4 research outputs found
An Esrrb and nanog cell fate regulatory module controlled by feed forward loop interactions
No full textCell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory 'dimensions' coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module
Analysis of Transcriptional Variability in a Large Human iPSC Library Reveals Genetic and Non-genetic Determinants of Heterogeneity
No full text(Cell Stem Cell 20, 518–532.e1–e9; April 6, 2017) In the originally published version of this manuscript, Caroline Hendry was inadvertently excluded from the author list. Her contributions to the work included the following: the development of SOPs for the relatively novel Sendai virus infection method for reprogramming; the design and implementation of studies to assess the variation among lines derived from a single donor (which included up to 14 lines per donor and analyses at both the iPSC stage and post-differentiation); and, together with S.L.D. and P.C., contributing to the development of an optimized workflow for the characterization of a large library of human iPSC lines, the creation and isolation of iPSCs, the performance of quality control for pluripotency markers, the serial passaging of each line for at least 12 passages, the verification that said lines were Sendai-free, the freezing down of cells at every split, the active maintenance of databases to catalogue the large number of lines and splits, and the preparation of these cells for RNA-seq analysis to be performed by members of the E.E.S. lab. All of these studies were performed to optimize the workflow that underpinned the project, and hence, the paper. All of the authors on the original author list, excluding those who are unable to be contacted and those who are deceased, have given their consent for Dr. Hendry's addition. The corrected author list is now included with this statement and the original manuscript online
High-throughput identification of small molecules that affect human embryonic vascular development
No full textBirth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature
Analysis of Transcriptional Variability in a Large Human iPSC Library Reveals Genetic and Non-genetic Determinants of Heterogeneity
No full textVariability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network, pathway, and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals, highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications.</p
