Shuai Li1,2,3,4, Huifang Zhao1,2,3, Xiaobo Han1,2,3,4, Bin Ni5, Lang He6, Omar Mukama1,2,3,4, Jean de Dieu Habimana4, Zuoxian Lin1,2,3,4, Rongqi Huang1,2,3,4, Hualin Huang1,2,3,4, Chao Tian1,2,3, Feng Tang1,2,3,6, Zhiyuan Li7,8,9,10,11,12. 1. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China. 2. Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China. 3. GZMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, China. 4. University of Chinese Academy of Sciences, Beijing, 100049, China. 5. NHC Key Laboratory of Birth Defect for Research and Prevention, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, 410008, Hunan, China. 6. Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China. 7. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China. li_zhiyuan@gibh.ac.cn. 8. Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China. li_zhiyuan@gibh.ac.cn. 9. GZMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, China. li_zhiyuan@gibh.ac.cn. 10. University of Chinese Academy of Sciences, Beijing, 100049, China. li_zhiyuan@gibh.ac.cn. 11. NHC Key Laboratory of Birth Defect for Research and Prevention, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, 410008, Hunan, China. li_zhiyuan@gibh.ac.cn. 12. Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China. li_zhiyuan@gibh.ac.cn.
Abstract
BACKGROUND: Neural stem cell (NSC) therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore, the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent. METHODS: We reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis, and in vitro as well as in vivo differentiation capabilities, etc.). RESULTS: Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 °C, 5% CO2) recovered their typical morphology, and retained their proliferation and differentiation abilities. CONCLUSIONS: In this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.
BACKGROUND: Neural stem cell (NSC) therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore, the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent. METHODS: We reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis, and in vitro as well as in vivo differentiation capabilities, etc.). RESULTS: Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 °C, 5% CO2) recovered their typical morphology, and retained their proliferation and differentiation abilities. CONCLUSIONS: In this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.
Entities:
Keywords:
Ambient temperature; Human induced pluripotent stem cells; Neurospheres; Transportation; hiPSC-derived human neural stem cells