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

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.

Project description:Glioblastoma multiforme (GBM) is an aggressive, heterogeneous and highly vascularized brain tumor. GBM is thought to arise from glioblastoma stem-like cells (GSCs) which are characterized as being either proneural or mesenchymal. The former isolates of GSCs are tight sphere forming and slow growing while mesenchymal GSCs are lose sphere forming, fast growing, highly invasive and when dominant yield poorer patient prognosis. GSCs are known to be plastic in nature and can therefore evolve from a proneural to a mesenchymal state. Here, we observed that factors secreted by endothelial cells (which make up the brain vasculature) alter several properties of GSCs resulting in the acquisition of a more mesenchymal and invasive phenotype coupled with changes at the level of secretory and cellular proteome. Thus, using mass spectrometry, quantitative proteomic analysis and GO term filters, we identified several mesenchymal traits in proneural GSCs exposed to endothelial cell secretome. Specifically, proneural cells treated with the conditioned media derived from human umbilical vein endothelial cells (HUVEC) upregulated the expression of mesenchymal proteins such as CD44 and VIM, while downregulating the expression of the proneural proteins such as NOTCH1, activated NOTCH intracellular domain (NICD), SOX2 and NESTIN, which were validated using flow cytometry (FACS) and western blots (WB). Using DAVID analysis tool, we detected the features of cellular proteome indicative of the activation of NFkB, Wnt and several other pathways in the proneural cells treated with HUVEC conditioned media. Using conditioned media fractionation through several centrifugation steps we identified the extracellular vesicles (EV) sedimented at 100,000 x g using ultracentrifugation, as the source of activity in endothelial conditioned media capable of triggering mesenchymal shift in proneural GSCs. EVs are heterogeneous membrane structures containing multiple bioactive macromolecules, which have the ability to carry multiple bioactive proteins, transfer them to recipient cells and alter their function, signalling and biological programmes. We compared the effects of EVs, soluble fraction and unfractionated conditioned media in terms of their ability to trigger mesenchymal changes in the phenotype of proneural GSCs. Once the cultures were established, the culture medium was removed and replaced with HUVEC-derived material (conditioned media, supernatant or EV fraction) and responses evaluated over 7 days by microscopical analysis of sphere structures, and biochemically by following the aforementioned proneural or mesenchymal markers (WB, FACS). We observed an upregulation of mesenchymal proteins, as well as downregulation of the proneural proteins, mentioned above. These effects recapitulated those of unfractionated conditioned media and were absent from target cells exposed to EV-depleted conditioned media. The data analysis of EV proteome included canonical markers and pathways of cellular vesiculation as well as markers and pathways of interest with regards to the biological effects associated with treatment of GSC recipient cells. In this regard we observed several EV related tetraspanin markers, which were validated using western blot including CD9, CD63, CD81 and a purity control, BIP. Although we identified several potential effectors associated with endothelial cell EVs that could impact proneural cell phenotype, we focused on MMPs for at least three reasons: (i) evidence in the literature (see text) indicated that MMPs may induce differentiation programs in neural stem cells; (ii) MMPs in EV cargo were relatively abundant and have been implicated in various biological processes; (iii) MMPs released from endothelial cells could be functionally involved in disrupting proneural cell sphere structures that we observed in the presence of endothelial cell secretome. We noted the expressions of MMP1, MMP2, MMP11 and MP14 in our HUVEC-EV mass spectrometry dataset, the activity of which was validated using the MMP activity assay kit from abcam (ab112146). Using GO terms we also detected a signal for NFkB pathway activation in the proteome of endothelial (HUVEC) conditioned media-treated proneural GSCs. NFkB activation is regarded as hallmark of mesenchymal phenotype in GSCs and GBM cells. We validated that the upregulation of NFkB, was also true for the proneural cells treated with HUVEC derived EVs. Moreover, upon blocking MMP expression in proneural cells treated with endothelial cell EVs, we inhibited the activation of NFkB activity thereby documenting that the initial effects of MMPs trigger a shift in cellular phenotype toward NfkB activation and mesenchymal reprogramming. Briefly, we compared GO terms of GSC157 cells treated with their own or HUVEC-derived EVs. Validation of the NFkB pathway activation was analysed using WB and immunofluorescence for levels of NFkB and phosphor-NFkB.

Project description:The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well-established that MLL-AF4 arises pre-natally during human development, its effects on hematopoietic development in utero remains unexplored. We have created a human-specific in vitro system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Differentiation and functional studies as well as clonal analyses and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. It enhances the specification of hemogenic precursors from hESCs and impairs further hematopoietic commitment of these precursors in favour of the endothelial cell fate. Similar to that reported in cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs), MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells in vitro or in vivo, indicating that additional events may be required to initiate leukemogenesis or that embryonic hematopoiesis is not the appropriate human cellular target for MLL-AF4-mediated leukemogenesis. This work illustrates how hESCs can provide unique insights into human development and further our understanding of how leukemic fusion genes known to arise pre-natally regulate human embryonic hematopoietic specification. MLL is involved in transcriptional regulation and most MLL translocations appear to result in increased expression of Hox genes and hematopoietic genes. We therefore assessed the impact of MLL-AF4 expression on the transcriptome of hESCs. Gene expression profiling performed in MLL-AF4 hESCs revealed that MLL-AF4 preferentially activates transcription. 1826 out of the 3001 genes (61%) expressed were up-regulated in MLL-AF4 hESCs. Human ESC samples were collected during the exponential cell growth phase and stabilized in RNA later. 500 ng of each total RNA sample was labelled with Cy3 using the Quick-Amp Labelling kit and hybridized with the Gene Expression Hybridization kit to a Whole Human Genome Oligo Microarray (Agilent Technologies) following the Manufacturer’s instructions. Each cell line was analyzed as independent duplicates. NEO-expressing (empty lentivector) hESC line was used as the baseline.

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