A Robust System Of Embryonic Stem Cells Differentiation Into Neural Epithelium Stem Cells And Cortical Projection Neurons

In the process of embryonic development, notochord induced the dorsal midline of ectoderm (neural ectoderm) to form the neural plate, the neuroepithelial stem cell of neural plate, arranged in radiated multilayer-circular, decreased gradually in thickness but increased in length, eventually forming a tubular structure and developed into neural tube (NT). Soon after the closing of neural tube, its lumen specializes into ventricle and its dorsal-most part individualizes into telencephalon. The neuroepithelial stem cells, located in the ventricle for proliferation amplification and then converted into radial glial stem cells, migrate along several stereotyped routes within embryonic tissues and finally settle and differentiate in various elected locations forming the cerebral cortex.During the development of nervous system, the dysplasia of neural tube will lead to neurological dysfunction, and developing a model of primate neural tube development is important to promote many NT disorder studies in model organisms. Here, we report a robust and stable systemto allow for clonal expansion of singlemonkey neuroepithelial stem cells (NESCs) to develop into miniature NT-like structures. Single NESCs can produce functional neurons in vitro, survive, and extensively regenerate neuron axons inmonkey brain. NT formation and NESC maintenance depend on highmetabolismactivity andWnt signaling. NESCs are regionally restricted to a telencephalic fate. Moreover, single NESCs can turn into radial glial progenitors (RGPCs). The transition is accuratelyregulated byWnt signaling through regulation ofNotch signaling and adhesionmolecules. Finally, using the "NESC-TO-NTs" system, we model the functions of folic acid (FA) on NT closure and demonstrate that FA can regulate multiple mechanisms to prevent NT defects. Our system is ideal for studying NT development and diseases.The cerebral cortex is one of the most complicated tissues in our brain, and its impairments result in neurodevelopmental and neurodegenerative disorders.which is comprised of six horizontal layers, the cell of first layer were Cajal-Retzius neurons, 2,3,4 layer were upper layer projection neurons, and 5,6 layer were deeper layer projection neurons. Accordingly to their distinct locations and synaptic connectivity patterns, deeper layer projection neurons are classified as corticofugal projection neurons (CfuPNs). Efforts to study development and function of corticofugal projection neurons (CfuPNs) in the human cerebral cortex for health and disease have been limited by the availability of highly enriched CfuPNs. Here, we develop a robust, two-step process for generating CfuPNs from human embryonic stem cells (hESCs): directed induction of neuroepithelial stem cells (NESCs) from hESCs and efficient differentiation of NESCs to about 80% of CfuPNs. NESCs or a NESC faithfully maintain unlimitedly self-renewal and self-organized abilities to develop into miniature neural tube-like structures. NESCs retain a stable propensity toward neuronal differentiation over culture as fate-restricted progenitors ofCfuPNs and interneurons. When grafted into mouse brains, NESCs successfully integrate into the host brains, differentiate into CfuPNs and effectively reestablish specific patterns of subcortical projections and synapse structures. Efficient generation of CfuPNsin vitro and in vivo will facilitate human cortex development and offer sufficient CfuPNs for cell therapy.