Alcohol consumption during pregnancy causes various developmental defects, including cognitive deficit. The impairment of the central nervous system in patients of fetal alcohol spectrum disorder (FASD) has been characterized intensively by non-invasive imaging and behavioral evaluations. While Ex vivo studies and animal models provide snapshots of the molecular responses elicited by alcohol, the model systems do not fully mimic the long gestational development of human brain. This proposal aims to develop a 3D in vitro model using induced pluripotent cells (iPSC)-derived cortical spheroids to study the effect of ethanol on cortical development, specifically on the outer subventricular zone (oSVZ). oSVZ is considered as an evolutionary adaptive changes of human corticogenesis to increase the diversity of progenitor population, neuronal production, and brain size. We are proposing to generate human iPSC-derived cortical spheroids in defined, differentiation-promoting culture medium that is optimized for test of alcohol and other chemicals that produce reactive oxidative stress. The development of stratified structure in the spheroids that were generated by our novel protocol mimics the early process of human corticogenesis, which includes the formation of oSVZ like structure. We will first match the developmental timeline of iPSC-derived cortical spheroids by immunohistochemistry and RNA sequencing to human fetal brain, to clearly define the window for in vitro FASD studies (Aim 1). Then we will incubate the iPSC-derived cortical spheroids in ethanol-containing medium for different periods of time to mimic the prenatal exposure. We will assess the effects of ethanol on cortical development by monitoring cell death, morphological change, cellular proliferation and the progenitor cell population with an emphasis on oSVZ (Aim 2). Lastly, leveraging CRISPR/Cas9 genome editing technique, we will demonstrate the applicability of our novel cortical spheroid system for investigation of molecular mechanisms underlying alcohol-mediated cell pathology during human brain development. We will select RBM39 as a target gene that was screened in our laboratory and functions defined in mouse cortex. This study will not only characterize a powerful in vitro platform for studying human cortical development under stress exposure, but also provide crucial insight into the mechanisms of FASD on cortical development at cellular and molecular levels.
Prenatal alcohol exposure has lasting impact on patient?s intellectual development and greatly reduces the quality of lives. It is of high importance to increase our understanding on the effect of alcohol use to developing brain since one in ten pregnant women reported alcohol use during pregnancy. This study proposes to validate our novel in vitro model using human iPSC-derived spheroids to investigate the cellular responses to ethanol exposure during early cortical development and the native molecular mechanism.
