Human IP₃ receptor triple knockout stem cells remain pluripotent despite altered mitochondrial metabolism

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ER Ca²⁺-release channels that control a broad set of cellular processes. Animal models lacking IP₃Rs in different combinations display severe developmental phenotypes. Given the importance of IP₃Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP₃R and triple IP₃R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP₃R isoforms, IP₃R1, IP₃R2, IP₃R3, failed to generate Ca²⁺ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP₃Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP₃Rs in human cell types of interest.