Rui Bai1,2, Yun Chang1,2, Amina Saleem1, Fujian Wu1,2, Lei Tian3, Siyao Zhang1,2, Ya'nan Li1,2, Shuhong Ma1,2, Tao Dong1,2, Tianwei Guo1,2, Youxu Jiang1,2, Yi You4,5, Wen-Jing Lu1,2, Hong Feng Jiang6,7,8, Feng Lan9,10,11,12. 1. Beijing Laboratory for Cardiovascular Precision Medicine, MOE Key Laboratory of Medical Engineering for Cardiovascular Diseases, MOE Key Laboratory of Remodeling-Related Cardiovascular Disease, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, 100029, China. 2. Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, 100029, China. 3. Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China. 4. Center for Clinical Translation and Innovation, Peking University Shenzhen Graduate School, Shenzhen, 518055, China. 5. Shenzhen Bay Laboratory, Shenzhen, 518055, China. 6. Beijing Laboratory for Cardiovascular Precision Medicine, MOE Key Laboratory of Medical Engineering for Cardiovascular Diseases, MOE Key Laboratory of Remodeling-Related Cardiovascular Disease, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, 100029, China. hfjiang@ccmu.edu.cn. 7. Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, 100029, China. hfjiang@ccmu.edu.cn. 8. Beijing Anzhen Hospital, Research Institute Building, Room 323, 2 Anzhen Road, Chaoyang District, Beijing, 100029, China. hfjiang@ccmu.edu.cn. 9. Beijing Laboratory for Cardiovascular Precision Medicine, MOE Key Laboratory of Medical Engineering for Cardiovascular Diseases, MOE Key Laboratory of Remodeling-Related Cardiovascular Disease, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, 100029, China. fenglan@ccmu.edu.cn. 10. Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, 100029, China. fenglan@ccmu.edu.cn. 11. State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. fenglan@ccmu.edu.cn. 12. Beijing Anzhen Hospital, Research Institute Building, Room 319, 2 Anzhen Road, Chaoyang District, Beijing, 100029, China. fenglan@ccmu.edu.cn.
Abstract
INTRODUCTION: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. METHODS: We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. RESULTS: HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express β-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. CONCLUSIONS: This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons.
INTRODUCTION:Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. METHODS: We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. RESULTS: HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express β-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. CONCLUSIONS: This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons.
Authors: A Iwanami; S Kaneko; M Nakamura; Y Kanemura; H Mori; S Kobayashi; M Yamasaki; S Momoshima; H Ishii; K Ando; Y Tanioka; N Tamaoki; T Nomura; Y Toyama; H Okano Journal: J Neurosci Res Date: 2005-04-15 Impact factor: 4.164
Authors: Y Ogawa; K Sawamoto; T Miyata; S Miyao; M Watanabe; M Nakamura; B S Bregman; M Koike; Y Uchiyama; Y Toyama; H Okano Journal: J Neurosci Res Date: 2002-09-15 Impact factor: 4.164
Authors: F H Gage; P W Coates; T D Palmer; H G Kuhn; L J Fisher; J O Suhonen; D A Peterson; S T Suhr; J Ray Journal: Proc Natl Acad Sci U S A Date: 1995-12-05 Impact factor: 11.205