Pannexin 1 Influences Lineage Specification of Human iPSCs

Abstract

Every single cell in the body communicates with nearby cells to locally organize activities with their neighbors and dysfunctional cell-cell communication can be detrimental during cell lineage commitment, tissue patterning and organ development. Pannexin channels (PANX1, PANX2, and PANX3) facilitate purinergic paracrine signaling through the passage of messenger molecules out of cells. PANX1 is widely expressed throughout the body and has recently been identified in human oocytes as well as 2 and 4-cell stage human embryos. Given its abundance across multiple adult tissues and its expression at the earliest stages of human development, we sought to understand whether PANX1 impacts human induced pluripotent stem cells (iPSCs) or plays a role in cell fate decisions. Western blot, immunofluorescence and flow cytometry reveal that PANX1 is expressed in iPSCs as well as all three germ lineages derived from these cells: ectoderm, endoderm, and mesoderm. PANX1 demonstrates differential glycosylation patterns and subcellular localization across the germ lineages. Using CRISPR-Cas9 gene ablation, we find that loss of PANX1 has no obvious impact on iPSC morphology, survival, or pluripotency gene expression. However, PANX1 gene knockout iPSCs exhibit apparent lineage specification bias under 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 increases representation of endodermal and mesodermal populations in PANX1 knockout cells. Importantly, PANX1 knockout iPSCs are fully capable of differentiating toward each specific lineage when exposed to the appropriate external signaling pressures, suggesting that although PANX1 influences germ lineage specification, it is not essential to this process.