ABSTRACT: Fetal hypoxia, characterized by reduced oxygen levels, adversely impacts fetal development and contributes to 23% of neonatal deaths worldwide. Notably, the developing brain, particularly in its immature stage, is highly susceptible to hypoxic insult, which increases the risk of neural defects, brain damage, and long-term cognitive impairments in offspring. Nevertheless, the specific cell types affected and the underlying molecular mechanisms that mediate hypoxic brain injury remain incompletely understood. In this study, we established a three-dimensional human brain organoid modeling fetal hypoxic brain injury. Utilizing single-cell transcriptomic analysis, we observed a global hypoxic response in cortical progenitors and neurons, characterized by enhanced HIF-1 signaling and increased expression of hypoxia-responsive genes following hypoxic insult. Under hypoxic conditions, glycolysis, reactive oxygen species pathway and oxidative phosphorylation were simultaneously upregulated. Furthermore, hypoxia led to a decrease in cell division and a suppression of neurotrophin signaling pathways. Regarding developmental stage, cortical progenitors exhibited minimal sensitivity to hypoxia, with ventral radial glia, outer radial glia, and intermediate progenitors (IPs) being largely unaffected. In contrast, glia progenitors displayed a slight tendency towards premature maturation. Conversely, hypoxia had a detrimental effect on the maturation of developing neurons, including glutamatergic and GABAergic neurons. Significantly, we identified two distinct populations of GABAergic neurons with differing characteristics. GABAergic neuron type 1 exhibited lower expression levels of GAD1 and GAD2 and greater resistance to hypoxia compared to GABAergic neuron type 2. Notably, GABAergic neuron type 2 was the most susceptible to hypoxia among all cell types, showing increased ferroptosis, disrupted axon development, and impaired synapse function, potentially due to decreased Hedgehog signaling. Additionally, specific responses to hypoxia in developing neurons included selective reduction in fatty acid synthesis in outer radial glia cells, glutamatergic neurons and GABAergic neuron type 1, selective downregulation of mTORC1 signaling in IPs, selective elevation of unfolded protein response in GABAergic neuron type 1, and selective activation of TNFα-signaling via NFκB in IPs, glutamatergic neurons and GABAergic neuron type 2. In summary, our study provides a comprehensive understanding of the distinct cellular responses among cortical progenitors and neurons in fetal hypoxic brain injury, providing potential therapeutic targets for this condition.
