Boundary cap neural crest stem cells exhibit remarkable resilience to environmental stressors associated with International Space Station mission

Boundary cap neural crest stem cells (BCs) previously demonstrated resilience during short-term microgravity exposure. In this study, BCs were sent to the International Space Station aboard Axiom Mission 3. Owing to launch regulations and weather delays, cells remained outside controlled culture conditions longer than anticipated, exceeding expected survival limits. Three BC populations were included: naive BCs (NBC), once-flown BCs (V15), and twice-flown BCs (V1415), cultured as neurospheres or within 3D-printed scaffolds. Corresponding ground controls were maintained under matched conditions. Only V1415 cells survived in both flight and ground groups, while NBC cells survived exclusively after spaceflight. V15 cells did not survive. All surviving cells exhibited reduced proliferation, contrasting with the enhanced proliferation reported after short-term microgravity exposure. Following 1 month of post-flight expansion, surviving cells retained the capacity to differentiate into neurons and glial cells without detectable electrophysiological impairment. Space-exposed NBC cultures showed increased glial differentiation, whereas V1415 flight cultures displayed a higher neuronal proportion than ground controls. BCs cultured in 3D-printed scaffolds exhibited robust survival and significantly increased proliferation after space exposure. Exosome analysis identified miRNAs associated with enhanced proliferation in flight samples. These findings demonstrate the remarkable stress resistance, developmental plasticity, and adaptability of BCs during prolonged spaceflight conditions.