Notch signaling and new therapeutic options in liver disease

Summary Notch signaling is a crucial determinant of cell fate decision during development and disease in several organs. Notch effects are strictly dependent on the cellular context in which it is activated. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis. Recent advances have shed light on Notch as a critical player in liver regeneration and repair, as well as in liver metabolism and inflammation and cancer. Notch signaling is finely regulated at several levels. The complexity of the pathway provides several possible targets for development of therapeutic agents able to inhibit Notch. Recent reports have shown that persistent activation of Notch signaling is associated with liver malignancies, particularly hepatocellular with stem cell features and cholangiocarcinoma. These novel findings suggest that interfering with the aberrant activation of the Notch pathway may have therapeutic relevance. However, further studies are needed to clarify the mechanisms regulating physiologic and pathologic Notch activation in the adult liver, to better understand the mechanistic role(s) of Notch in liver diseases and to develop safe and specific therapeutic agents

Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells.

Funder: Cambridge Hospitals National Institute for Health Research Biomedical Research CenterFunder: Wellcome Trust and Medical Research Council - Cambridge Stem Cell InstituteFunder: European Research CouncilRB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional redundancy of RB family proteins (RB, RBL1 and RBL2) renders it difficult to investigate their roles during early development, especially in human. Here, we address this problem by generating human embryonic stem cells lacking RB family proteins. To achieve this goal, we first introduced frameshift mutations in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then integrated the shRNA-expression cassette to knockdown RB upon tetracycline treatment. The resulting RBL1/2_dKO+RB_iKD cells remain pluripotent and efficiently differentiate into the primary germ layers in vitro even in the absence of the RB family proteins. In contrast, we observed that subsequent differentiation into foregut endoderm was impaired without the expression of RB, RBL1 and RBL2. Thus, it is suggested that RB proteins are dispensable for the maintenance and acquisition of cell identities during early development, but they are essential to generate advanced derivatives after the formation of primary germ layers. These results also indicate that our RBL1/2_dKO+RB_iKD cell lines are useful to depict the detailed molecular roles of RB family proteins in the maintenance and generation of various cell types accessible from human pluripotent stem cells

Transcript levels of representative genes in the pluripotent tWTA and dKOA cells measured by qRT-PCR.

Box-plot elements are same as described in Fig 1. n = 7 from three different experiments. To determine statistical significance between CTRL and TET sample groups from the same time point, Student’s t-test and Mann-Whitney test were performed based on Shapiro-Wilk normality test. No mark: p-value ≥ 0.05, ****: p-value (JPG)</p

RNA-seq analyses of samples from endoderm day 3.

A, Number of DEGs identified in each comparison (FC > 1). B, Gene ontology enrichment analysis on the DEGs detected between tWT CTRL and dKO TET (black box in A). (JPG)</p

RNA-seq analyses of tWT and dKO cells in endoderm differentiation.

A, Number of DEGs identified in each comparison (FC > 1). 349 among 485 DEGs identified in tWT CTRL vs dKO TET were unique in this comparison. B, Gene ontology enrichment analysis on the DEGs detected between tWT CTRL and dKO TET on endoderm day 6 (black box in A). C, Heatmap graphs showing the fold change in the representative gene expression between tWT CTRL and dKO TET cells on endoderm day6. Starred genes were found uniquely in this comparison. D, Genes upregulated or downregulated in dKO TET over tWT CTRL were compared between endoderm day 3 and day6. Limited number of genes were found in both timepoints.</p

Endoderm differentiation of tWT and dKO hESCs.

A, A schematic drawing of the endoderm differentiation. B, Relative expression levels of representative genes during the endoderm differentiation. Box-plot elements are described in Fig 1. n = 7 from three different experiments. To determine statistical significance between CTRL and TET sample groups on the same time point, Student’s t-test and Mann-Whitney test were performed based on Shapiro-Wilk normality test. No mark: p-value ≥ 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001, ****: p-value < 0.0001. C, Immunofluorescent images of tWT and dKO cells on endoderm differentiation day 8. Scale bars represent 100 μm. D, Cell cycle profiling of the tWT and dKO cells during the endoderm differentiation. S and G2/M phases are shown in supplemental figures. NS: p-value ≥ 0.05, *: p-value < 0.05 (Ordinary one-way ANOVA and Kruskal-Wallis test were used after Shapiro-Wilk normality test). n = 3 or 5.</p

Gene expression analyses of the RB family in the wild type H9 hESCs during directed <i>in vitro</i> differentiation.

A, Transcript levels measured by qRT-PCR. Box-plot elements: centre line is median; box limits are upper and lower quartiles; whiskers are minimum and maximum; cross is mean. n = 5 or 6 from two or three different experiments. NS: p-value ≥ 0.05, *: p-value < 0.05, **: p-value < 0.01, ****: p-value < 0.0001 (Ordinary one-way ANOVA and Kruskal-Wallis test were used based on Shapiro-Wilk normality test). B, Western blot analysis of RB, RBL1 and RBL2 proteins. α-TUBLIN was used as a loading control. Star indicates non-specific bands stained with the RBL2 antibody.</p

Analyses of the established tWT and dKO hESCs in the pluripotent stem cell culture condition.

A, Bright field images of tWT and dKO cells with or without tetracycline. Scale bars represent 100 μm. B, Transcript levels of representative genes in the pluripotent tWT and dKO cells measured by qRT-PCR. Box-plot elements are same as described in Fig 1. n = 7 from three different experiments. To determine statistical significance between CTRL and TET sample groups on the same time point, Student’s t-test and Mann-Whitney test were performed based on Shapiro-Wilk normality test. No mark: p-value ≥ 0.05, ****: p-value 4 cells were seeded as single cells and treated immediately with tetracycline. Bars represent means from six samples. Ordinary one-way ANOVA test were used.</p

S2 Fig -

A, Relative expression levels of neural marker genes in neuroectoderm 6 day. NS: p-value ≥ 0.05, ****: p-value Fig 1. n = 7 from three different experiments. To determine statistical significance between CTRL and TET sample groups on the same time point, Student’s t-test and Mann-Whitney test were performed based on Shapiro-Wilk normality test. No mark: p-value ≥ 0.05, *: p-value (JPG)</p

Sequences of the oligonucleotides used in this work.

(XLSX)</p