Project description:All data are derived from the same batch of human ES cells (line H9, WA-09). With the exception of the “UD” file all files represent neural cell types derived from the undifferentiated hESCs. Following mechanical isolation, rosettes were maintained for various passages in the presence of defined growth factors and morphogen factors as indicated below at the R-NSC stage. Cells at the NSCFGF/EGF stage were derived from mechanically isolated neural rosettes that were purified and long-term expanded as attached monolayer cultures in serum free medium in the presence of FGF2/EGF. Keywords: differentiation of human ES cells (line H9, WA-09).
Project description:All data are derived from the same batch of human ES cells (line H9, WA-09). With the exception of the â??UDâ?? file all files represent neural cell types derived from the undifferentiated hESCs. Following mechanical isolation, rosettes were maintained for various passages in the presence of defined growth factors and morphogen factors as indicated below at the R-NSC stage. Cells at the NSCFGF/EGF stage were derived from mechanically isolated neural rosettes that were purified and long-term expanded as attached monolayer cultures in serum free medium in the presence of FGF2/EGF. Experiment Overall Design: All data are derived from the same batch of human ES cells (line H9, WA-09). With the exception of the â??UDâ?? file all files represent neural cell types derived from the undifferentiated hESCs. Following mechanical isolation, rosettes were maintained for various passages in the presence of defined growth factors and morphogen factors as indicated below at the R-NSC stage. Cells at the NSCFGF/EGF stage were derived from mechanically isolated neural rosettes that were purified and long-term expanded as attached monolayer cultures in serum free medium in the presence of FGF2/EGF.
Project description:The purpose of this study was to examine global gene expression during the differentiation of human embryonic stem cells to more restricted neural progenitors. We developed an adherent differentiation protocol with completely defined media that produced 2 distinct classes of neuroepithelia based on timing, morphology and expression of known neural markers. The first stage of differentiation is undifferentiated human ES cells (hESCs). The second stage is aggregates of these hESCs after 6 days of separation from supportive mouse embryonic fibroblasts. The third stage is a primitive anterior neuroepithelia that arises after 10 days of differentiation and is marked by a columnar morphology, expression of Pax6 and anterior neural patterning genes. The fourth stage peaks at 17 days when cells form rosettes that resemble the neural tube and express the pan-neural marker Sox1.
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.
Project description:The purpose of this study was to examine global gene expression during the differentiation of human embryonic stem cells to more restricted neural progenitors. We developed an adherent differentiation protocol with completely defined media that produced 2 distinct classes of neuroepithelia based on timing, morphology and expression of known neural markers. The first stage of differentiation is undifferentiated human ES cells (hESCs). The second stage is aggregates of these hESCs after 6 days of separation from supportive mouse embryonic fibroblasts. The third stage is a primitive anterior neuroepithelia that arises after 10 days of differentiation and is marked by a columnar morphology, expression of Pax6 and anterior neural patterning genes. The fourth stage peaks at 17 days when cells form rosettes that resemble the neural tube and express the pan-neural marker Sox1. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
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:Primitive neural stem cells (NSCs) could be derived from pluripotent mouse embryonic stem (ES) cells, and then differentiate into definitive-type neural stem cells which resemble NSCs obtained from the central nervous system. Hence, primitive NSCs define an early stage of neural induction and provide a model to understand the mechanism that controls initial neural commitment. In this study, we performed microarray assay to analyze the global transcriptional profiles in mouse ES cell-derived primitive and definitive NSCs and to depict the molecular changes during the multi-staged neural differentiation process.
Project description:The purpose of this study was to examine global gene expression during the differentiation of human embryonic stem cells to more restricted neural progenitors. We developed an adherent differentiation protocol with completely defined media that produced 2 distinct classes of neuroepithelia based on timing, morphology and expression of known neural markers. The first stage of differentiation is undifferentiated human ES cells (hESCs). The second stage is aggregates of these hESCs after 6 days of separation from supportive mouse embryonic fibroblasts. The third stage is a primitive anterior neuroepithelia that arises after 10 days of differentiation and is marked by a columnar morphology, expression of Pax6 and anterior neural patterning genes. The fourth stage peaks at 17 days when cells form rosettes that resemble the neural tube and express the pan-neural marker Sox1. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Using regression correlation
Project description:Primitive neural stem cells (NSCs) could be derived from pluripotent mouse embryonic stem (ES) cells, and then differentiate into definitive-type neural stem cells which resemble NSCs obtained from the central nervous system. Hence, primitive NSCs define an early stage of neural induction and provide a model to understand the mechanism that controls initial neural commitment. In this study, we performed microarray assay to analyze the global transcriptional profiles in mouse ES cell-derived primitive and definitive NSCs and to depict the molecular changes during the multi-staged neural differentiation process. Primitive NSCs derived directly from ESCs in Lif (p-NSC_L), primitive NSCs that were sub-cultured in the presence of Lif and FGF (p-NSC_LF), as well as definitive NSCs derived from primitive NSCs in medium containing FGF and EGF, were collected for RNA extraction and hybridization on Affymetrix microarrays. Mouse ESCs and NSCs obtained from mouse embryonic brain (E11.5) were included for controls. For each cell type, we collected two biological replicate samples for microarray analysis.