Short Duration Electrical Stimulation to Enhance Neurite Outgrowth and Maturation of Adult Neural Stem Progenitor Cells

Abstract

New therapies are desperately needed for human central nervous system (CNS) regeneration to circumvent the lack of innate regenerative ability following traumatic injuries. Previously attempted therapies have been stymied by barriers to CNS regeneration largely because of protective mechanisms such as the blood brain barrier, inhibitory molecules, and glial scar formation. The application of electric stimulation (ES) has shown promise for enhancing peripheral nervous system regeneration, but is in its infancy in CNS regeneration. The objective of this study is to better understand how short duration ES can be harnessed to direct adult neural stem progenitor cell (NSPC) neurogenesis, neurite extension, and maturation. Herein, NSPCs were exposed to physiological levels of electrical stimulation of 0.53 or 1.83 V/m (applied power supply setting of 1.2 and 2.5 V) of direct current (DC) for 10 min/days for 2 days with a total differentiation time of 3 days. Culturing conditions consisted of either mitogenic growth factors or the neuronal differentiation factor interferon-γ (IFN-γ). Stimulated NSPCs showed lengths that were over five times longer than unstimulated controls (112.0 ± 88.8 μm at 0.53 V/m vs. 21.3 ± 8.5 μm for 0 V/m with IFN-γ) with the longest neurites reaching up to 600 µm. Additionally, ES resulted in mature neuronal morphologies and signs of differentiation through positive βIII tubulin, neuronal nuclei (NeuN), and better organized filamentous-actin (f-actin) staining with growth cone formation. Additionally, the neurites and soma of stimulated NSPCs showed increases in intracellular Ca(2+) during stimulation, signifying the presence of functional neurons capable of electrical conductance and communication with other cells. Our study demonstrates that short stimulation times (10 min/ day) result in significant neurite extension of stem cells in a quick time frame (3 days). This ES modality is potentially advantageous for promoting axon re-growth at an injury site using delivered adult stem cells; however, significant work still remains to understand both the delivery approach of cells as well as ES application in vivo.

Human Neural Tissues from Neural Stem Cells Using Conductive Biogel and Printed Polymer Microelectrode Arrays for 3D Electrical Stimulation

“…Notwithstanding the need to further investigate the interaction between the insulating layer and cells presently used, any putative negative effects were resolved by ES. Our findings are in keeping with other reports of the effects of ES on rate and fate determination of differentiating NSCs, including our own research, albeit it through the use of CP films for 2D planar ES rather than 3D ES of tissues presently described …”

“…Cellular responses include galvanotaxis, increased intracellular calcium concentration, extracellular matrix assembly, and associated cell proliferation and differentiation . Several recent reports have shown that electrical stimulation (ES) can be used to modulate fate determination of differentiating human neural stem cell (NSCs), including the promotion of neuronal versus glial cell induction and increased neuritogenesis . From our own research, we have demonstrated the use of biphasic electrical current via a conductive polymer (CP), polypyrrole, to promote neurite outgrowth and synaptogenesis of rat primary cortical neurons, as well as differentiation of human NSCs to neurons with longer neurites and increased branching, and concomitant lower induction of neuroglia …”

Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards βIII-Tubulin+ Neurons

“…Short duration electrical stimulation at physiological level (0.53 or 1.83 V/m) was effective in enhancing neurite outgrowth and maturation of adult neural stem progenitor cells [25]. Ariza et al [26] found that the NPCs treated with a 437 V/m direct current (DC) EF aligned perpendicularly to the field vector had a greater tendency to differentiate into neurons, but not into oligodendrocytes or astrocytes, compared to controls.…”

Transspinal direct current stimulation modulates migration and proliferation of adult newly born spinal cells in mice

“…However, it has not been investigated how an applied EF may influence the movements of migratory cells such as adult-born spinal neural cells in vivo. An applied EF also affects cell differentiation, as well as the maturation of isolated adult-born neural cells by causing neural cells to differentiate into neurons, but not glia (11,40). Thus, while the effects of an EF on isolated stem/precursor cells is well established, the effects of an EF on adult-born cells in vivo remain unknown.…”