Abstract
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.
Highlights
The connexin (Cx) family of gap junction-forming proteins facilitates the direct exchange of small messenger molecules between adjacent cells
Given that aberrant gap junction intercellular communication (GJIC) is linked to numerous diseases [8], we investigated the expression of 11 connexin isoforms in human control induced pluripotent stem cells (iPSCs) and in their subsequent directed differentiation to the three embryonic germ layers: ectoderm, mesoderm, and definitive endoderm (Figure 1 and Table 5)
Ectoderm differentiation was accompanied by a 1.451 ± 0.3001 fold increase in Cx45 transcripts (p < 0.05) and 30.68 ± 20.94 fold increase in Cx62 (p < 0.01) transcripts compared to undifferentiated iPSCs (Figure 1)
Summary
The connexin (Cx) family of gap junction-forming proteins facilitates the direct exchange of small messenger molecules between adjacent cells. Cx proteins form hexameric hemichannels at the cell surface which dock to those on neighboring cells and facilitate gap junction intercellular communication (GJIC) [2,3]. Homomeric and homotypic channels arise from the assembly of a single connexin isoform, while heteromeric and heterotypic channels involve the intermixing of related connexin isoforms within a single hemichannel or gap junction channel. Multiple Cxs can be expressed in the same cell types, increasing communication complexity [7]. This high level of Cx isoform intermixing creates a complex hierarchy of channel selectivity to exquisitely regulate cell signaling
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