Abstract
Various solutions are utilized widely for the isolation, harvesting, sorting, testing and transplantation of neural stem cells (NSCs), whereas the effects of harvesting media on the biological characteristics and repair potential of NSCs remain unclear. To examine some of these effects, NSCs were isolated from cortex of E14.5 mice and exposed to the conventional harvesting media [0.9% saline (Saline), phosphate-buffered saline (PBS) or artificial cerebrospinal fluid (ACSF)] or the proliferation culture medium (PCM) for different durations at 4°C. Treated NSCs were grafted by in situ injection into the lesion sites of traumatic brain injury (TBI) mice. In vitro, harvesting media-exposed NSCs displayed time-dependent reduction of viability and proliferation. S phase entry decreased in harvesting media-exposed cells, which was associated with upregulation of p53 protein and downregulation of cyclin E1 protein. Moreover, harvesting media exposure induced the necrosis and apoptosis of NSCs. The levels of Fas-L, cleaved caspase 3 and 8 were increased, which suggests that the death receptor signaling pathway is involved in the apoptosis of NSCs. In addition, exposure to Saline did not facilitate the neuronal differentiation of NSCs, suggesting that Saline exposure may be disadvantageous for neurogenesis. In vivo, NSC-mediated functional recovery in harvesting media-exposed NSC groups was notably attenuated in comparison with the PCM-exposed NSC group. In conclusion, harvesting media exposure modulates the biological characteristics and repair potential of NSCs after TBI. Our results suggest that insight of the effects of harvesting media exposure on NSCs is critical for developing strategies to assure the successful long-term engraftment of NSCs.
Highlights
Traumatic brain injury (TBI) remains a major cause of morbidity, mortality and long-term disability in children and young adults [1,2]
As treatment was prolonged longer than 8 h, harvesting media exposure resulted in a significant decrease in viable cell in contrast to exposure to proliferation culture medium (PCM) (Saline, phosphate buffered saline (PBS) and artificial cerebrospinal fluid (ACSF), p,0.001 versus PCM on 8, 12, 16 and 24 h; n = 5 per group per time point) and this decrease occurred in a time-dependent manner (p,0.001; n = 5 per group per time point)
When neural stem cells (NSCs) were exposed to the harvesting media for more than 8 h, the proportion of viable cells was lowest in the Saline treatment group, followed in order by the PBS group, and the ACSF group (ACSF, p,0.001 versus Saline or PBS on 8, 12, 16 and 24 h; PBS, p,0.001 versus Saline on 12, 16 and 24 h n = 5 per group per time point)
Summary
Traumatic brain injury (TBI) remains a major cause of morbidity, mortality and long-term disability in children and young adults [1,2]. Transplanted NSCs have the capacity to migrate long distances to lesion sites and to improve functional recovery after brain injury. Under appropriate conditions, they can differentiate into neuronal and glial lineages and induce the regeneration of damaged brain tissue [4,5]. In the current study, aiming to optimize the NSC transplantation regimen, maximize the NSC therapeutic potential and develop strategies to assure successful long-term engraftment of NSCs, we investigated the effects of harvesting media exposure on the biological properties and repair function of NSCs. We found that exposure to harvesting media modulated the viability and proliferation of NSCs in a time dependent manner and attenuated the repair potential of NSCs for TBI
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