Project description:These arrays were conducted in order to confirm the generation of astrocytes from human embryonic stem cells and to determine expression differences between astrocyte subtypes. Experiment Overall Design: Human embryonic stem cells were differentated in vitro to neuroepithelial cells (day-17), neurons (day-50), or astrocytes (day-180) after specification with the morphogens retinoic acid (RA) or FGF8 from days 10-21.
Project description:The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Initial expansion of the progenitor cell pool is followed by the generation of neurons of all the cortical layers and later, astrocytes and oligodendrocytes. However, the regulatory pathways that control the expansion and maintenance of the neuroepithelial progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-b1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate to lower and upper layer cortical neurons both in vitro and in vivo. Identifying the mechanisms that drive the self-renewal and fate of cNESCs decision of neuroepithelial stem cells is key to developing new stem cell-based therapeutic approaches to treat neurological conditions.
Project description:The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Initial expansion of the progenitor cell pool is followed by the generation of neurons of all the cortical layers and later, astrocytes and oligodendrocytes. However, the regulatory pathways that control the expansion and maintenance of the neuroepithelial progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-b1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate to lower and upper layer cortical neurons both in vitro and in vivo. Identifying the mechanisms that drive the self-renewal and fate of cNESCs decision of neuroepithelial stem cells is key to developing new stem cell-based therapeutic approaches to treat neurological conditions. In this study, we performed Oxford Nanopore technologies long-read RNA-sequencing (lrRNA-seq) of human cNESCs and of the human embryonic stem cells used to generate them. Two hESC cell lines were differentiated to cNESCs, which were then maintained in either six factors (6F : BMPRi, TGFBRi, GSK3i, FGF, TANKYRASEi, AKTi) or 4 factors (6F-2F : BMPRi, TGFBRi, GSK3i, FGF). All samples (hESCs, cNESCs in 6 factors, cNESCs in 4 factors) are submitted to lrRNA-seq as duplicates.
Project description:Comparing gene expressing profile of human embryonic stem cells and neuroepithelial cells differentiated from human embryonic stem cells.
Project description:Neural progenitor cells (hNPC) derived from the developing human brain can be expanded in culture and subsequently differentiated into neurons and glia. They provide an interesting source of tissue for both modeling brain development and future cellular replacement therapies. It is becoming clear that hNPC are regionally and temporally specified depending on which brain region they were isolated from and its developmental stage. We show here that hNPC derived from the developing cortex (hNPCCTX) and ventral midbrain (hNPCVM) have similar morphological characteristics and express the progenitor cell marker nestin. However, hNPCCTX cultures were highly proliferative and produced large numbers of neurons, while hNPCVM divided slowly and produced less neurons but more astrocytes. Microarray analysis revealed a similar expression pattern for some stemness markers between the two growing cultures, overlaid with a regionally specific profile that identified some important differentially expressed neurogenic transcription factors. By over expressing one of these, the transcription factor ASCL1, we were able to regain neurogenesis from hNPCVM cultures which produced larger neurons with more neurites than hNPCCTX, but no fully mature dopamine neurons. Thus hNPC are regionally specified and can be induced to undergo neurogenesis following genetic manipulation. While this restores neuronal production with a region specific phenotype, it does not restore full neurochemical maturation which may require additional factors. Experiment Overall Design: G001 and G002 are neural progenitor cells derived from human (German) embryos 8-10 wk old. Experiment Overall Design: After abortion,the cortex and ventral midbrain have been dissected and grown as neurospheres. Experiment Overall Design: 4 samples total: G001CTX, G001VM, G002CTX, G002VM
Project description:Neural progenitor cells (hNPC) derived from the developing human brain can be expanded in culture and subsequently differentiated into neurons and glia. They provide an interesting source of tissue for both modeling brain development and future cellular replacement therapies. It is becoming clear that hNPC are regionally and temporally specified depending on which brain region they were isolated from and its developmental stage. We show here that hNPC derived from the developing cortex (hNPCCTX) and ventral midbrain (hNPCVM) have similar morphological characteristics and express the progenitor cell marker nestin. However, hNPCCTX cultures were highly proliferative and produced large numbers of neurons, while hNPCVM divided slowly and produced less neurons but more astrocytes. Microarray analysis revealed a similar expression pattern for some stemness markers between the two growing cultures, overlaid with a regionally specific profile that identified some important differentially expressed neurogenic transcription factors. By over expressing one of these, the transcription factor ASCL1, we were able to regain neurogenesis from hNPCVM cultures which produced larger neurons with more neurites than hNPCCTX, but no fully mature dopamine neurons. Thus hNPC are regionally specified and can be induced to undergo neurogenesis following genetic manipulation. While this restores neuronal production with a region specific phenotype, it does not restore full neurochemical maturation which may require additional factors.