Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human ES cells, EBs grown in suspension (d6), PEL (d10) stage and neural rosettes (d17)

ABSTRACT: Human embryonic stem cells not only provide a continuous cell source for potential cell therapy but also offer a system to unveil events of embryonic development in humans. This proposal will examine how the earliest neural cells, neuroepithelia, are specified from the naÃ¯ve ES cells, and test the hypothesis that neural specification in humans employs a similar mechanism as in other vertebrates. We will first re-create in culture the developmental events of the first 2-3 weeks of human embryonic development during which ES cells will be differentiated through the stages of embryoid bodies, primitive ectoderm cells, neural tube-like rosette cells. The stage-specific events will be defined by DNA microarray analysis along with the characteristic morphologic changes. This study will lead to an optimized procedure for generating enriched neural precursor cells, which will lay the groundwork for potential use of human ES cells in the treatment of neurological injuries and diseases. Aim 1: Establish a stepwise neural differentiation system from human ES cells. Aim 2. Determine the mechanism of Neural Specification in humans. Aim 3: Define the identity and function of ES cell-derived neural precursor cells. ES cells offer an alternative approach to study early developmental events in humans. This however requires re-creating the neural differentiation process in culture. Cell fate specification is determined by interactions between environmental factors and intrinsic signals. Our preliminary data suggest that the temporal pattern of neural differentiation and, to some degree, the spatial organization of neural precursors from human ES cells recapitulate in vivo neural development. We hypothesize that the intrinsic neural specification program may be preserved in culture, which offers a controlled system to examine the effect of extrinsic signals on neural specification. Distinctive morphological changes along the neural differentiation pathway are presumably accompanied by molecular changes. DNA microarray analysis will be used to determine the gene expression pattern by cells at each of the given stages. By analogy with early embryonic development and using morphological and antigenic markers, we can now subdivide the human ES cell neural differentiation process into four identifiable stages: ES, EB, PEL cells, and neural rosette cells. This definition is based on the assumption that early human development is the same as in other species, and employs the limited known markers from mouse ES cells. We will systematically investigate the molecular profile of cells at each of the neural differentiation stages using DNA microarray analysis. Total RNAs will be extracted from cells at the following developmental stages: ES cells, EBs grown in suspension (d6), PEL (d10) stage and neural rosettes (d17). Since the neural rosette culture contains non-neural lineage cells, we will separate the neural rosette cells from the surrounding non-neural cells through differential enzymatic response and differential adhesion. Three independent biological replicates consisting of three pooled experiments will be run for each of the four developmental time points, for a total of twelve chips.

ORGANISM(S): Homo sapiens

SUBMITTER: Elizabeth Salomon

PROVIDER: E-GEOD-9940 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human embryonic stem cells not only provide a continuous cell source for potential cell therapy but also offer a system to unveil events of embryonic development in humans. This proposal will examine how the earliest neural cells, neuroepithelia, are specified from the naïve ES cells, and test the hypothesis that neural specification in humans employs a similar mechanism as in other vertebrates. We will first re-create in culture the developmental events of the first 2-3 weeks of human embryonic development during which ES cells will be differentiated through the stages of embryoid bodies, primitive ectoderm cells, neural tube-like rosette cells. The stage-specific events will be defined by DNA microarray analysis along with the characteristic morphologic changes. This study will lead to an optimized procedure for generating enriched neural precursor cells, which will lay the groundwork for potential use of human ES cells in the treatment of neurological injuries and diseases. Aim 1: Establish a stepwise neural differentiation system from human ES cells. Aim 2. Determine the mechanism of Neural Specification in humans. Aim 3: Define the identity and function of ES cell-derived neural precursor cells. ES cells offer an alternative approach to study early developmental events in humans. This however requires re-creating the neural differentiation process in culture. Cell fate specification is determined by interactions between environmental factors and intrinsic signals. Our preliminary data suggest that the temporal pattern of neural differentiation and, to some degree, the spatial organization of neural precursors from human ES cells recapitulate in vivo neural development. We hypothesize that the intrinsic neural specification program may be preserved in culture, which offers a controlled system to examine the effect of extrinsic signals on neural specification. Distinctive morphological changes along the neural differentiation pathway are presumably accompanied by molecular changes. DNA microarray analysis will be used to determine the gene expression pattern by cells at each of the given stages. By analogy with early embryonic development and using morphological and antigenic markers, we can now subdivide the human ES cell neural differentiation process into four identifiable stages: ES, EB, PEL cells, and neural rosette cells. This definition is based on the assumption that early human development is the same as in other species, and employs the limited known markers from mouse ES cells. We will systematically investigate the molecular profile of cells at each of the neural differentiation stages using DNA microarray analysis. Total RNAs will be extracted from cells at the following developmental stages: ES cells, EBs grown in suspension (d6), PEL (d10) stage and neural rosettes (d17). Since the neural rosette culture contains non-neural lineage cells, we will separate the neural rosette cells from the surrounding non-neural cells through differential enzymatic response and differential adhesion. Three independent biological replicates consisting of three pooled experiments will be run for each of the four developmental time points, for a total of twelve chips. Keywords: time-course

Project description:This experiment is part of the FunGenES project (FunGenES - "Functional Genomics in Embryonic Stem Cells" partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at the "Instituto de Medicina Molecular,Faculdade de Medicina de Lisboa", Portugal. Goal of the experiment is the in vitro generation and characterisation of neurons from embryonic stem (ES) cells. This is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages try to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors (NPs) to their terminal differentiation into neurons and glial cells, is required. In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer system [1]. We established improved conditions to routinely produce highly homogeneous cultures of NPs, which display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. We show that the transition to neuronal production is linked to the organization of NPs into neural tube-like rosettes, where these cells divide and give rise to neurons. In order to characterize the global gene activity correlated with each particular stage of neural differentiation, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five synexpression groups that correlate with successive stages in the path from ES cells to neurons. This resulted in the identification of unique gene signatures for each cell stage, predicting in addition some of the molecular pathways associated with the transitions between these stages. Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

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Transcription profiling of human ES cells, EBs grown in suspension (d6), PEL (d10) stage and neural rosettes (d17)

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