Connexin 43 Gene Ablation Does Not Alter Human Pluripotent Stem Cell Germ Lineage Specification

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.

Inhibition of Gap Junctional Intercellular Communication Upregulates Pluripotency Gene Expression in Endogenous Pluripotent Muse Cells

“…In this study, we focused on the role of Cx43 in Muse cells treated with these three different GJIC inhibitors that work by very different biochemical mechanisms to inhibit functional GJIC at the posttranscriptional level, as well as in GJA1 siRNA-transfected Muse cells. Other studies, using iPS cells (Cx43 knockout ( GJA1 −/− )) iPSCs, generated using CRISPR-Cas9 gene ablation), maintained characteristics typical of iPSCs and successfully differentiated into cells of ectoderm, mesoderm, or endoderm lineages [ 49 ]. While these studies were well executed, the interpretation of these results is complicated and might not apply to normal adult organ-specific adult stem cells or normal Muse cells.…”