Dynamic regulation of connexins in stem cell pluripotency

Esseltine, Jessica L.; Brooks, Courtney; Edwards, Nicole; Subasri, Mathushan; Sampson, Jacinda B.; Séguin, Cheryle A.; Betts, Dean H.; Laird, Dale W.

doi:10.1002/stem.3092

Cited by 19 publications

(28 citation statements)

References 60 publications

(136 reference statements)

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“…Further studies are needed to examine the implication of these findings. For example, connexin expression in human pluripotent stem cells (hPSCs) has been found to be dramatically different between the pluripotent “naïve state” and the “primed state” 58 . Sonoporation may provide a tool to determine whether the changes in GJIC can serve as a functional biomarker for the pluripotency continuum in stem cells.…”

Section: Discussionmentioning

confidence: 99%

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Fan

Xue

et al. 2020

Sci Rep

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Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell–cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells.

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Section: Discussionmentioning

confidence: 99%

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Fan

Xue

et al. 2020

Sci Rep

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“…To examine the role of Cx43 in REKs, the Gja1 gene encoding rat Cx43 was ablated using CRISPR-Cas9 following strategies we previously described [ 36 , 37 ]. Briefly, cells successfully expressing the gRNA were single-cell fluorescence activated cell sorted and plated for single colony cell growth.…”

Section: Methodsmentioning

confidence: 99%

Comparative Analysis of Cx31 and Cx43 in Differentiation-Competent Rodent Keratinocytes

Shao

White

et al. 2020

Biomolecules

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When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

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“…HEI-OC1 cells were subjected to CRISPR-Cas9 ablation of the Gja1 gene, as we have previously described 43 . Two gRNAs were engineered using the Sanger Institute CRISPR finder (http://www.sanger.ac.uk/htgt/wge/) (mouse Gja1: Sanger sgRNA ID: 324658622 (5′-CGCTGTAACACTCAACAACC-3′) and Sanger sgRNA ID: 324658605 (5′-AAGCCTACTCCACGGCCGGA-3′)).…”

Section: Crispr-cas9 Gene Ablationmentioning

confidence: 99%

“…Two gRNAs were engineered using the Sanger Institute CRISPR finder (http://www.sanger.ac.uk/htgt/wge/) (mouse Gja1: Sanger sgRNA ID: 324658622 (5′-CGCTGTAACACTCAACAACC-3′) and Sanger sgRNA ID: 324658605 (5′-AAGCCTACTCCACGGCCGGA-3′)). HEI-OC1 cells expressing the reporter green fluorescent protein were sorted using fluorescence-activated cell sorting (FACS), and at least two Cx43-ablated clones were confirmed and used for each experiment as we previously described 43 .…”

Section: Crispr-cas9 Gene Ablationmentioning

confidence: 99%

“…Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed as we recently described 43 . The following primers were used: 18s rRNA, the house keeping gene (forward, 5′-GTAACCCGTTGA ACCCCATT; reverse, 5′-CCATCCAATCGGTAGTAGC G), manganese superoxide dismutase (MnSOD) (forward, 5′-GGCCAAGGGAGATGTTACAA; reverse, 5′-CCTTG GACTCCCACAGACAT), glutathione peroxidase (GPx1) (forward, 5′-GTCCACCGTGTATGCCTTCT; reverse 5′-CCTCAGAGAGACGCGACATT), catalase (forward, 5′-GGAGGCGGGAACCCAATAG; reverse, 5′-GTGTGCC ATCTCGTCAGTGAA, Bcl-2 (forward, 5′-CCTGTGGA TGACTGAGTACC; reverse, 5′-GAGACAGCCAGGAG AAATCA), Bax (forward, 5′-GTTTCATCCAGGATCGA GCA; reverse, 5′-CATCTTCTTCCAGATGGTGA).…”

Section: Quantitative Reverse Transcriptase Polymerase Chain Reactionmentioning

confidence: 99%

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Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication

et al. 2020

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Cisplatin is a very effective chemotherapeutic, but severe and permanent hearing loss remains a prevalent side effect. The processes underpinning cisplatin-induced ototoxicity are not well understood. Gap junction channels composed of connexin (Cx) subunits allow for the passage of small molecules and ions between contacting neighboring cells. These specialized channels have been postulated to enhance cisplatin-induced cell death by spreading "death signals" throughout the supporting cells of the organ of Corti. This study sought to investigate the role of Cx43 in cisplatininduced ototoxicity using organotypic cochlear cultures from control and two Cx43-mutant mouse strains harboring either a moderate (Cx43 I130T/+) or severe (Cx43 G60S/+) reduction of Cx43 function. Cochlear cultures from Cx43-mutant mice with a severe reduction in Cx43-based gap junctional intercellular communication (GJIC) had an enhanced number of hair cells that were positive for cleaved caspase 3, a marker of active apoptosis, after cisplatin treatment. In cisplatin-treated organotypic cochlear cultures, there was a decrease in the co-localization of Cx26 and Cx30 compared with untreated cultures, suggesting that cisplatin causes reorganization of connexin composition in supporting cells. Both Cx26 and Cx30 protein expression as well as GJIC were decreased in organotypic cochlear cultures treated with the gap-junction blocker carbenoxolone. When cisplatin and carbenoxolone were coadministered, there were no differences in hair cell loss compared with cisplatin treatment alone. Using cisplatintreated control and Cx43-ablated organ of Corti derived HEI-OC1 mouse cells, we found that greatly reducing GJIC led to preferential induction of an ER stress pathway. Taken together, this study strongly suggests that inhibition of GJIC in organ of Corti cells does not lead to differential susceptibility to cisplatin-induced ototoxicity. Although cisplatin causes the same degree of cell death in gap junction competent and incompetent cochlear cells, the engagement of the mitochondrial dysregulation and ER stress differs.

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Dynamic regulation of connexins in stem cell pluripotency

Cited by 19 publications

References 60 publications

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Comparative Analysis of Cx31 and Cx43 in Differentiation-Competent Rodent Keratinocytes

Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication

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