Roles of gangliosides in mouse embryogenesis and embryonic stem cell differentiation

Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the ma ternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).     

The potential use of mesenchymal stem cells in hematopoietic stem cell transplantation

In the last 10 years, mesenchymal stem cells (MSCs) have emerged as a therapeutic approach to regenerative medicine, cancer, autoimmune diseases, and many more due to their potential to differentiate into various tissues, to repair damaged tissues and organs, and also for their immunomodulatory properties. Findings in vitro and in vivo have demonstrated immune regulatory function of MSCs and have facilitated their application in clinical trials, such as those of autoimmune diseases and chronic inflammatory diseases. There has been an increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT), including hematopoietic stem cell engraftment and the prevention and treatment of graft-versus-host disease (GVHD), and their therapeutic potential has been reported in numerous clinical trials. Although the safety of clinical application of MSCs is established, further modifications to improve their efficacy are required. In this review, we summarize advances in the potential use of MSCs in HSCT. In addition, we discuss their use in clinical trials of the treatment of GVHD following HSCT, the immunomodulatory capacity of MSCs, and their regenerative and therapeutic potential in the field of HSCT.     

Asparagus racemosus mediated silver chloride nanoparticles induce apoptosis in glioblastoma stem cells in vitro and inhibit Ehrlich ascites carcinoma cells growth in vivo

Glioblastoma multiforme (GBM) is a type of brain tumor that is most aggressive, proliferates rapidly and intensive invasion is governed by cell migration and destruction of the extracellular matrix. In the present study, we evaluated the antiproliferative efficacy of the synthesized silver chloride nanoparticles (AgCl-NPs) from Asparagus racemosus root extract against human glioblastoma stem cells (GSCs) and Ehrlich ascites carcinoma (EAC) cells. Biosynthesis of A. racemosus-AgCl-NPs was confirmed by color change, UV–visible spectroscopy and characterized by transmitted electron microscope, energy dispersive spectroscopy, x-ray powder diffraction and Fourier-transform infrared spectroscopy. The A. racemosus-AgCl-NPs inhibited GSCs and EAC cells growth with the IC₅₀ values of 4.8 and 4.69 µg/ml, respectively. A. racemosus-AgCl-NPs induced apoptosis in GSCs which was confirmed by annexin V/PI assay, various genes expression, and caspase-3 protein expression as detected by the immunofluorometric assay. The expression level of the TLR9, NFκB, TNFα, p21 and IKK genes were increased consequently with the decrease of PARP, EGFR, NOTCH2, mTOR and STAT3 genes in GSCs as examined by real-time PCR. The cell cycle arrest at G₀/G₁ phase was detected by flow cytometry. In addition, A. racemosus-AgCl-NPs caused significant inhibition of EAC cells growth, reduced tumor burden, increased the survival of EAC-bearing mice and restored the hematological parameters when compared with the control mice. The synthesized AgCl-NPs inhibited the proliferation of GSCs in vitro with the induction of apoptosis and inhibited the growth of EAC cells in vivo in mice by reducing the tumor burden and increasing the survival periods.     

The roles of glycosphingolipids in the proliferation and neural differentiation of mouse embryonic stem cells

Glycosphingolipids including gangliosides play important regulatory roles in cell proliferation and differentiation. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs (shRNAs) to evaluate the roles of glycosphingolipids in proliferation and neural differentiation of mouse embryonic stem cells (mESCs). HPTLC/immunofluorescence analyses of shRNA-transfected mESCs revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM3 and GD3. Furthermore, MTT and Western blot/immunofluorescence analyses demonstrated that inhibition of the Ugcg expression in mESCs resulted in decrease of cell proliferation (P < 0.05) and decrease of activation of the ERK1/2 (P < 0.05), respectively. To further investigate the role of glycosphingolipids in neural differentiation, the embryoid bodies formed from Ugcg-shRNA transfected mESCs were differentiated into neural cells by treatment with retinoic acid. We found that inhibition of Ugcg expression did not affect embryoid body (EB) differentiation, as judged by morphological comparison and expression of early neural precursor cell marker, nestin, in differentiated EBs. However, RT-PCR/immunofluorescence analyses showed that expression of microtubule- associated protein 2 (MAP-2) for neurons and glial fibrillary acidic protein (GFAP) for glial cells was decreased in neural cells differentiated from the shRNA-transfected mESCs. These results suggest that glycosphingolipids are involved in the proliferation of mESCs through ERK1/2 activation, and that glycosphingolipids play roles in differentiation of neural precursor cells derived from mESCs.     

Study of uptake and loss of silica nanoparticles in living human lung epithelial cells at single cell level

The toxicology of nanomaterials is a blooming field of study, yet it is difficult to keep pace with the innovations in new materials and material applications. Those applications are quickly being introduced in research, industrial, and consumer settings. Even though the cytotoxicity of many types of nanoparticles has been demonstrated, the behavior of those particles in a biological environment is not yet fully known. This work characterized the following over time: protein adsorption on silica particle surfaces, the internalization of particles in human lung carcinoma (A549) cells when coated with different specific proteins or no proteins at all, and the cellular loss of particles following the removal of extracellular particles. Proteins were shown to quickly saturate the particle surface, followed by a competitive process of particle agglomeration and protein adsorption. Uptake of particles peaked at 8–10 h, and it was determined that, in this system, the charge of the protein-coated particles changed the rate of uptake if the charge difference was great enough. Cells internalized particles lacking any adsorbed proteins with approximately 3 times the rate of protein-coated particles with the same charge. Although particles exited cells over time, the process was slower than uptake and did not near completion within 24 h. Finally, analysis at the single cell level afforded observations of particle agglomerates loosely associated with cell membranes when serum was present in the culture medium, but in the absence of serum, particles adhered to the dish floor and formed smaller agglomerates on cell surfaces. Although data trends were easily distinguished, all samples showed considerable variation from cell to cell. Figure Silica-capped fluorescent semiconductor nanoparticles as internalized by human lung epithelial cells and adsorbed to a glass substrate in protein-free culture medium.     

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.