Autism Spectrum Disorder (ASD) constitutes a group of complex, lifelong neurodevelopmental disorder that is characterized by deficiencies in social interaction, verbal communication, and repetitive patterns of behavior. Clinical symptomology varies in severity, with some individuals presenting with more pronounced behavioral deficits than others. The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) describes a spectrum that includes three levels of ASD diagnosis based on the severity of these behavioral deficits, and the amount of parental support that the individual requires. ASD is one of the most prevalent neurodevelopmental disorders, affecting 1 in every 36 children within the United States, with males being 4 times more likely to be diagnosed with ASD than females. Converging studies have attributed genetic heritability as the major contributor to ASD development, although there are multiple environmental risk factors and immune system disorders that are known to cause ASD development. Despite the progress made in understanding ASD, there is not yet a known universal cause or etiology for all ASD cases. Most cases, about 80%, have no known cause and are termed idiopathic ASD. The remaining 20% of syndromic cases have a defined cause which includes genetic origins and environmental toxin exposure. The most prevalent monogenic cause of ASD, Fragile X syndrome, accounts for up to 5% of all cases and is caused by a mutation in the FMR1 gene of the X chromosome. Other rare genetic conditions that cause ASD include Tuberous Sclerosis, Retts syndrome, and Phelan-McDermid syndrome. In conjunction with genetic risk factors, certain environmental factors are known contributors to promoting ASD development. Environmental exposure to Valproic Acid (VPA), air pollution, pesticides, and maternal immune system disorders have also all been associated with ASD development. This diverse range of causes, combined with varying degrees of severity in clinical symptomology across the ASD population, make it challenging to understand the underlying etiology and to treat the condition.
At the moment, there is no cure or effective therapy for treating any of the core ASD symptoms. Pharmaceutical-based intervention strategies, like risperidone, only help to alleviate secondary associated symptoms such as anxiety and irritability. Moreover, stem cell-based treatments for ASD, while promising, are scarce with only a small number of human clinical trials providing varying degrees of success, due in part to the heterogeneity of the ASD patient population and the lack of understanding of the pathogenic mechanisms that underlie ASD. Despite this, recent insights into the etiology of ASD have provided scientists with a better understanding and approach for designing more precise and effective targeted therapies for ASD. Many of these insights converge on the theory of excitation/inhibition(E/I), in which ASD brains are characterized by an imbalance in the ratio between the amount of excitatory and inhibitory signaling. Cellular and molecular level alterations in both the GABA and Glutamatergic signaling systems contribute to promoting these deficits. This imbalance is believed to cause hyper-excitability in cortical circuits which contributes to promoting the core behavioral deficits observed in ASD patients. Notable cellular alterations of the GABA system in ASD, and related neurodevelopmental conditions like Fragile X syndrome, have been characterized recently. In human ASD studies, findings have revealed a significant reduction of Parvalbumin+ (PV+) GABAergic interneurons across three different areas in the prefrontal cortex (PFC) of ASD brains: Brodmann areas (BA) 9, BA 46, and BA 47. Moreover, when we conducted a similar study on Fragile X Postmortem Brains (Chapter 2) we found a significant cortical-wide reduction of PV+ interneurons in BA22,BA4, BA46 and BA24. As the most populous interneuron population in the cerebral cortex, the PV+ interneuron, with its fast-spiking electrophysiological properties, is critical for regulating inhibitory cortical signaling in part through the generation of gamma oscillations. Abnormal cortical gamma wave oscillations were shown to be altered in children with ASD during a stimulus-related electroencephalogram (EEG) test. This suggests that PV+ cell dysregulation may contribute to decreased inhibitory signaling, which is believed to exacerbate an E/I signaling imbalance and ASD-associated behavior deficits
Therefore, designing a cell replacement therapy that specifically targets this cellular component of the GABA signaling system is a very promising approach for alleviating core ASD behavioral deficits. We propose a human-based stem cell transplant therapy where the aim is to transplant human-derived interneuron precursor cells into the brains of VPA rats. VPA-exposed rats, better known as VPA rats, are one of the most used rodent models to study the etiology of ASD. While there are multiple ASD mouse and rat models, we choose to work with the VPA rat model due to its high reliability and phenotypic reproducibility in the field of ASD research. We first developed and characterized our own VPA rat model of ASD (Chapter 3) that presents with similar behavioral and anatomical alterations seen in humans with ASD. We then reprogramed human induced pluripotent stem cells (hIPSC’s) into Medial Ganglionic Eminence (MGE) cells (PV+ GABA precursor cells) in vitro and transplanted them into the prefrontal cortex of 6-week-old VPA rats. We hypothesized that substituting the lost population of PV+ cells in VPA rats would improve E/I signal balance and in turn improve behavioral deficits. Our findings showed that MGE transplants were able to survive, migrate, and mature up to 15 months post transplants. Moreover, our MGE transplants differentiated into mature neuronal lineages, including GABAergic PV+-expressing cells. In line with this, our findings revealed rescue in social and anxiety-like behavioral deficits, and a trend towards improvement in repetitive-like behaviors, 1-month post-transplant. Collectively, our hIPSC-based MGE transplant therapy was effective in alleviating select core and secondary behavioral deficits in VPA-treated rats.
The clinical use of hiPSCs is an intriguing therapeutic approach as it can provide an autologous approach for administering a potential stem cell therapy for patients with ASD. Specifically, hIPSCs can be collected, reprogrammed into GABA precursor cells in vitro, and then transplanted back into the same individual in an autologous fashion, where the possibility of immune rejection would be substantially minimized relative to other stem cell therapeutic approaches. The first autologous human iPSC transplant therapy was conducted in 2014 for macular degeneration. While there are no active trials for neurodevelopmental disorders like ASD, developing the framework for a potential future hIPSC-based trial is imperative and promising. To better understand the rationale behind establishing this targeted therapeutic approach for ASD, it is fundamental to review the current literature that contributed to the framework and design of this novel study. In Chapter 1, we discuss PV+ cell function and PV+ cell-associated pathology in ASD and other neurodevelopmental disorders. In Chapter 2, we will discuss our recent neuropathological findings in human Fragile X brains, which align with our previous findings in ASD, and detail the significant reduction of PV+ cells across multiple cortical areas. In Chapter 3, we will discuss the creation, validity, and characterization of our Valproic Acid Rat model of ASD as a tool to further study ASD etiology. Lastly, in Chapter 4, we will discuss our findings summarizing the therapeutic efficacy of our hiPSC-based MGE therapy in improving behavioral deficits in a VPA rat model of ASD.
