Associate Professor University of Illinois at Urbana-Champagin, United States
Introduction:: In vitro neural networks derived from neural stem cells (NSCs) have gained attention recently for various applications, ranging from brain-on-a-chip devices to therapies. However, there is still a need to fabricate electrophysiologically active neural networks. Several factors, including growth factors and the extracellular microenvironment, influence the differentiation and subsequent neural network of NSCs. Among these factors, the basic fibroblast growth factor (bFGF) promotes NSC migration and survival. Therefore, we investigated the potential of bFGF to accelerate neural network formation and enhance electrophysiological activity using mouse cortical NSCs.
Materials and Methods:: Mouse cortical NSCs were cultured in Matrigel with a seeding density of 0.5M cells/cm2. The NSCs were then differentiated for two weeks using differentiation media, either with or without bFGF. Immunofluorescent images of the two conditions were captured to assess neural outgrowth and branching, and neurons were reconstructed using IMARIS neural tracing. The reconstructed neurons were analyzed for neurite length and Sholl intersection. Additionally, calcium flux imaging was performed to examine neural activities, including the calcium flux peak and synchronicity.
Results, Conclusions, and Discussions:: The bFGF(+) condition exhibited higher neurite outgrowth and branching than the bFGF(-) condition. A cell cluster was formed in the bFGF(-) condition, and neurite outgrowth and branching were not observed (Figure 1A). Among the bFGF(+) condition, those treated with a higher concentration of bFGF showed longer neurite length and a more significant number of Sholl intersections over a broader range of distances from the soma (Figure 1B). Furthermore, the calcium flux imaging results revealed that the bFGF(+) condition exhibited higher calcium rises and greater synchronicity of calcium flux (Figure 1C-E).
Our study supports the hypothesis that bFGF treatment of NSCs accelerates neural branching and the formation of neural networks, resulting in enhanced intracellular calcium flux activities. Further investigations are underway to determine the optimal dose and extracellular microenvironment for the rapid and efficient fabrication of neural networks physiologically close to in vivo neural networks.
Acknowledgements (Optional): : This work was partially supported by grants from the National Institutes of Health (R61 HL159948) and the National Science Foundation Expedition 'Mind in Vitro' Award (IIS-2123781).
