RNA sequencing (RNA-SEQ) of Human endothelial cells (HUVEC) in LFS, sFRP2OE, and WT conditioned media
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE102729 | GEO | 2018/12/22
REPOSITORIES: GEO
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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
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Project description:Background: Factors secreted from the placenta into maternal and fetal circulation are important for maternal and fetal development during pregnancy. Maternal hematopoietic stem cells maintain maternal blood supply. The placenta is an early site of fetal hematopoiesis, and placental hematopoietic stem cells are involved in the early stages of fetal blood cell differentiation. Cross-talk between placental cells and hematopoietic stem cells represents an important but understudied phenomenon of pregnancy. The impact of toxicant exposure on placental-immune cell communication is poorly understood. The goals of this study were to 1) determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells in vitro and 2) if monoethylhexyl phthalate (MEHP), the major metabolite of the pollutant diethylhexyl phthalate, modifies these effects. Methods: Using in vitro cell line models of hematopoietic stem cells (K-562) and placental syncytiotrophoblasts (differentiated BeWo), we treated K-562 cells for 24 hours with media conditioned by incubation with BeWo cells, media conditioned with BeWo cells treated with 10 µM MEHP, or unconditioned controls (n = 4 replicates for each group). We extracted K-562 cell RNA and performed RNA sequencing. We then conducted differential gene expression and pathway analysis by treatment group and accounted for multiple comparisons using false discovery rate (FDR). Results: K-562 cells treated with BeWo cell conditioned media differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), relative to vehicle control. Cells treated with BeWo media upregulated TPM4 2.4 fold (FDR=1.8x10-53) and S1PR3 3.3 fold (FDR=1.6x10-40) compared to controls. Upregulated genes were enriched for biological processes including stem cell maintenance (“somatic stem cell population maintenance”), cell proliferation (“positive regulation of endothelial cell proliferation”) and immune processes (“myeloid leukocyte cytokine production”). Downregulated genes were enriched for protein translation (“mitochondrial translation”) and transcriptional regulation (“RNA processing”). Cells treated with media from BeWo cells treated with MEHP upregulated (FDR<0.05) eight genes, including genes involved in fat/lipid metabolism (PLIN2, fold-change: 1.4; CPT1A, fold-change: 1.4) and iron uptake (TFRC, fold-change: 1.3), relative to the BeWo conditioned media treatment. Conclusion: Hematopoietic stem cells are responsive to media that has been conditioned by placental cells, potentially impacting processes related to stem cell maintenance and proliferation, which may represent placental-immune communication important for development. The metabolite MEHP only had a modest impact on these responses at the single concentration tested. Future directions will investigate components of placental cell media (hormones, microvesicles, and proteins) contributing to hematopoietic cell signals.
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