Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

Chênais, Nathalie; Lareyre, Jean‐Jacques; Bail, Pierre‐Yves Le; Labbé, Catherine

doi:10.1016/j.yexcr.2015.04.011

Cited by 9 publications

(12 citation statements)

References 57 publications

(87 reference statements)

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“…This homogeneity resulted from the two-steps plating procedure that was set up in this study. The population obtained with this faster new method was similar to the one described previously in explant culture 23 . The objective of using adherent cells was to validate the permeabilization conditions in a culture system that would later be the most suitable for treating the cells with egg extract.…”

Section: Resultsmentioning

confidence: 80%

“…After 24 hours, the faster adhering epithelial cells were discarded and the supernatant, enriched with the slower adhering mesenchymal cells was collected. These cells have previously been shown to be the most suitable for nuclear transfer 11,23 . After filtration and washing, the cells were seeded at 0.25.10 6 cells/well in 24 well plates on 1.3 cm 2 glass coverslips coated with 10 µg/mL human fibronectin (Sigma F0556) and cultured for 2 days (about 60–70% confluence).…”

Section: Methodsmentioning

confidence: 99%

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Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

Chênais

Lorca²,

Morin³

et al. 2019

Sci Rep

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Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus- egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

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Section: Resultsmentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

Chênais

Lorca²,

Morin³

et al. 2019

Sci Rep

Self Cite

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show abstract

“…Several indicators in our study showed that indeed, the treated fin cells were not entirely changed in their transcriptomic profile. Namely, although several collagens underwent a reduced expression in the treated cells, col1a1a abundantly expressed in fin cells [71] remained highly expressed after cell treatment. Furthermore, we failed to detect any re-expression of the canonical pluripotency markers that are pou2, sox2, nanog and c-myc .…”

Section: Discussionmentioning

confidence: 99%

“…Confirmation of the expression pattern of several marker genes, namely col1a1a, nanog, pou2, sox2, c-myca1 and c-myca2 , were achieved by RTqPCR analysis according to [71] . The Ct values were normalized using the endogenous 18S rRNA control and the target mRNA relative abundance was calculated according the formula: 2 −ΔCt with ΔCt = mean Ct ( target gene ) – mean Ct ( 18S rRNA ).…”

Section: Methodsmentioning

confidence: 99%

Reprogramming of fish somatic cells for nuclear transfer is primed by Xenopus egg extract

Chênais

Cam

Guillet

et al. 2022

Preprint

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Somatic cell reprogramming in vitro prior to nuclear transfer is one strategy expected to improve clone survival during development. In this study, we investigated the reprogramming extent of fish fin somatic cells after in vitro exposure to Xenopus egg extract and subsequent culture. Using a cDNA microarray approach, we observed drastic changes in the gene expression profile of the treated cells. Several actors of the TGFβ and Wnt/β-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove somatic cells towards a mesenchymal-epithelial transition (MET), the hallmark of somatic reprogramming in induced pluripotent stem cells (iPSCs). However, treated cells were also characterized by a strong decrease in de novo lipid biosynthesis metabolism, the lack of re-expression of pou2 and nanog pluripotency markers, and absence of DNA methylation remodeling of their promoter region. In all, this study showed that Xenopus egg extract treatment initiated an in vitro reprogramming of fin somatic cells in culture. Although not thorough, the induced changes have primed the somatic chromatin for a better embryonic reprogramming upon nuclear transfer.

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“…Building on the in vitro approach, it became possible to study the effects of chemical carcinogens, heat shock, or ionizing radiation. For these investigations, many workers chose easily accessible tissues such as the gills, 13 fins, 14,15 skin, 16,17 and scales, 18 all of which form rapid outgrowths of primary cells. Excellent summaries of fish-derived cell lines are available.…”

Section: History Of Fish Cells In Vitromentioning

confidence: 99%

The Last Half Century of Fish Explant and Organ Culture

LeClair

2021

Zebrafish

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Explants are three-dimensional tissue fragments maintained outside the organism. The goals of this article are to review the history of fish explant culture and discuss applications of this technique that may assist the modern zebrafish laboratory. Because most zebrafish workers do not have a background in tissue culture, the key variables of this method are deliberately explained in a general way. This is followed by a review of fishspecific explantation approaches, including presurgical husbandry, aseptic dissection technique, choice of media and additives, incubation conditions, viability assays, and imaging studies. Relevant articles since 1970 are organized in a table grouped by organ system. From these, I highlight several recent studies using explant culture to study physiological and embryological processes in teleosts, including circadian rhythms, hormonal regulation, and cardiac development.

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Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

Cited by 9 publications

References 57 publications

Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

Reprogramming of fish somatic cells for nuclear transfer is primed by Xenopus egg extract

The Last Half Century of Fish Explant and Organ Culture

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