Human neural stem cells derived from fetal human brain communicate each other and rescue ischemic neurons through tunneling nanotubes

Pre-clinical trials have demonstrated the neuroprotective effects of transplanted human neural stem cells (hNSCs) during the post-ischemic phase. However, the exact neuroprotective mechanism remains unclear. Tunneling nanotubes (TNTs) are long plasma membrane bridges that physically connect distant cells, enabling the intercellular transfer of mitochondria, contributing to post-ischemic repair processes. Whether hNSCs communicate through TNTs and their role in post-ischemic neuroprotection remain unknown. In this study, non-immortalized hNSC lines derived from fetal human brain tissues were examined to explore these possibilities and assess the post-ischemic neuroprotection potential of these hNSCs. Using Tau-STED super-resolution confocal microscopy, live cell time-lapse fluorescence microscopy, electron microscopy, and direct or non-contact homotypic co-cultures, we demonstrated that hNSCs generate nestin-positive TNTs in both 3D neurospheres and 2D cultures, though which they transfer functional mitochondria. Co-culturing hNSCs with human neurons revealed heterotypic TNTs allowing mitochondrial transfer from hNSCs to neurons. To investigate the role of heterotypic TNTs in post-ischemic neuroprotection, neurons were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R) with or without hNSCs in direct or non-contact co-cultures. Compared to normoxia, OGD/R neurons became apoptotic with impaired electrical activity. When OGD/R neurons were co-cultured in direct contact with hNSCs, heterotypic TNTs enabled the transfer of functional mitochondria from hNSCs to OGD/R neurons, rescuing them from apoptosis and restoring the bioelectrical profile toward normoxic neurons. This complete neuroprotection did not occur in the non-contact co-culture. In summary, our data reveal the presence of a functional TNTs network containing nestin within hNSCs, demonstrate the involvement of TNTs in post-ischemic neuroprotection mediated by hNSCs, and highlight the strong efficacy of our hNSC lines in post-ischemic neuroprotection.