genes selected based on whole genomics DNA microarray profiles of 24 (non-)genotoxic reference chemicals covering different modes of action in metabolically competent human HepaRG™ cells. In the present study, new prediction models for genotoxicity were developed based on an extended reference dataset of 38 chemicals including existing as well as newly generated gene expression data. Both unsupervised and supervised machine learning algorithms were used, but as unsupervised machine learning did not clearly distinguish between groups, the performance of two supervised machine learning algorithms, i.e., support vector machine (SVM) and random forest (RF), was evaluated. More specifically, the predictive accuracy was compared, the sensitivity to outliers for one or more biomarker genes was assessed, and the prediction performance for 10 misleading positive chemicals exposed at their IC10 concentration was determined. In addition, the applicability of both prediction models on a publicly available gene expression dataset, generated with RNA-sequencing, was investigated. Overall, the RF and SVM models were complementary in their classification of chemicals for genotoxicity. To facilitate data analysis, an online application was developed, combining the outcomes of both prediction models. This research demonstrates that the combination of gene expression data with supervised machine learning algorithms can contribute to the ongoing paradigm shift towards a more human-relevant in vitro genotoxicity testing strategy without the use of experimental animals.

Toward a Demand-Driven, Collaborative Data Agenda for Adolescent Mental Health.

PURPOSE: Existing datasets and research in the field of adolescent mental health do not always meet the needs of practitioners, policymakers, and program implementers, particularly in the context of vulnerable populations. Here, we introduce a collaborative, demand-driven methodology for the development of a strategic adolescent mental health research agenda. Ultimately, this agenda aims to guide future data sharing and collection efforts that meet the most pressing data needs of key stakeholders. METHODS: We conducted a rapid literature search to summarize common themes in adolescent mental health research into a "topic map". We then hosted two virtual workshops with a range of international experts to discuss the topic map and identify shared priorities for future collaboration and research. RESULTS: Our topic map identifies 10 major themes in adolescent mental health, organized into system-level, community-level, and individual-level categories. The engagement of cross-sectoral experts resulted in the validation of the mapping exercise, critical insights for refining the topic map, and a collaborative list of priorities for future research. DISCUSSION: This innovative agile methodology enables a focused deliberation with diverse stakeholders and can serve as the starting point for data generation and collaboration practices, both in the field of adolescent mental health and other topics.

Loss of CEACAM1 in endothelial cells causes hepatic fibrosis.

OBJECTIVES: Hepatocytic CEACAM1 plays a critical role in NASH pathogenesis, as bolstered by the development of insulin resistance, visceral obesity, steatohepatitis and fibrosis in mice with global Ceacam1 (Cc1) deletion. In contrast, VECadCre+Cc1fl/fl mice with endothelial loss of Cc1 manifested insulin sensitivity with no visceral obesity despite elevated NF-κB signaling and increased systemic inflammation. We herein investigated whether VECadCre+Cc1fl/fl male mice develop hepatic fibrosis and whether this is mediated by increased production of endothelin1 (ET1), a transcriptional NF-κB target. METHODS: VECadCre+Et1.Cc1fl/fl mice with combined endothelial loss of Cc1/Et1 genes were generated. Histological and immunohistochemical analyses were conducted on their livers and on liver tissue biopsies from adult patients undergoing bariatric surgery or from patients with NASH diagnosis receiving liver transplant. RESULTS: Hepatic fibrosis and inflammatory infiltration developed in VECadCre+Cc1fl/fl liver parenchyma. This was preceded by increased ET1 production and reversed with combined endothelial loss of Et1. Conditioned media from VECadCre+Cc1fl/fl, but not VECadCre+Et1.Cc1fl/fl primary liver endothelial cells activated wild-type hepatic stellate cells; a process inhibited by bosentan, an ETAR/ETBR dual antagonist. Consistently, immunohistochemical analysis of liver biopsies from patients with NASH showed a decline in endothelial CEACAM1 in parallel with increased plasma endothelin1 levels and progression of hepatic fibrosis stage. CONCLUSIONS: The data demonstrated that endothelial CEACAM1 plays a key role in preventing hepatic fibrogenesis by reducing autocrine endothelin1 production.

Essential requirements for the governance and management of data trusts, data repositories, and other data collaborations.

Introduction: Around the world, many organisations are working on ways to increase the use, sharing, and reuse of person-level data for research, evaluation, planning, and innovation while ensuring that data are secure and privacy is protected. As a contribution to broader efforts to improve data governance and management, in 2020 members of our team published 12 minimum specification essential requirements (min specs) to provide practical guidance for organisations establishing or operating data trusts and other forms of data infrastructure. Approach and Aims: We convened an international team, consisting mostly of participants from Canada and the United States of America, to test and refine the original 12 min specs. Twenty-three (23) data-focused organisations and initiatives recorded the various ways they address the min specs. Sub-teams analysed the results, used the findings to make improvements to the min specs, and identified materials to support organisations/initiatives in addressing the min specs. Results: Analyses and discussion led to an updated set of 15 min specs covering five categories: one min spec for Legal, five for Governance, four for Management, two for Data Users, and three for Stakeholder & Public Engagement. Multiple changes were made to make the min specs language more technically complete and precise. The updated set of 15 min specs has been integrated into a Canadian national standard that, to our knowledge, is the first to include requirements for public engagement and Indigenous Data Sovereignty. Conclusions: The testing and refinement of the min specs led to significant additions and improvements. The min specs helped the 23 organisations/initiatives involved in this project communicate and compare how they achieve responsible and trustworthy data governance and management. By extension, the min specs, and the Canadian national standard based on them, are likely to be useful for other data-focused organisations and initiatives.

Identifying and addressing data asymmetries so as to enable (better) science.

As a society, we need to become more sophisticated in assessing and addressing data asymmetries-and their resulting political and economic power inequalities-particularly in the realm of open science, research, and development. This article seeks to start filling the analytical gap regarding data asymmetries globally, with a specific focus on the asymmetrical availability of privately-held data for open science, and a look at current efforts to address these data asymmetries. It provides a taxonomy of asymmetries, as well as both their societal and institutional impacts. Moreover, this contribution outlines a set of solutions that could provide a toolbox for open science practitioners and data demand-side actors that stand to benefit from increased access to data. The concept of data liquidity (and portability) is explored at length in connection with efforts to generate an ecosystem of responsible data exchanges. We also examine how data holders and demand-side actors are experimenting with new and emerging operational models and governance frameworks for purpose-driven, cross-sector data collaboratives that connect previously siloed datasets. Key solutions discussed include professionalizing and re-imagining data steward roles and functions (i.e., individuals or groups who are tasked with managing data and their ethical and responsible reuse within organizations). We present these solutions through case studies on notable efforts to address science data asymmetries. We examine these cases using a repurposable analytical framework that could inform future research. We conclude with recommended actions that could support the creation of an evidence base on work to address data asymmetries and unlock the public value of greater science data liquidity and responsible reuse.

Lipofection of Non-integrative CRISPR/Cas9 Ribonucleoproteins in Male Germline Stem Cells: A Simple and Effective Knockout Tool for Germline Genome Engineering.

Gene editing in male germline stem (GS) cells is a potent tool to study spermatogenesis and to create transgenic mice. Various engineered nucleases already demonstrated the ability to modify the genome of GS cells. However, current systems are limited by technical complexity diminishing application options. To establish an easier method to mediate gene editing, we tested the lipofection of site-specific Cas9:gRNA ribonucleoprotein (RNP) complexes to knockout the enhanced green fluorescent protein (Egfp) in mouse EGFP-GS cells via non-homologous end joining. To monitor whether gene conversion through homology-directed repair events occurred, single-stranded oligodeoxynucleotides were co-lipofected to deliver a Bfp donor sequence. Results showed Egfp knockout in up to 22% of GS cells, which retained their undifferentiated status following transfection, while only less than 0.7% EGFP to BFP conversion was detected in gated GS cells. These data show that CRISPR/Cas9 RNP-based lipofection is a promising system to simply and effectively knock out genes in mouse GS cells. Understanding the genes involved in spermatogenesis could expand therapeutic opportunities for men suffering from infertility.

Improved Precision-Cut Liver Slice Cultures for Testing Drug-Induced Liver Fibrosis.

In vitro models of human liver disease often fail to mimic the complex 3D structures and cellular organizations found in vivo. Precision cut liver slices (PCLS) retain the complex physiological architecture of the native liver and therefore could be an exceptional in vitro liver model. However, the production of PCLS induces a spontaneous culture-induced fibrogenic reaction, limiting the application of PCLS to anti-fibrotic compounds. Our aim was to improve PCLS cultures to allow compound-induced fibrosis induction. Hepatotoxicity in PCLS cultures was analyzed by lactate dehydrogenase leakage and albumin secretion, while fibrogenesis was analyzed by qRT-PCR and western blot for hepatic stellate cell (HSC) activation markers and collagen 6 secretion by enzyme-linked immunosorbent assays (ELISA). We demonstrate that supplementation of 3 mm mouse PCLS cultures with valproate strongly reduces fibrosis and improves cell viability in our PCLS cultures for up to 5 days. Fibrogenesis can still be induced both directly and indirectly through exposure to TGFß and the hepatotoxin acetaminophen, respectively. Finally, human PCLS cultures showed similar but less robust results. In conclusion, we optimized PCLS cultures to allow for drug-induced liver fibrosis modeling.

Inhibition of the Protein Arginine Methyltransferase PRMT5 in High-Risk Multiple Myeloma as a Novel Treatment Approach.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Subsets of patients have high-risk features linked with dismal outcome. Therefore, the need for effective therapeutic options remains high. Here, we used bio-informatic tools to identify novel targets involved in DNA repair and epigenetics and which are associated with high-risk myeloma. The prognostic significance of the target genes was analyzed using publicly available gene expression data of MM patients (TT2/3 and HM cohorts). Hence, protein arginine methyltransferase 5 (PRMT5) was identified as a promising target. Druggability was assessed in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5-inhibitor EPZ015938. EPZ015938 strongly reduced the total symmetric-dimethyl arginine levels in all cell lines and lead to decreased cellular growth, supported by cell line dependent changes in cell cycle distribution. At later time points, apoptosis occurred, as evidenced by increased AnnexinV-positivity and cleavage of PARP and caspases. Transcriptome analysis revealed a role for PRMT5 in regulating alternative splicing, nonsense-mediated decay, DNA repair and PI3K/mTOR-signaling, irrespective of the cell line type. PRMT5 inhibition reduced the expression of upstream DNA repair kinases ATM and ATR, which may in part explain our observation that EPZ015938 and the DNA-alkylating agent, melphalan, have combinatory effects. Of interest, using a low-dose of mTOR-inhibitor, we observed that cell viability was partially rescued from the effects of EPZ015938, indicating a role for mTOR-related pathways in the anti-myeloma activity of EPZ015938. Moreover, PRMT5 was shown to be involved in splicing regulation of MMSET and SLAMF7, known genes of importance in MM disease. As such, we broaden the understanding of the exact role of PRMT5 in MM disease and further underline its use as a possible therapeutic target.