Publication Date

5-2018

Advisor(s)

Laura Grabel

Department

Biology

Abstract

The selective loss or dysfunction of GABAergic inhibitory interneurons creates an excitation/inhibition imbalance in the hippocampal and cortical neural circuits and characterizes numerous neurodegenerative diseases, including temporal lobe epilepsy (TLE). TLE is a seizure disorder that may be associated with aberrant neurogenesis, mossy fiber sprouting, synaptic reorganization, hilar interneuron loss and dysfunction. Our long-term goal is to replenish lost or dysfunctional hippocampal inhibitory interneuron subtypes through cell replacement therapy (CRT) using a human embryonic stem cell (hESC) source. During embryonic development, cortical inhibitory interneuron progenitors arise predominantly from a transient ventral forebrain zone known as the medial ganglionic eminence (MGE) and are characterized by the expression of NKX2.1.

We first optimized an adherent monolayer protocol for the generation of NKX2.1+ neural progenitors from hESCs, using sonic hedgehog treatment. MGE-like progenitors derived using this protocol were FACS-isolated and extensively characterized using transcriptome profiling and in vitro long-term differentiation (Chen et al., 2016). To test the differentiation potential of the NKX2.1 cells in vitro, we utilized a co-culture system with mouse cortical astrocytes; deriving an enriched population of interneurons in which 75% of the MAP2-positive cells were also GABA-positive after 8 weeks (Chen et al., 2016).

We next investigated the ability of human ESC-derived neural progenitors (hESNPs) to mature, correct behavioral deficits, and suppress seizures in epileptic mice. We show that hESNPs grafted into the hippocampus of epileptic mice matured and expressed the neuronal markers Hu, NeuN, and the inhibitory neurotransmitter GABA by twelve weeks posttransplantation. Additionally, a subset of mature transplanted neurons expressed the inhibitory interneuron subtypes somatostatin (SST), parvalbumin (PV), calbindin (CB), or calretinin (CR) by twenty-four weeks after transplantation. Some mice developed neural tumors containing a mixed population of mature neurons and neural stem cells. Using patch clamp analyses, grafted cells exhibited five different types of firing patterns including highly adapting action potentials (APs), fast spiking, bursting with accommodating, and nonaccommodating patterns. Cells also showed inhibitory post-synaptic currents (IPSCs) and excitatory post-synaptic currents (EPSCs). Mice with transplanted cells exhibited significant improvement in the Morris Water Maze spatial memory task as early as six weeks posttransplant, with significantly shorter latencies to the escape platform, and improved search strategies. The transplanted cells did not however suppress recurring seizures in TLE mice, as EEG analyses over a 4-week period showed no significant differences in the average number of seizures per day, seizure durations or seizure severity in mice that received transplants when compared to controls with media injections.

Overall, our findings indicate that transplantation of interneuron progenitors does have the potential to repair some aspects of TLE-induced deficits, like spatial memory deficits. However, more extensive long-term studies looking directly at the synaptic targets are required to address functional integration of the transplanted cells for seizure suppression.

Available for download on Saturday, June 01, 2019

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