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 therapy for SCI by transplantation. In our study, we aimed to establish a chemically defined culture system by using serum-free medium and ascorbic acid (AA) to generation and expansion of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably.
Methods: We induce hESC/iPSC to neurospheres by using a newly established induction system in vitro in our study. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction systems, including conventional N2 medium with gelatin-coated (coated) and N2+AA medium without pre-coated (AA) were characterized by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons and the microelectrode array (MEA) recording system was used to evaluate the functionality of neurons differentiated from lt-NES cells. Moreover, the mechanism of AA-induced lt-NES cells was explored through RNA-seq and the use of inhibitors.
Results: HESCs/iPSCs were efficiently induced to neurospheres by using a newly established induction system in vitro. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction system (coated vs AA) both expressed neural pluripotency-associated genes PAX6, NESTIN, SOX1, SOX2. After long-term cultivation, we found that they both can maintain the long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retain the ability to differentiate into general functional neurons that highly express β-tubulin. 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 pathways.
Conclusions: Taken together, this new chemically defined culture system is stable and effective to generate and culture the lt-NES cells induced by hESCs/iPSCs using serum-free medium combined with ascorbic acid (AA). The lt-NES cells under this culture system can maintain the long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Keywords: Spinal cord injury, Neurospheres, Ascorbic acid, lt-NES cells, Human pluripotent stem cells.