Abstract
My work investigated the effects of VPA on MGE-derived cells in the rat cortex and the targeted differentiation of hiPSCs to MGE cells. VPA exposure in utero is a known risk factor for the development of ASD. MGE cells give rise to inhibitory cortical neurons—a neuronal subtype that is often diminished in ASD. ASD refers to a group of heterogeneous behaviorally defined neurodevelopmental disorders affecting 1 in 68 children in the U.S. There is no known cure for ASD and treatment options are limited. Dozens of genes are currently strongly associated with ASD, but no physiological or genetic tests are currently available for the diagnosis of ASD. Given the complex etiologies of ASD and the deficit of treatment options, further investigation is needed. In this study, we used the VPA-induced rat model of ASD to study neuronal phenotypes with an emphasis on cortical inhibitory neurons. In many cases of ASD, there is an improper ratio of neuronal excitation and inhibition—ASD brains have a decrease in the number of inhibitory neurons in the cerebral cortex. Prenatal exposure to VPA, an anti-epileptic drug, is associated with an increased prevalence of ASD in humans. Indeed, VPA-exposed rodent models—which exhibit an increase in ASD-like behaviors—have a decrease in inhibitory neuron protein markers. One aim of my project was to further characterize the brains of VPA-exposed rats. Inhibitory cortical neurons are derived from the MGE, a transitory structure of the embryonic brain. Another aim of my project was to investigate whether hiPSCs could serve as an efficient source of MGE cells for future research into transplantation as a corrective therapy for individuals with ASD. Stem cell transplantation has shown potential to treat epilepsy and neurodegenerative diseases such as Parkinson’s in rodent models. Our lab is currently studying if the transplantation of MGE cells into the cortex of adult rats can improve ASD symptoms. There are currently no studies that have addressed this question. For this project, MGE cells were isolated from embryonic rat or produced via targeted differentiation from hiPSCs using treatment with SHH agonist and dual SMAD inhibitors. MGE cells were assessed for MGE cell markers, Nkx2.1 and Lhx6, via immunocytochemistry to ensure the correct cells were harvested or produced. To induce our ASD rat model, rats were exposed to 400 mg/kg of VPA at one of two gestational time points, E12 and E15, during a critical window in the development of cortex-bound interneurons. To characterize changes in the number and location of MGE-derived cells in the cortex of the postnatal ASD rat, cells were visualized via immunohistochemistry at P1, P6, P12, and P30. MGE-derived cells were stained for MGEderived cell markers, Lhx6 and PV. To characterize changes in the numbers of BCs and ChCs in the P30 ASD rat cortex, cells were visualized via immunohistochemistry. Cells were stained for inhibitory neuron subtype markers, PV and VVA. Changes in the numbers of PV+/VVA+ BCs and PV+/VVA- ChCs in the cerebral cortex were quantified using Stereo Investigator software following fluorescent microscopy. I found that our VPA-induced rat model of ASD supports previous studies showing that human ASD cases had fewer PV+/VVA- ChCs in the cortex. I also found that while there was a decrease in the number of PV+ cells and changes in the location of PV+ cells in the cortex, further investigation is needed to determine if VPA exposure causes changes in Lhx6+ cells. I was also able to reproduce a targeted differentiation protocol, successfully producing human GFP-MGE cells. Current preliminary transplantation tests show GFP-hiPSC-MGE survival. Future research will determine if MGE cell transplantation can ameliorate ASD neuronal and behavioral symptoms in rats.