Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of mouse Embryonic stem cells at different stages of embryoid body differentiation

ABSTRACT: Haematopoietic and endothelial cells derive from a subset of Flk-1 expressing cells. In vitro differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) recapitulate several aspects of early embryonic development, including the formation of a subset of Flk-1 expressing cells. By day 4 of EB differentiation, mesodermal lineages with distinct lineage potentials can be identified based on the expression of the two surface markers Flk-1 and Pdgfra. Cells expressing Flk-1 alone (Flk-1⁺/Pdgfrα⁻) are enriched for haematoendothelial precursors, whereas double-positive cells (Flk-1⁺/Pdgfrα⁺) are enriched for primitive/cardiac mesodermal lineages. Here we performed RNA sequencing at multiple stages of EB differentiation to identify genes potentially important for mesoderm specification towards the haematoendothelial lineages. Mouse ESCs were differentiated to embryoid bodies for 4 days, dissociated, stained for the Flk-1 and Pdgfra markers and Flk-1⁺/Pdgfrα⁻ and Flk-1⁺/Pdgfrα⁺ populations were sorted by FACS. RNA sequencing was performed at multiple stages of EB differentiation: day 0 (D0), day 2 (D2), day 4 (D4) and the two sorted populations (D4 Flk-1⁺/Pdgfrα⁻ and D4 Flk-1⁺/Pdgfrα⁺).

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Giulia Picco

PROVIDER: E-MTAB-15595 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The outcome for children with high-grade gliomas (HGG) remains dismal, with a two-year survival rate of only 10-30%. Approximately half of pediatric HGGs are diffuse intrinsic pontine glioma (DIPG), a brainstem tumor that arises almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet derived growth factor receptor alpha (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs. To determine whether the PDGFRA is also targeted by more subtle mutations not detected by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic activating mutations were identified in 14.4% (13/90) of non-brainstem pediatric HGGs and 4.7% (2/43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. 40% of tumors with mutation showed concurrent amplification, while 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro, and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 non-brainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFR in childhood HGG. To better understand the consequence of PDGFRα mutation in pediatric gliomagenesis, retroviral constructs expressing wild-type PDGFRα or six selected PDGFRα mutants that affect different regions of the receptor were generated for functional studies. p53-null primary mouse astrocyte (PMA) cultures were chosen as a relevant cellular background to assess PDGFRα function.

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RNA-seq of mouse Embryonic stem cells at different stages of embryoid body differentiation

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