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我们开发了一套无血清、化学成分限定且低成本的培养体系，可用于搅拌式悬浮生物反应器内神经外胚层干细胞（NESCs）的大规模扩增。该稳定可控的三维培养体系能够支持高质量、均一的神经外胚层干细胞球进行长期扩增。此类神经外胚层干细胞球未来可高效分化为皮层神经元，应用于细胞治疗、疾病模型构建及药物筛选领域。整体实验设计如下：首先对神经外胚层干细胞培养基进行优化，随后将悬浮培养体系从培养板扩增至搅拌式生物反应器，以60 rpm的搅拌转速实现神经外胚层干细胞球的规模化生产。扩增过程中，对神经外胚层干细胞球的质量开展评估。通过从神经外胚层干细胞培养基中移除碱性成纤维细胞生长因子（bFGF）及两种通路抑制剂，验证了神经外胚层干细胞球向皮层神经元分化的潜能。分别采用细胞免疫荧光染色、全转录组测序及单细胞RNA测序分析，对神经外胚层干细胞球及其分化细胞的特征、身份属性、纯度与均一性进行鉴定。