Pannexin1 in human pluripotent stem cells and its influence on early cell fate decisions

Noori (Frohlich), Rebecca J. (2021) Pannexin1 in human pluripotent stem cells and its influence on early cell fate decisions. Masters thesis, Memorial University of Newfoundland.

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Abstract

Human development requires cells to work together within their environment to successfully differentiate and organize into complex tissues. Pannexin1 (PANX1) is a channel-forming protein that facilitates cell-cell communication by conveying small messenger molecules across cellular membranes. PANX1 is expressed in the early embryo and persists in most adult tissues. Dysfunctional PANX1 activity has been implicated in several human pathologies including oocyte cell death and neurological disease. Given its expression in the early embryo and its link to disease, we investigated PANX1’s involvement in cell fate specification to the embryonic germ layers and during complex, 3-dimensional human cerebral organoid development. Using immunofluorescence, flow cytometry and quantitative RT-PCR, we find that human induced pluripotent stem cells (iPSCs) express PANX1. iPSCs subjected to CRISPR-Cas9 PANX1 genetic ablation show no changes in cellular morphology, pluripotency gene expression, proliferation, or apoptosis. However, spontaneously differentiated PANX1 knockout iPSCs have enhanced ability to generate mesoderm and endoderm germ layers compared to control. Furthermore, PANX1 is dynamically expressed and localized across different neural cell types at distinct stages of human cerebral organoid development. Taken together, our results suggest that PANX1-mediated cell communication actively participates in cell fate specification and human cerebral organoid development.

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