A protocol to assess cell cycle and apoptosis in human and mouse pluripotent cells

Edel, Michael J.; Menchon, Cristina; Vaquero, Jose Miguel Andres; Belmonte, Juan Carlos Izpisúa

doi:10.1186/1478-811x-9-8

Cited by 9 publications

(5 citation statements)

References 21 publications

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“…In this work, by performing live imaging experiments of FUCCI mESCs in the naive ground state, we directly measured the cell cycle length, the length of the G1 phase and the length of the S/G2/M phases of hundreds of individual cells. Contrary to other methods that rely on measuring indirect variables on the overall population such as propidium iodide staining or nucleoside analog incorporation 35 , our analysis allowed to capture the complete distribution of these variables at the single cell level, providing a more detailed characterization of the cell cycle dynamics. Our results suggest that mESCs grown in the naive ground state display a similar proliferation rate than the previously reported for cells cultured in FBS/LIF.…”

Section: Discussionmentioning

confidence: 99%

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency

Waisman

Sevlever

Costa

et al. 2019

Sci Rep

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Mouse embryonic stem cells (mESCs) can be maintained as homogeneous populations in the ground state of pluripotency. Release from this state in minimal conditions allows to obtain cells that resemble those of the early post-implantation epiblast, providing an important developmental model to study cell identity transitions. However, the cell cycle dynamics of mESCs in the ground state and during its dissolution have not been extensively studied. By performing live imaging experiments of mESCs bearing cell cycle reporters, we show here that cells in the pluripotent ground state display a cell cycle structure comparable to the reported for mESCs in serum-based media. Upon release from self-renewal, the cell cycle is rapidly accelerated by a reduction in the length of the G1 phase and of the S/G2/M phases, causing an increased proliferation rate. Analysis of cell lineages indicates that cell cycle variables of sister cells are highly correlated, suggesting the existence of inherited cell cycle regulators from the parental cell. Together with a major morphological reconfiguration upon differentiation, our findings support a correlation between this in vitro model and early embryonic events.

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Section: Discussionmentioning

confidence: 99%

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency

Waisman

Sevlever

Costa

et al. 2019

Sci Rep

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“…After washing with PBS, DilC signal was analyzed using FACS Calibur (BD Biosciences). The percentage of apoptotic cells was determined as the number of viable cells with decreased DilC intensity, as reported before (Edel et al., 2011). As positive control, apoptosis was induced with Actinomycin D (ActD) at a final concentration of 20 μM and 40 μM.…”

Section: Methodsmentioning

confidence: 99%

Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors

et al. 2013

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C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression. Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, re-activation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets.

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“…For the measurement of cell cycle, cells exposed were fixed and stained with 2.5 mg/mL PI in the presence of 0.5 mg/mL RNase A (Sigma) [49], immediately before measurement on a Beckman Coulter flow cytometer (Indianapolis, IN). DNAploidy histogram analysis was performed with FlowJo using the Dean-Jett-Fox algorithm model that fits Gaussian curves to the G0/G1 and G2/M and a polynomial curve to the S phase [50].…”

Section: Maintenance and Differentiation Of Es Cells And Ethanol Treamentioning

confidence: 99%

Ethanol Alters the Balance of Sox2, Oct4, and Nanog Expression in Distinct Subpopulations During Differentiation of Embryonic Stem Cells

Ogony

Malahias

Vadigepalli

et al. 2013

Stem Cells and Development

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The transcription factors Sox2, Oct4, and Nanog regulate within a narrow dose-range embryonic stem (ES) cell pluripotency and cell lineage commitment. Excess of Oct4 relative to Sox2 guides cells to mesoendoderm (ME), while abundance of Sox2 promotes neuroectoderm (NE) formation. Literature does not address whether ethanol interferes with these regulatory interactions during neural development. We hypothesized that ethanol exposure of ES cells in early differentiation causes an imbalance of Oct4 and Sox2 that diverts cells away from NE to ME lineage, consistent with the teratogenesis effects caused by prenatal alcohol exposure. Mouse ES cells were exposed to ethanol (0, 25, 50, and 100 mM) during retinoic acid (10 nM)-directed differentiation to NE for 0-6 days, and the expression of Sox2, Oct4, and Nanog was measured in single live cells by multiparametric flow cytometry, and the cellular phenotype was characterized by immunocytochemistry. Our data showed an ethanol dose-and time-dependent asymmetric modulation of Oct4 and Sox2 expression, as early as after 2 days of exposure. Single-cell analysis of the correlated expression of Sox2, Oct4, and Nanog revealed that ethanol promoted distinct subpopulations with a high Oct4/Sox2 ratio. Ethanol-exposed cells differentiated to fewer b-III tubulin-immunoreactive cells with an immature neuronal phenotype by 4 days. We interpret these data as suggesting that ethanol diverted cells in early differentiation from the NE fate toward the ME lineage. Our results provide a novel insight into the mode of ethanol action and opportunities for discovery of prenatal biomarkers at early stages.

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A protocol to assess cell cycle and apoptosis in human and mouse pluripotent cells

Cited by 9 publications

References 21 publications

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency

Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors

Ethanol Alters the Balance of Sox2, Oct4, and Nanog Expression in Distinct Subpopulations During Differentiation of Embryonic Stem Cells

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