Project description:A fully defined media for human embryonic stem cells (hESCs) was designed after examining the phosphorylation status of 42 receptor tyrosine kinases (RTKs) on hESCs exposed to mouse embryonic fibroblast conditioned medium (CM). Activation of the insulin and insulin-like growth factor-1 receptors (IR, IGF1R) as well as EGFR family members including ERBB2 and ERBB3 led to the design of a simple defined media which supported robust long-term growth of multiple hESC lines and allowed massive expansion of undifferentiated cells. Illumina bead arrays were used to demonstrate the maintenance of gene expression profiles characteristic of pluripotent cells in cultures grown in CM or defined media. Keywords: Comparison of cells in different culture conditions
Project description:Embryonic stem cells from B6 and NOD backgrounds were derived freshly in the presence of 2i. After 3-5 passages on feeders, ES cells were cultured in 2i media without any feeders for several passages. In order to identify differentially expressed genes and proteins, we performed RNA-Seq and mass spectromety analysis respectively. Among the differentially expressed genes, we identified several important players in innate and adaptive immunity. Several of these genes had been linked to onset of type-1 diabetes. Proteomics analysis was able to quantitative differences in protein expression among the B6 and NOD ES cell lines.
Project description:Human embryonic stem cells can be maintained in a basic Serum Replacement (Invitrogen) based medium that has been conditioned on mouse embryonic fibroblasts (MEFs), yielding MEF-CM. Ligands secreted into the medium by the MEFs include Activin A, TGFß1, and Gremlin. This experiment served the purpose of identifying the short-term effects of MEF-CM and its substitute UM_GTA (unconditioned medium plus Activin A, TGFb1, and Gremlin) on gene expression in human embryonic stem cells. Keywords: Media / growth factor stimulation experiment
Project description:In this study we investigated the effects of breast cancer cell conditioned media on human lymphatic endothlial cells. For this, we cultured the three different breast cancer cell lines MDA-MB-231, MCF-7 and T47D in endothelial cell media and transferred this conditioned media to primary, lymph-node derived human lymphatic endothelial cells. These cells were then collected and RNA-sequencing was performed from them.
Project description:In this study, we evalauted the transcriptionl profiles of human primordial germ cell-like cell (hPGCLCs) cultured for an extended period on STO feeders in FR10 media for an additional 10 days (D4C10). The hPGCLCs were obtained from two different human embryonic stem cell line (hESCs), UCLA 1 (XX) and UCLA2 (XY) for the RNA sequencing data. For whole genome bisulfite sequencing, we evaluate primed UCLA2 hESCs, directly out of the aggragate hPGCLCs (Day 4= D4) and extended culture hPGCLCs D4C10. Our D4C10 hPGCLCs were compared to prior RNA-sequencing data generated in our laboraty, which revealed that D4C10 hPGCLCs retain germline identy and closely resemble D4 hPGCLCs at the transcriptional level. Our WGBS data revealed that extended culture hPGCLCs at D4C10 have a decrease in CG methlyation, as compared to the starting hESCs and D4 hPGCLCs.
Project description:The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder-free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation towards hematopoietic lineage than hESCs grown in standard human foreskin fibroblast (HFF)-conditioned media (HFF-CM). We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. Human ESCs grown in MSC-CM showed a decrease of 20% in global DNA methylation and a promoter DNA methylation signature consisting in 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition towards hematopoietic specification. Total DNA isolated by standard procedures from human embryonic stem cells (hESC) cultured in different conditioned media
