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Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists

View Article: PubMed Central - PubMed

ABSTRACT

High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate “universal” marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage.

No MeSH data available.


Related in: MedlinePlus

Validation of ALDH1A1-siRNA transfection in DMSC23 cells.(a) Validation of siRNA uptake with fluorescent Allstars NC-AF488 siRNA. DMSC23 cells were transfected with a fluorescent (FITC) siRNA, Allstars NC-AF488, to visualise siRNA uptake. DMSC23 cells showing uptake of Allstars NC-AF488 (FITC). (b) Control with the omission of Allstars NC-AF488. Reactions were carried out in three independent experiments. Cell nuclei were counter-stained with DAPI (blue). Magnification is 200X and scalebar is 100 μm. (c) Example of gating parameters for Annexin V apoptosis assay. FSC versus SSC dot plot showing the gate used for this analysis. Dots in the lower left portion of the plot represent cellular debris. FSC: forward scatter, SSC: side scatter. (d). Dual-staining plot for apoptosis detection with Propidium Iodide/PI on the Y-axis and Annexin V/FITC on the X-axis. Events in Q2: PI positive and FITC positive show cells undergoing late apoptosis. Events in Q3: PI negative and FITC negative show viable cells. Events in Q4: PI negative and FITC positive show cells undergoing early apoptosis. (e) Quantification of early and late apoptosis using Annexin V staining following ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis shows the percentage of cells undergoing apoptosis and the X-axis the different sample groups. Data are presented as mean ± SEM from three independent experiments. One-way ANOVA test, p-value = 0.5219 (late apoptosis group), p-value = 0.6396 (early apoptosis group). (f ) xCELLigence system proliferation assay following 72-hour ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis represents the cell index at the 72 hrs timepoint and X-axis represents the different sample groups. Data are presented as mean ± SEM from triplicates of three independent experiments. NC (non-specific siRNA control) and mock (non-siRNA control). One-way ANOVA test, p-value = 0.1496.
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f2: Validation of ALDH1A1-siRNA transfection in DMSC23 cells.(a) Validation of siRNA uptake with fluorescent Allstars NC-AF488 siRNA. DMSC23 cells were transfected with a fluorescent (FITC) siRNA, Allstars NC-AF488, to visualise siRNA uptake. DMSC23 cells showing uptake of Allstars NC-AF488 (FITC). (b) Control with the omission of Allstars NC-AF488. Reactions were carried out in three independent experiments. Cell nuclei were counter-stained with DAPI (blue). Magnification is 200X and scalebar is 100 μm. (c) Example of gating parameters for Annexin V apoptosis assay. FSC versus SSC dot plot showing the gate used for this analysis. Dots in the lower left portion of the plot represent cellular debris. FSC: forward scatter, SSC: side scatter. (d). Dual-staining plot for apoptosis detection with Propidium Iodide/PI on the Y-axis and Annexin V/FITC on the X-axis. Events in Q2: PI positive and FITC positive show cells undergoing late apoptosis. Events in Q3: PI negative and FITC negative show viable cells. Events in Q4: PI negative and FITC positive show cells undergoing early apoptosis. (e) Quantification of early and late apoptosis using Annexin V staining following ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis shows the percentage of cells undergoing apoptosis and the X-axis the different sample groups. Data are presented as mean ± SEM from three independent experiments. One-way ANOVA test, p-value = 0.5219 (late apoptosis group), p-value = 0.6396 (early apoptosis group). (f ) xCELLigence system proliferation assay following 72-hour ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis represents the cell index at the 72 hrs timepoint and X-axis represents the different sample groups. Data are presented as mean ± SEM from triplicates of three independent experiments. NC (non-specific siRNA control) and mock (non-siRNA control). One-way ANOVA test, p-value = 0.1496.

Mentions: The efficiency of the siRNA transfection was evaluated by transfecting the DMSC23 cells with nonsilencing (NC) siRNA which is directly conjugated to AF488 (FITC fluorescence). Figure 2a shows transfection of siRNAs in DMSC23 cells, as evidenced from the FITC fluorescence around the cell nuclei due to the siRNAs being taken up by the cells. More than 90% of the cells were transfected by qualitative assessment of at least three fields of view. The negative control, where Allstars NC-AF488 siRNA was omitted, showed no significant FITC fluorescence surrounding the cell nuclei, which were counterstained with DAPI (Fig. 2b).


Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists
Validation of ALDH1A1-siRNA transfection in DMSC23 cells.(a) Validation of siRNA uptake with fluorescent Allstars NC-AF488 siRNA. DMSC23 cells were transfected with a fluorescent (FITC) siRNA, Allstars NC-AF488, to visualise siRNA uptake. DMSC23 cells showing uptake of Allstars NC-AF488 (FITC). (b) Control with the omission of Allstars NC-AF488. Reactions were carried out in three independent experiments. Cell nuclei were counter-stained with DAPI (blue). Magnification is 200X and scalebar is 100 μm. (c) Example of gating parameters for Annexin V apoptosis assay. FSC versus SSC dot plot showing the gate used for this analysis. Dots in the lower left portion of the plot represent cellular debris. FSC: forward scatter, SSC: side scatter. (d). Dual-staining plot for apoptosis detection with Propidium Iodide/PI on the Y-axis and Annexin V/FITC on the X-axis. Events in Q2: PI positive and FITC positive show cells undergoing late apoptosis. Events in Q3: PI negative and FITC negative show viable cells. Events in Q4: PI negative and FITC positive show cells undergoing early apoptosis. (e) Quantification of early and late apoptosis using Annexin V staining following ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis shows the percentage of cells undergoing apoptosis and the X-axis the different sample groups. Data are presented as mean ± SEM from three independent experiments. One-way ANOVA test, p-value = 0.5219 (late apoptosis group), p-value = 0.6396 (early apoptosis group). (f ) xCELLigence system proliferation assay following 72-hour ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis represents the cell index at the 72 hrs timepoint and X-axis represents the different sample groups. Data are presented as mean ± SEM from triplicates of three independent experiments. NC (non-specific siRNA control) and mock (non-siRNA control). One-way ANOVA test, p-value = 0.1496.
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f2: Validation of ALDH1A1-siRNA transfection in DMSC23 cells.(a) Validation of siRNA uptake with fluorescent Allstars NC-AF488 siRNA. DMSC23 cells were transfected with a fluorescent (FITC) siRNA, Allstars NC-AF488, to visualise siRNA uptake. DMSC23 cells showing uptake of Allstars NC-AF488 (FITC). (b) Control with the omission of Allstars NC-AF488. Reactions were carried out in three independent experiments. Cell nuclei were counter-stained with DAPI (blue). Magnification is 200X and scalebar is 100 μm. (c) Example of gating parameters for Annexin V apoptosis assay. FSC versus SSC dot plot showing the gate used for this analysis. Dots in the lower left portion of the plot represent cellular debris. FSC: forward scatter, SSC: side scatter. (d). Dual-staining plot for apoptosis detection with Propidium Iodide/PI on the Y-axis and Annexin V/FITC on the X-axis. Events in Q2: PI positive and FITC positive show cells undergoing late apoptosis. Events in Q3: PI negative and FITC negative show viable cells. Events in Q4: PI negative and FITC positive show cells undergoing early apoptosis. (e) Quantification of early and late apoptosis using Annexin V staining following ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis shows the percentage of cells undergoing apoptosis and the X-axis the different sample groups. Data are presented as mean ± SEM from three independent experiments. One-way ANOVA test, p-value = 0.5219 (late apoptosis group), p-value = 0.6396 (early apoptosis group). (f ) xCELLigence system proliferation assay following 72-hour ALDH1A1-siRNA transfection of DMSC23 cells. The Y-axis represents the cell index at the 72 hrs timepoint and X-axis represents the different sample groups. Data are presented as mean ± SEM from triplicates of three independent experiments. NC (non-specific siRNA control) and mock (non-siRNA control). One-way ANOVA test, p-value = 0.1496.
Mentions: The efficiency of the siRNA transfection was evaluated by transfecting the DMSC23 cells with nonsilencing (NC) siRNA which is directly conjugated to AF488 (FITC fluorescence). Figure 2a shows transfection of siRNAs in DMSC23 cells, as evidenced from the FITC fluorescence around the cell nuclei due to the siRNAs being taken up by the cells. More than 90% of the cells were transfected by qualitative assessment of at least three fields of view. The negative control, where Allstars NC-AF488 siRNA was omitted, showed no significant FITC fluorescence surrounding the cell nuclei, which were counterstained with DAPI (Fig. 2b).

View Article: PubMed Central - PubMed

ABSTRACT

High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate “universal” marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage.

No MeSH data available.


Related in: MedlinePlus