From cytogenomic to epigenomic profiles: monitoring the biological behavior of in vitro cultured human bone marrow mesenchymal stem cells

ABSTRACT Aim To evaluate and compare the cytotoxicity of various concentrations of sodium hypochlorite on immortalized human bone marrow mesenchymal stem cells (MSCs). Materials and methods The 5.25 percent sodium hypochlorite (NaOCl) at concentrations of 0.5, 0.1, 0.025, 0.0125, and 0.005 mg/ml were used to assess the cytotoxic effect on MSCs. Immortalized human bone marrow mesenchymal stem cells (hTERT-MSCs) were exposed to NaOCl at 5 different concentrations. Cell viability was assessed by 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alamarBlue assays. The cell morphology changes were assessed with scanning electron microscopy (SEM) after exposure to 2, 4, and 24 hour incubation. The ethidium bromide/acridine orange (EB/ AO) fluorescent stain was applied to the cells in the 8-chamber slides after they were incubated with the testing agents for 2 and 4 hours to detect live and dead cells. The observations were quantitatively and qualitatively analyzed. Results The cell viability study using MTT assay and AB assay showed significant reduction with varying concentration at 2 and 4 hours incubation period. The cell viability decreased with the higher percentage of NaOCl. The exposure time also revealed an inverse relation to the cell viability. The SEM analysis showed reduction in the number of cells and morphological alterations with 0.5 mg/ml at 2 and 4 hours compared to 0.025 mg/ml NaOCl. Destruction of the cells with structural alterations and lysis was evident under fluorescence microscope when the cells were exposed to 0.5 mg/ml NaOCl. Conclusion Within the limitations of this in vitro study it can be concluded that NaOCl is toxic to the human bone marrow MSCs. The cell lysis was evident with higher concentration of sodium hypochlorite. From the observations, it can be concluded that a lower concentration of NaOCl may be used as endodontic irrigant due to its cytotoxic properties. Further studies are man datory to evolve a consensus on the optimal concentration of sodium hypochlorite to be used as endodontic irrigant. How to cite this article Alkahtani A, Alkahtany SM, Anil S. An in vitro Evaluation of the Cytotoxicity of Varying Concentrations of Sodium Hypochlorite on Human Mesenchymal Stem Cells. J Contemp Dent Pract 2014;15(4):473-481.

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The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles

Stem Cells International ◽

10.1155/2016/5656701 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 14

Author(s):

Angela Bentivegna ◽

Gaia Roversi ◽

Gabriele Riva ◽

Laura Paoletta ◽

Serena Redaelli ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Differentiation Potential ◽

Marrow Mesenchymal Stem Cells

Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-termin vitroculture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions forin vitroexpansion in order to minimize these epigenetic changes and to standardize safer procedures.

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