Project description:During in vitro differentiation, pluripotent stem cells undergo extensive remodeling of their gene expression profiles. While studied extensively at the transcriptome level, much less is known about protein dynamics, which might differ significantly from their mRNA counterparts. Here, we present deep proteome-wide measurements of protein levels during the differentiation of embryonic stem cells.
Project description:Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating Retinoic Acid Receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites. We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation. We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggests that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation. The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid. Here, ChIP-seq is used to profile the genome-wide occupancy of RAR isofroms both immediately prior to and during exposure of the cells to retinoic acid. ChIP-seq is also used to profile the genomic occupancy of Pol2 with phosphorylated serine 5 (Pol2-S5P) and phosphorylated serine 2 (Pol2-S2P) after exposure to retinoic acid.
Project description:Metabolism is vital to cellular function and tissue homeostasis during human lung development. In utero, embryonic pluripotent stem cells undergo endodermal differentiation towards a lung progenitor cell fate that can be mimicked in vitro using induced human pluripotent stem cells (hiPSCs) to study genetic mutations. To identify differences between wild type and surfactant protein B (SFTPB)-deficient cell lines during endoderm specification towards lung, we used an untargeted metabolomics approach to evaluate the developmental changes in metabolites. We found that the metabolites most enriched during the differentiation from pluripotent stem cell to lung progenitor cell, regardless of cell line, were sphingomyelins and phosphatidylcholines, two important lipid classes in fetal lung development. The SFTPB mutation had no metabolic impact on early endodermal lung development. The identified metabolite signatures during lung progenitor cell differentiation may be utilized as biomarkers for normal embryonic lung development.
Project description:Genome-wide binding profiles of nuclear FGFR1, RXRα and Nur77 in pluripotent mouse Embryonic Stem Cells and during Retinoic Acid-induced differentiation
Project description:The aim of this study is to profile gene expression dynamics during the in vitro differentiation of embryonic stem cells into ventral motor neurons. Expression levels were profiled using Affymetrix microarrays at six timepoints during in vitro differentiation: ES cells (Day 0), embryoid bodies (Day 2), retinoid induction of neurogenesis (Day 2 +8hours of exposure to retinoic acid), neural precursors (Day 3), progenitor motor neurons (Day 4), postmitotic motor neurons (Day 7). The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog agonist. Here, gene expression patterns are profiled at various defined stages during the differentiation process using Affymetrix expression arrays.
