Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells
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RESEARCH
Open Access
Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells Shumei Zhao1†, Kui Duan1†, Zongyong Ai1, Baohua Niu1, Yanying Chen1, Ruize Kong1 and Tianqing Li1,2*
Abstract Background: Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into cortical neurons for disease modeling and regenerative medicine. However, these procedures are hard to provide sufficient cells for their applications. Using a combination of small-molecules and growth factors, we previously identified one condition which can rapidly induce hPSCs into neuroepithelial stem cells (NESCs). Here, we developed a scalable suspension culture system, which largely yields high-quality NESC-spheres and subsequent cortical neurons. Methods: 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. Results: The optimized culture system is more conducive to large-scale suspension production of NESCs. These largely expanded NESC-spheres maintain unlimited self-renewal ability and NESC state by retaining their uniform sizes, high cell vitalities, and robust expansion abilities. After long-term expansion, NESC-spheres preserve high purity, homogeneity, and normal diploid karyotype. These expanded NESC-spheres on a large scale have strong differentiation potential and effectively produce mature cortical neurons. Conclusions: 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. Keywords: Human pluripotent stem cells, Neuroepithelial stem cells, Large-scale suspension culture, Cortical neurons
* Correspondence: [email protected] † Shumei Zhao and Kui Duan contributed equally to this work. 1 Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China 2 Xi’an ChaoYue Stem Cell Co, Ltd, Xi’an, China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropri
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