Intercellular communication during human pluripotent stem cell lineage specification

Christopher, Grace A. (2021) Intercellular communication during human pluripotent stem cell lineage specification. Masters thesis, Memorial University of Newfoundland.

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During embryonic germ layer development, cells must communicate with each other and their environment to ensure proper lineage specification and tissue development. Proteins of the connexin (Cx) or pannexin (PANX) protein families mediate cellular communication. Cx’s facilitate direct cell-cell communication through gap junction channels, while PANXs release small molecules into the extracellular space for autocrine or paracrine signalling. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been no comprehensive expression analyses or genetic ablation studies on either Cxs or PANXs during human pluripotent stem cell (PSC) specification to the three embryonic germ lineages. I screened the mRNA profile and select protein expression of human Cx and PANX isoforms in undifferentiated human induced pluripotent stem cells and after directed differentiation into the three embryonic germ lineages: ectoderm, endoderm and mesoderm. RT-qPCR analysis of control iPSCs revealed upregulation of Cx62 in iPSC-derived ectoderm cells, Cx45 upregulation in ectoderm and mesoderm cells, and enrichment of Cx30.3, Cx31, Cx32, Cx36, Cx37 and Cx40 in endoderm. However, Cx43 and PANX1 remain highly expressed in iPSCs as well as cells of each germ lineage. Despite high expression levels, both the Cx43 and the PANX1 CRISPR-Cas9 knockout iPSCs each successfully differentiated into cells of all three germ layers, suggesting that Cx43 and PANX1 are dispensable during directed iPSC lineage specification. This is the first comprehensive analysis of Cx and PANX isoform expression throughout iPSC germ layer segregation. Future studies will reveal whether other Cx or PANX isoforms compensate for the loss of Cx43 and/or PANX1 in the generated knockout iPSCs cells during germ lineage specification.

Item Type: Thesis (Masters)
Item ID: 15164
Additional Information: Includes bibliographical references (pages 97-127).
Keywords: Connexin, Pannexin, Stem Cell, Development, Lineage Specification
Department(s): Medicine, Faculty of > Biomedical Sciences
Date: October 2021
Date Type: Submission
Digital Object Identifier (DOI):
Medical Subject Heading: Induced Pluripotent Stem Cells; Cell Communication; Connexins

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