Polycomb group RING finger protein 5 influences several developmental signaling pathways during the in vitro differentiation of mouse embryonic stem cells.

Polycomb group (PcG) RING finger protein 5 (PCGF5) is a core component of the so-called Polycomb repressive complex 1.5 (PRC1.5), which is involved in epigenetic transcriptional repression. To explore the developmental function of Pcgf5, we generated Pcgf5 knockout (Pcgf5-/- ) mouse embryonic stem cell (mESC) lines with the help of CRISPR/Cas9 technology. We subjected the Pcgf5-/- and wild-type (WT) mESCs to a differentiation protocol toward mesodermal-cardiac cell types as aggregated embryoid bodies (EBs) and we found that knockout of Pcgf5 delayed the generation of the three germ layers, especially the ectoderm. Further, disruption of Pcgf5 impacted the epithelial-mesenchymal transition during EB morphogenesis and differentially affected the gene expression of essential developmental signaling pathways such as Nodal and Wnt. Finally, we also unveiled that loss of Pcgf5 induced the repression of genes involved in the Notch pathway, which may explain the enhancement of cardiomyocyte maturation and the dampening of ectodermal-neural differentiation observed in the Pcgf5-/- EBs.

Natural Histogel-Based Bio-Scaffolds for Sustaining Angiogenesis in Beige Adipose Tissue.

In the treatment of obesity and its related disorders, one of the measures adopted is weight reduction by controlling nutrition and increasing physical activity. A valid alternative to restore the physiological function of the human body could be the increase of energy consumption by inducing the browning of adipose tissue. To this purpose, we tested the ability of Histogel, a natural mixture of glycosaminoglycans isolated from animal Wharton jelly, to sustain the differentiation of adipose derived mesenchymal cells (ADSCs) into brown-like cells expressing UCP-1. Differentiated cells show a higher energy metabolism compared to undifferentiated mesenchymal cells. Furthermore, Histogel acts as a pro-angiogenic matrix, induces endothelial cell proliferation and sprouting in a three-dimensional gel in vitro, and stimulates neovascularization when applied in vivo on top of the chicken embryo chorioallantoic membrane or injected subcutaneously in mice. In addition to the pro-angiogenic activity of Histogel, also the ADSC derived beige cells contribute to activating endothelial cells. These data led us to propose Histogel as a promising scaffold for the modulation of the thermogenic behavior of adipose tissue. Indeed, Histogel simultaneously supports the acquisition of brown tissue markers and activates the vasculature process necessary for the correct function of the thermogenic tissue. Thus, Histogel represents a valid candidate for the development of bioscaffolds to increase the amount of brown adipose tissue in patients with metabolic disorders.

Loss of Emp2 compromises cardiogenic differentiation in mouse embryonic stem cells.

Isolated mouse embryonic stem cells (mESCs) retain the capacities to self-renew limitlessly and to give rise to all tissues of an adult mouse. A precise understanding of the relationships, mechanisms of action and functions of novel genes involved in mESCs differentiation is crucial to expand our knowledge of vertebrate development. The epithelial membrane protein 2 (EMP2) is a membrane-spanning protein found in epithelial and endothelial cell-cell junctions that has been implicated in the regulation of cell proliferation and migration in normal and tumor tissues. In this study, Emp2 was disrupted in mESCs using the CRISPR/Cas9 technology. We subsequently assessed Emp2 functions by using mouse embryoid bodies (EBs) capable of forming the three germ layers of an embryo in vitro and by further analyzing the emergence of the future cardiac tissue in these EB models. We found that when Emp2 is disrupted, expression of pluripotency markers was up-regulated and/or longer retained in EBs. Additionally, the formation of each germ layer was variously affected during gastrulation and in particular, the formation of mesoderm was delayed. Besides, we discovered that Emp2 was involved in the regulation of the epithelial-mesenchymal transition (EMT) process and in the differentiation of cells into functional cardiomyocytes.

Morphological observation of embryoid bodies completes the in vitro evaluation of nanomaterial embryotoxicity in the embryonic stem cell test (EST).

The wide and frequent use of engineered nanomaterials (NMs) raises serious concerns about their safety for human health. Our aim is to evaluate the embryotoxic potential of silver, uncoated and coated zinc oxide, titanium dioxide and silica NMs through the embryonic stem cell test (EST). EST is a validated in vitro assay that permits classification of chemicals into three classes (non, weakly or strongly embryotoxic). Because of the peculiar physico-chemical characteristics of NMs, we first adapted and simplified the differentiation protocol. To verify the efficiency of this adapted protocol we screened 3 well-characterized chemicals (5-fluorouracil, hydroxyurea and saccharin). Next, we assessed the embryotoxic potential of NMs. Our data showed that silver NM is classified as a strong embryotoxic compound, while coated and uncoated zinc oxide, titanium and silica NMs as weak embryotoxic compounds. In addition, we observed daily the formation and growth of embryoid bodies (EBs). We showed that multiple EBs formed in each well starting from 50 µg/ml of SiO2 while EB formation was inhibited starting from 20 µg/ml of ZnO NMs. This has never been reported with chemicals and could pose a risk of wrongly evaluating the NMs embryotoxic potential. For NMs, morphological observation of EBs can provide valuable information on early differentiation effects. Finally, we suggest that the prediction model should be revised for the assessment of NMs embryotoxicity.

Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies.

Whole-mount in situ hybridization (WISH) is a technique widely used in developmental biology to study the localization of RNA sequences in intact tissues or whole organisms. In this chapter we present a detailed protocol that was optimized for gene expression analysis in early stage mouse embryos (5.5-10.5 days post-coitum) and embryoid bodies formed by differentiating embryonic stem cells and can be used for the detection of up to two distinct RNA sequences simultaneously. The initial steps of the procedure are the generation of the labeled riboprobe(s) and the embryo or embryoid body preparation, which can be completed in less than 2 days. The actual WISH procedure, comprised of the hybridization, the post-hybridization washes, and the immunological staining, can be completed in 3 days.