Embryonic stem cells are devoid of macropinocytosis, a trafficking pathway for activin A in differentiated cells.

Ligand-receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the selection of interaction partners by the complex along the trafficking route. We report that, in differentiated cells, activin A-receptor complexes are internalised via clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurs via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route for activin A in differentiated cells, and that MP is not active in hESCs and is induced as cells differentiate.

G6PD and ACSL3 are synthetic lethal partners of NF2 in Schwann cells.

Neurofibromatosis Type II (NFII) is a genetic condition caused by loss of the NF2 gene, resulting in activation of the YAP/TAZ pathway and recurrent Schwann cell tumors, as well as meningiomas and ependymomas. Unfortunately, few pharmacological options are available for NFII. Here, we undertake a genome-wide CRISPR/Cas9 screen to search for synthetic-lethal genes that, when inhibited, cause death of NF2 mutant Schwann cells but not NF2 wildtype cells. We identify ACSL3 and G6PD as two synthetic-lethal partners for NF2, both involved in lipid biogenesis and cellular redox. We find that NF2 mutant Schwann cells are more oxidized than control cells, in part due to reduced expression of genes involved in NADPH generation such as ME1. Since G6PD and ME1 redundantly generate cytosolic NADPH, lack of either one is compatible with cell viability, but not down-regulation of both. Since genetic deficiency for G6PD is tolerated in the human population, G6PD could be a good pharmacological target for NFII.

ERBIN is a new SARA-interacting protein: competition between SARA and SMAD2 and SMAD3 for binding to ERBIN.

SARA, an early endosomal protein, plays a key role in TGFß signalling, as it presents SMAD2 and SMAD3 for phosphorylation by the activated TGFß receptors. Here, we show that ERBIN is a new SARA-interacting protein that can be recruited by SARA to early endosomes. ERBIN was recently shown to bind and segregate phosphorylated SMAD2 and SMAD3 (SMAD2/3) in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. SARA binds to ERBIN using a new domain, which we have called the ERBID (ERBIN-binding domain), whereas ERBIN binds to SARA using a domain (amino acids 1208-1265) that also interacts with SMAD2 and SMAD3, which we have called the SSID (SARA- and SMAD-interacting domain). We additionally show that SARA competes with SMAD2/3 for binding to ERBIN. In agreement, overexpression of SARA or the ERBID peptide reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Taken together, these data suggest that the response of cells to TGFß and activin A can be influenced by the relative concentrations of SARA, ERBIN and SMAD2/3.

Tissue Engineering Using Vascular Organoids From Human Pluripotent Stem Cell Derived Mural Cell Phenotypes.

Diffusion is a limiting factor in regenerating large tissues (100-200 µm) due to reduced nutrient supply and waste removal leading to low viability of the regenerating cells as neovascularization of the implant by the host is a slow process. Thus, generating prevascularized tissue engineered constructs, in which endothelial (ECs) and mural (MCs) cells, such as smooth muscle cells (SMCs), and pericytes (PCs), are preassembled into functional in vitro vessels capable of rapidly connecting to the host vasculature could overcome this obstacle. Toward this purpose, using feeder-free and low serum conditions, we developed a simple, efficient and rapid in vitro approach to induce the differentiation of human pluripotent stem cells-hPSCs (human embryonic stem cells and human induced pluripotent stem cells) to defined SMC populations (contractile and synthetic hPSC-SMCs) by extensively characterizing the cellular phenotype (expression of CD44, CD73, CD105, NG2, PDGFRß, and contractile proteins) and function of hPSC-SMCs. The latter were phenotypically and functionally stable for at least 8 passages, and could stabilize vessel formation and inhibit vessel network regression, when co-cultured with ECs in vitro. Subsequently, using a methylcellulose-based hydrogel system, we generated spheroids consisting of EC/hPSC-SMC (vascular organoids), which were extensively phenotypically characterized. Moreover, the vascular organoids served as focal starting points for the sprouting of capillary-like structures in vitro, whereas their delivery in vivo led to rapid generation of a complex functional vascular network. Finally, we investigated the vascularization potential of these vascular organoids, when embedded in hydrogels composed of defined extracellular components (collagen/fibrinogen/fibronectin) that can be used as scaffolds in tissue engineering applications. In summary, we developed a robust method for the generation of defined SMC phenotypes from hPSCs. Fabrication of vascularized tissue constructs using hPSC-SMC/EC vascular organoids embedded in chemically defined matrices is a significant step forward in tissue engineering and regenerative medicine.

Generation of human induced pluripotent stem cells in defined, feeder-free conditions.

Herein, we describe a modified protocol for the generation of human induced pluripotent stem cells (hiPS) and expansion under defined, serum free and feeder free conditions. These cells exhibit a high level of plasticity towards various differentiation pathways both in vitro and in vivo. Ultimately, hiPS-derived lines achieved high standards of three dimensional differentiations on biomaterial scaffolds and promoted in vivo regeneration of complex organs, such as Anterior Cruciate Ligament (in swine ACL-rupture models) and other tissues as well.

Generation of stem cell-based bioartificial anterior cruciate ligament (ACL) grafts for effective ACL rupture repair.

In the present study, we combined stem cell technology with a non-absorbable biomaterial for the reconstruction of the ruptured ACL. Towards this purpose, multipotential stromal cells derived either from subcutaneous human adipose tissue (hAT-MSCs) or from induced pluripotent stem cells (iPSCs) generated from human foreskin fibroblasts (hiPSC-MSCs) were cultured on the biomaterial for 21days in vitro to generate a 3D bioartifical ACL graft. Stem cell differentiation towards bone and ligament at the ends and central part of the biomaterial was selectively induced using either BMP-2/FGF-2 or TGF-ß/FGF-2 combinations, respectively. The bioartificial ACL graft was subsequently implanted in a swine ACL rupture model in place of the surgically removed normal ACL. Four months post-implantation, the tissue engineered ACL graft generated an ACL-like tissue exhibiting morphological and biochemical characteristics resembling those of normal ACL.

Non-CpG cytosine methylation of p53 exon 5 in non-small cell lung carcinoma.

Non-CpG methylation of cytosine residues, a mechanism associated with regulation of gene expression, has not been investigated in human cancer until now. Analysis of the p53 exon 5 mutation spectrum in mutation databases for lung cancer reveals frequent GC>AT transitions, several of which occur at non-CpG sequences. To investigate the involvement of cytosine methylation in this mutagenesis process, we analyzed the methylation profile of p53 exon 5, in lung carcinoma. In this report, we present evidence that extensive clustered non-CpG methylation is observed in three regions of this exon, namely the sequences spanning codons 156-159, 175-179 and the 3' splice site, as well as in scattered CpA sequences. This methylation pattern was verified using direct methylation sequencing, and a two-stage methylation-specific PCR assay (MSP), designed for the detection of methylation in a GC rich region (oligo C sequence, of codons 175-179) of exon 5. The results from this MSP assay reveal that DNA from cancerous specimens was more heavily methylated in non-CpG cytosines, compared to that from non-cancerous lung tissue of cancer patients (14/19 cancerous and 6/19 non-cancerous, respectively). DNA isolated from human leucocytes and some non-cancerous specimens (2/19) was free of non-CpG methylation. Careful analysis of the mutations reported in p53 mutation databases also provides corroborating evidence that the high incidence of GC>AT mutations in the p53 gene, observed in lung cancer, might also be related to non-CpG methylation, as well as to the overall increase of methylation sites in this locus.

The RhoD to centrosomal duplication.

The main functional roles attributed to the centrosome, the major microtubule organizing center (MTOC) of metazoans, are related to cell locomotion, sensory perception and division. The role of vesicular trafficking in the regulation of the centrosome cycle has been largely unexplored. Recently, however, several studies have indicated the involvement of molecules and/or complexes of the trafficking routes in centrosome positioning, duplication and regulation. Functional screens have revealed communication between the outer nuclear envelope, the Golgi apparatus, the endosomal recycling compartment and centrosomes, while other studies underline the involvement of the ESCRT complex proteins in centrosome function. In this commentary, we discuss our recent study, which shows the involvement of an endosomal Rho protein, namely RhoD, in centrosome duplication and possible links between the centrosome's structural and functional integrity to vesicular trafficking.