Project description:To investigate the interplay of X-chromosome status and meiosis during primordial germ cell (PGC) maturation, we differentiated mouse embryonic stem cells (ESCs) via epiblast-like cells (EpiLCs) into primordial germ cell like cells (PGCLCs) and further into meiotic germ cells. During the differentiation of PGCLCs, we assessed the kinetics of X-chromosome inactivation and reactivation. We generated allele-specific total RNA-seq datasets to assess gene inactivation and reactivation dynamics, as well as allele-specific single-cell RNA-seq using SMART-Seq v5 to investigate the interplay of meiosis and X-chromosome status in germ cells.
Project description:We performed an integrated analysis of RNA and proteins at the transition between naïve ES cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators were specifically released from translational inhibition mediated by RNA-Induced Silencing Complex (RISC). This suggests that, prior to differentiation, the propensity of ES cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators was reinstated following acute inactivation of RISC, and it correlated with loss of stemness markers and activation of early cell differentiation markers in treated ES cells. Comparison between EpiSC derived directly from mouse embryonic stem cells and from Epiblast-Like Aggregates
Project description:We performed an integrated analysis of RNA and proteins at the transition between naïve ES cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators were specifically released from translational inhibition mediated by RNA-Induced Silencing Complex (RISC). This suggests that, prior to differentiation, the propensity of ES cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators was reinstated following acute inactivation of RISC, and it correlated with loss of stemness markers and activation of early cell differentiation markers in treated ES cells. Cytoplasmic RNA-protein complexes from mouse embryonic stem cells at different stages of neural in vitro differentiation were immunoprecipitated with anti-Argonaute antibody and analyzed by microarray hybridization
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:We performed an integrated analysis of RNA and proteins at the transition between naïve ES cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators were specifically released from translational inhibition mediated by RNA-Induced Silencing Complex (RISC). This suggests that, prior to differentiation, the propensity of ES cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators was reinstated following acute inactivation of RISC, and it correlated with loss of stemness markers and activation of early cell differentiation markers in treated ES cells. We evaluated ribosome occupancy of mRNAs in embryonic stem cells and Epiblast-Like Aggregates by means of polysome profiling with a modified Translating Ribosome Affinity Purification protocol (TRAP).
Project description:We performed an integrated analysis of RNA and proteins at the transition between naïve ES cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators were specifically released from translational inhibition mediated by RNA-Induced Silencing Complex (RISC). This suggests that, prior to differentiation, the propensity of ES cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators was reinstated following acute inactivation of RISC, and it correlated with loss of stemness markers and activation of early cell differentiation markers in treated ES cells. We evaluated global miRNA profiles of embryonic stem cells cultured in either 10%FCS+LIF or 2i+LIF and of Epiblast-Like Aggregates derived from ES cells in 10%FCS+LIF or 2iL+LIF.
Project description:<p>During development of the human brain, multiple cell types with diverse regional identities are generated. Here we report a system to generate early human brain forebrain and mid/hindbrain cell types from human embryonic stem cells (hESCs), and infer and experimentally confirm a lineage tree for the generation of these types based on single-cell RNA-Seq analysis. We engineered <i>SOX2<sup>Cit/+</sup></i> and <i>DCX<sup>Cit/Y</sup></i> hESC lines to target progenitors and neurons throughout neural differentiation for single-cell transcriptomic profiling, then identified discrete cell types consisting of both rostral (cortical) and caudal (mid/hindbrain) identities. Direct comparison of the cell types were made to primary tissues using gene expression atlases and fetal human brain single-cell gene expression data, and this established that the cell types resembled early human brain cell types, including preplate cells. From the single-cell transcriptomic data a Bayesian algorithm generated a unified lineage tree, and predicted novel regulatory transcription factors. The lineage tree highlighted a prominent bifurcation between cortical and mid/hindbrain cell types, confirmed by clonal analysis experiments. We demonstrated that cell types from either branch could preferentially be generated by manipulation of the canonical Wnt/beta-catenin pathway. In summary, we present an experimentally validated lineage tree that encompasses multiple brain regions, and our work sheds light on the molecular regulation of region-specific neural lineages during human brain development.</p>