Abstracts

Rationale: Epilepsy occurs due to an imbalance between neuronal excitation and inhibition. Loss of inhibitory interneurons causes reduced inhibition in neuronal networks and can lead to epilepsy. Cell-based therapy to replace lost inhibitory interneurons holds promise as a treatment of some types of epilepsy. Using a human neural stem cell line (hNSC), which is able to provide a renewable source of neural precursor cells in which the vast majority of neurons are inhibitory interneurons, we aimed to confirm the functional integration of grafted cells within host brain circuitries.Methods: hNSCs were serially passaged with growth factors using an established non-adherent culturing procedure known as the Neurosphere Assay (Delleyrolle and Reynolds, 2009) and transfected with Lentivirus encoding green fluorescent protein (GFP). To avoid rejection by the host immune system, NOD scid IL2 receptor gamma chain knockout (NSG) mouse, an immunodeficient mouse, was utilized as the host for hNSC transplantation. GFP+ hNSCs were collected, triturated into single-cell suspensions, and transplanted into the neocortex of P1 NSG mouse pups (~200,000/animal). Four weeks later, whole cell recordings were performed on cortical slices. GFP+ hNSCs were identified using fluorescence microscopy. The firing properties, spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) of hNSC-derived fast- and regular-spiking neurons (FS and RS) in layer IV to V of somatosensory cortex were recorded and compared to host (mouse) neurons.Results: The majority of GFP+, hNSC-derived neurons had round, fusiform or ovoid soma that were characteristic of interneurons and were initially chosen for recording from FS interneurons. A small proportion of neurons had triangular soma and a main apical dendritic trunk that were initially chosen for recording from pyramidal RS neurons. Nine GFP+, donor-derived neurons demonstrated fast and non-adapting firing patterns (FS neurons) during depolarizing currents (300 pA), and seven of them were parvalbumin (PV)-positive interneurons. Four pyramidal GFP+ neurons demonstrated regular and adapting firing patterns (RS neurons). The frequency of action potential firing of GFP+ FS and RS neurons was not significantly different from FS and RS host neurons (Table 1). The frequency and amplitude of sEPSCs and sIPSCs from GFP+ FS and RS neurons and host neurons were quantified and compared. In FS and RS neurons, there were no differences between the host and grafted GFP+ neurons (Table 1). Conclusions: Four weeks after transplantation, hNSC-derived neurons in mouse neocortex have firing properties and synaptic activities similar to host neurons. Our results suggest that GFP+ hNSC neurons are competent to integrate functionally into host neuronal networks. This provides promising data on the potential for hNSCs to serve as therapeutic agents in diseases with abnormal neuronal circuitry such as epilepsy. This work was supported by the Citizens United for Epilepsy Research (CURE), the McKnight Brain Research Foundation, the Densch Foundation, the Wells Fund and Mead Johnson Nutrition.