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During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell–cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.

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, 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 knockout iPSCs exhibit apparent lineage specification bias during 2-dimensional and 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 significantly decreases the proportion of ectodermal cells within spontaneously differentiated cultures, while endodermal and mesodermal representation is increased 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.Graphical 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.