Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived astrocytes

Abstract

Abstract The P2X7 receptor (P2X7R) is a cation-permeable ionotropic receptor activated by extracellular adenosine 5’-triphosphate (ATP) which has been implicated in numerous diseases of the CNS, including epilepsy. Activation of the P2X7R can trigger diverse responses including the release of pro-inflammatory cytokines, modulation of neurotransmission, cell proliferation or cell death. There have been conflicting reports on the cellular identity of P2X7R-expressing cells in the brain. Expression of P2X7Rs is well documented on microglia and oligodendrocytes but the presence of P2X7Rs on astrocytes remains debated. Furthermore, most functional studies on P2X7R responses have used cells from rodents or immortalised cell lines expressing human P2X7Rs. To assess the endogenous and functional expression of P2X7Rs in human astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into GFAP and S100 β-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining on hiPSC-derived astrocytes and P2X7R protein expression was also confirmed by Western blot analysis. Importantly, stimulation with the potent nonselective P2X7R agonist BzATP or endogenous agonist ATP induced robust calcium rises in hiPSC-derived astrocytes which were blocked by the selective P2X7R antagonists AFC-5128 or JNJ-47965567. Our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived astrocytes and support their in vitro utility in investigating the role of the P2X7R and drug screening in disorders of the CNS.