Neurotrophin-induced migration and neuronal differentiation of multipotent astrocytic stem cells in vitro

Abstract

Hypoxic ischemic encephalopathy (HIE) affects 2-3 per 1000 full-term neonates. Up to 75% of newborns with severe HIE die or have severe neurological handicaps. Stem cell therapy offers the potential to replace HIE-damaged cells and enhances the autoregeneration process. Our laboratory implanted Multipotent Astrocytic Stem Cells (MASCs) into a neonatal rat model of hypoxia-ischemia (HI) and demonstrated that MASCs move to areas of injury in the cortex and hippocampus. However, only a small proportion of the implanted MASCs differentiated into neurons. MASCs injected into control pups did not move into the cortex or differentiate into neurons. We do not know the mechanism by which the MASCs moved from the site of injection to the injured cortex. We found neurotrophins present after the hypoxic-ischemic milieu and hypothesized that neurotrophins could enhance the migration and differentiation of MASCs. Using a Boyden chamber device, we demonstrated that neurotrophins potentiate the in vitro migration of stem cells. NGF, GDNF, BDNF and NT-3 increased stem cell migration when compared to a chemokinesis control. Also, MASCs had increased differentiation toward neuronal phenotypes when these neurotrophins were added to MASC culture tissue. Due to this finding, we believed neurotrophins could guide migration and differentiation of stem cell transplants after brain injury.

Figure 1. Migrated MASC (Mean and SEM) under different conditions (y axis).

Panel A and B: GDNF at 10, 50 and 100 ng/mL concentrations during 1 and 3 days, respectively. Panel C and D: BDNF at 10, 50 and 100 ng/mL concentrations during 1 and 3 days. Panel E and F: NT-3 at 50, 100 and 150 ng/mL during 1 and 3 days, respectively. Panel F and G: NGF at 200, 300 and 400 ng/mL during 1 and 3 days, respectively. Each panel includes negative control (no neurotrophins added), chemokinesis (random movement control) and positive control (10% FBS). Statistically significant differences between chemokinesis and specific concentrations of neurotrophin were noted with an asterisk (p<0.05). The bars over the groups show statistically significant differences between different neurotrophins’ concentrations.

Figure 2. Percentage β-3 tubulin positive cells (Mean and SEM) under different neurotrophins conditions.

Panel A: GDNF at 10, 50 and 100 ng/mL concentrations. Panel B: BDNF at 10, 50 and 100 ng/mL concentrations. Panel C: NT-3 at 50, 100 and 150 ng/mL. Panel D: NGF at 200, 300 and 400 ng/mL. The asterisks (**) represent statistically significant differences between the negative control and specific neurotrophin (p<0.01). The only significant difference between different concentrations of neurotrophins existed between NGF 200 and 400 ng/mL.