P2X purinergic receptors are required for correct cortical development in human brain organoids

The human neocortex represents a crucial evolutionary advance, the formation of which requires the tight and precise orchestration of both intracellular and extracellular signals. Structures grown in three-dimensional cultures, specifically human-induced pluripotent stem cells (hIPSCs)-derived cerebral organoids (COs), have been fundamental to study the signals that regulate the formation of the cortex, overcoming the limitations of 2D cultures. Amongst these, purinergic signaling driven by extracellular ATP and other nucleotides may encode crucial intercellular communications that govern central nervous system (CNS) development. The ATP that accumulates in the extracellular milieu can interact with both ionotropic P2X and metabotropic P2Y receptors on cells to exert its modulating effects. Although widely studied in different animal models, little is known about the expression and function of this signaling system in the human cortex. Thus, here we analyzed the expression of P2X receptor subunits comprehensively throughout the entire process of CO development, confirming that P2X receptors are functional in ventricular structures of the human cortex. Specifically, we detected the expression of P2X1, P2X4, and P2X6 in CO, showing distinct distributions in Nestin⁺ radial glial cells and/or DCX⁺ newborn neurons. Significantly, we also show how prolonged pharmacological inhibition of P2X activity affects CO development, resulting in smaller organoids with fewer and less well-organized cortical ventricles. Altogether, our findings point to a relevant role of purinergic signaling during the formation of the human cerebral cortex.