Neurons of the neocortex are generated by stem cells called radial glial cells. These polarized cells extend a short apical process toward the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport is unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in varicosities of the basal fiber. CAMSAP family members accumulate in these varicosities, where they control microtubule growth. Double knockdown of CAMSAP1 and 2 leads to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we reveal that this organization is conserved in basal radial glial cells, a related progenitor cell population associated with human brain size expansion.
Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications.
19Neurons of the neocortex are generated by neural progenitors called radial glial cells. 20These polarized cells extend a short apical process towards the ventricular surface and a long 21 basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is 22 organized in these cells to support long-range transport in unknown. Using subcellular live 23 imaging within brain tissue, we show that microtubules in the apical process uniformly emanate 24 for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, 25 reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing 26 centers localized in swellings of the basal fiber. We characterize their distribution and 27 demonstrate that they accumulate the minus end stabilizing factor CAMSAP3 and TGN-related 28 membranes, from which the majority of microtubules grow. Finally, using live imaging of 29 human fetal cortex, we show that this organization is conserved in basal radial glial (bRG) cells, 30 a highly abundant progenitor cell population associated with human brain size expansion. 31 32 33
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