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Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

García CP, Videla Richardson GA, Dimopoulos NA, Fernandez Espinosa DD, Miriuka SG, Sevlever GE, Romorini L, Scassa ME - PLoS ONE (2016)

Bottom Line: However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types.We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs.Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratorios de Investigación Aplicada a Neurociencias, LIAN-CONICET, Fundación FLENI, Ruta 9, Km 53, (B1625XAF) Escobar, Buenos Aires, Argentina.

ABSTRACT
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.

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hESCs-derived NP and differentiated-neuronal-like cells phenotypic characterization.(a) Representative images of NP stained with primary antibodies against nestin and DCX (left panel). Representative flow cytometry histograms overlays of H9 hESCs-derived NP are shown to visualize CD133 expression relative to isotype control (right panel) (b) Representative images of NP and differentiated neuronal-like counterparts stained with primary antibodies against MAP-2, MAP-5 and Tuj1. The nuclei were counterstained with DAPI. The scale bars represent 100 μm.
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pone.0152607.g001: hESCs-derived NP and differentiated-neuronal-like cells phenotypic characterization.(a) Representative images of NP stained with primary antibodies against nestin and DCX (left panel). Representative flow cytometry histograms overlays of H9 hESCs-derived NP are shown to visualize CD133 expression relative to isotype control (right panel) (b) Representative images of NP and differentiated neuronal-like counterparts stained with primary antibodies against MAP-2, MAP-5 and Tuj1. The nuclei were counterstained with DAPI. The scale bars represent 100 μm.

Mentions: Although recent progresses in stem cells biology fostered the characterization of hESCs responses to diverse genotoxic stresses, these are still far from being understood in full detail. Thus, to gain insight into the responses achieved by pluripotent cells upon DNA damage, we sought to compare how two different types of human pluripotent stem cells (hESCs and hiPSCs) respond to the DSBs inducing agent CPT, a well known inhibitor of DNA-topoisomerase I complexes. Taking into account that hESCs offer an accessible and manageable platform to model the early stages of human development, we generated hESCs-derived NP to further characterize the cellular responses triggered by genotoxic stress in hESCs undergoing neural differentiation. To do so, we subjected H9 hESCs to a neural differentiation protocol. We determined that the hESCs-derived NP express the stemness markers nestin and CD133 and the neuronal migration protein, doublecortin (DCX) (Fig 1a). These NP further differentiate into process-bearing neuronal-like cells that stain positively for microtubule-associated proteins MAP-2 and MAP-5 and neuron-specific class III β-tubulin (Tuj1) (Fig 1b, bottom panel).


Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

García CP, Videla Richardson GA, Dimopoulos NA, Fernandez Espinosa DD, Miriuka SG, Sevlever GE, Romorini L, Scassa ME - PLoS ONE (2016)

hESCs-derived NP and differentiated-neuronal-like cells phenotypic characterization.(a) Representative images of NP stained with primary antibodies against nestin and DCX (left panel). Representative flow cytometry histograms overlays of H9 hESCs-derived NP are shown to visualize CD133 expression relative to isotype control (right panel) (b) Representative images of NP and differentiated neuronal-like counterparts stained with primary antibodies against MAP-2, MAP-5 and Tuj1. The nuclei were counterstained with DAPI. The scale bars represent 100 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816327&req=5

pone.0152607.g001: hESCs-derived NP and differentiated-neuronal-like cells phenotypic characterization.(a) Representative images of NP stained with primary antibodies against nestin and DCX (left panel). Representative flow cytometry histograms overlays of H9 hESCs-derived NP are shown to visualize CD133 expression relative to isotype control (right panel) (b) Representative images of NP and differentiated neuronal-like counterparts stained with primary antibodies against MAP-2, MAP-5 and Tuj1. The nuclei were counterstained with DAPI. The scale bars represent 100 μm.
Mentions: Although recent progresses in stem cells biology fostered the characterization of hESCs responses to diverse genotoxic stresses, these are still far from being understood in full detail. Thus, to gain insight into the responses achieved by pluripotent cells upon DNA damage, we sought to compare how two different types of human pluripotent stem cells (hESCs and hiPSCs) respond to the DSBs inducing agent CPT, a well known inhibitor of DNA-topoisomerase I complexes. Taking into account that hESCs offer an accessible and manageable platform to model the early stages of human development, we generated hESCs-derived NP to further characterize the cellular responses triggered by genotoxic stress in hESCs undergoing neural differentiation. To do so, we subjected H9 hESCs to a neural differentiation protocol. We determined that the hESCs-derived NP express the stemness markers nestin and CD133 and the neuronal migration protein, doublecortin (DCX) (Fig 1a). These NP further differentiate into process-bearing neuronal-like cells that stain positively for microtubule-associated proteins MAP-2 and MAP-5 and neuron-specific class III β-tubulin (Tuj1) (Fig 1b, bottom panel).

Bottom Line: However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types.We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs.Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratorios de Investigación Aplicada a Neurociencias, LIAN-CONICET, Fundación FLENI, Ruta 9, Km 53, (B1625XAF) Escobar, Buenos Aires, Argentina.

ABSTRACT
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.

Show MeSH
Related in: MedlinePlus