Large-scale Production Of Human Neuroepithelial Stem Cells And Purification Of NESC-derived Exosomes

The emergence of human embryonic stem cells(ESCs)and induced pluripotent stem cells(i PSCs)provides a renewable source of cells.Neural stem cells derived from pluripotent stem cells can be used for cell therapy of neurological diseases,but traditional adherent culture cannot meet the needs of cell therapy.Using a combinatorial small-molecules and growth factors,we previously identified one condition which can rapidly induce ESCs into neuroepithelial stem cells(NESCs).Here,we have established a stable and repeatable large-scale suspension culture system of NESCs,which can be used for cell therapy,disease modeling and drug screening.We first optimized the NESCs medium in low-attachment plates and compared the quality of NESC-spheres in the two pre-optimized and post-optimized culture systems.The optimized culture system is more conducive to the suspension culture of NESCs.Subsequently,the suspension culture system supported expansion from a petri dish to a stirred suspension bioreactor,producing large-scale NESC-spheres,with a rate of 60 rpm,cells can stably maintain self-renewal.Through transcriptome RNA-seq and single cell RNA-seq analysis,NESC-spheres can stably maintain identity,with high purity and homogeneity.Therefore,we established a serum-free,simple composition,and lowercost suspension culture system that can be used for large-scale production of NESCs.The NESC-spheres from this system: uniform sphere size,high cell viability(> 95%),and strong proliferation ability;high purity and homogeneity,normal diploid karyotype;maintain self-renewal;stably maintain the characteristics of NESCs,capable of selfreorganization to form a neural tube-like structure.Abnormal development or damage of the cerebral cortex may lead to neurological diseases.Once the cortex is damaged,it is difficult to regenerate new nerves.NESCs was induced to spontaneously differentiate by removing the growth factors and small molecules in the medium,confirming that NESC-spheres have a strong differentiation potential,and more than 90% of cells can differentiate into neurons.Long-term expansile suspension culture does not affect its ability to differentiate into cortical neurons,and it can differentiate into different types of mature cortical neurons.Exosomes have a variety of biological functions,carrying nucleic acids,proteins,and lipids,and can mediate the exchange of information between cells.They have been widely used in the diagnosis and treatment of diseases.However,the purification of exosomes requires a large number of cells,which restricts the application of exosomes.Here,we have established a system for large-scale production of NESCs,so we want to test whether the system can be used to produce exosomes.Subsequently,we used ultracentrifugation and size exclusion chromatography to extract the exosomes secreted by NESC-spheres in suspension culture system.The exosomes extracted by these two methods have a complete double-layer phospholipid membrane structure with a size between 40?120 nm,and both express CD63 and TSG101.Single-particle nanoflow analysis showed that the exosomes extracted by size exclusion chromatography had higher particle concentration.Thus,exosomes extracted by size exclusion chromatography have higher purity,which will be used in the study of exosome function and clinical transformation in the future.