Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells

We developed a serum-free, defined, and low-cost culture system for large-scale expansion of NESCs in stirred suspension bioreactors. The stable and controllable 3D system supports long-term expansion of highquality and homogeneous NESC-spheres. These NESC-spheres can be used to efficiently give rise to cortical neurons for cell therapy, disease modeling, and drug screening in future. Overall design: The NESC medium was first optimized, and the suspension culture system was then enlarged from plates to stirred bioreactors for large-scale production of NESC-spheres by a stirring speed of 60 rpm. During the expansion, the quality of NESC-spheres was evaluated. The differentiation potential of NESC-spheres into cortical neurons was demonstrated by removing bFGF and two pathway inhibitors from the NESC medium. Cellular immunofluorescence staining, global transcriptome, and single-cell RNA sequencing analysis were used to identify the characteristics, identities, purities, or homogeneities of NESC-spheres or their differentiated cells, respectively