Fig 1.
Work flow for generating Rho-0 Cells from human mesenchymal stem cells (hMSCs).
d4t = Stavudine; mtDNA = mitochondrial DNA; Q-PCR = quantitative polymerase chain reaction; 12S-rRNA = mitochondrial12S ribosomal gene; RNAseP = nuclear gene.
Table 1.
List of drugs analyzed to deplete mitochondrial DNA (mtDNA) in human mesenchymal stem cell lines (hMSCs).
Fig 2.
3a6 cell line response to different substances capable of depleting the levels of mitochondrial DNA.
All mtDNA copy numbers are expressed as percentages comparing each value with untreated cells valued at 100% (a). Treatment with ethidium bromide (EtBr) at three different concentrations (1 mM, 100 nM and 500 nM) for 240 hours. The maximum effectiveness of this treatment was at 100 nM. (b). Treatment with Rhodamine 6g at 1, 3 and 5 μg/ml for 24, 72, 120 and 168 hours. (c). Treatment with 1-methyl-4-phenylpyridinium (MPP+) at 25 μM, 0.5 mM and 1 mM for 72 hours. (d). Treatment with Zidovudine (AZT) at 20 μM and 0.5 mM, for 96, 144 and 216 hours. AZT increased mtDNA content. (e). Treatment with Stavudine (d4t) at 5, 10 and 100 μM and 0.5 mM for 96 and 144 hours. The analysis of mtDNA copy numbers reflect that treatment with the highest concentration (0.5 mM) for 144 hours decreased the levels of mtDNA in treated cells nearly 98% compared to those of untreated cells. 8x104 cells were plated in each experiment for treatment with a reagent. The figures represent at least three independent experiments.
Table 2.
Summary of the most effective reagent reducing mitochondrial DNA (mtDNA) copy number in 3a6 human mesenchymal stem cells (hMSCs).
Fig 3.
(a). The mitochondrial DNA (mtDNA) copy number of 15 clones isolated by limit dilution from 3a6 cells treated with Stavudine (d4t) at 0.5 mM for 240 hours. Data for clones are expressed as percentages of untreated cells [without treatment (wt), value 100%]. (b). mtDNA copy number of two selected clones, one with low mtDNA (C-3; 1.57%) and the other with a high value for this parameter (C-10; 0.80%). Both clones were re-treated with d4t at 0.5 mM for 192 hours with mtDNA copy number expressed as a % of that of the 3a6 parental line. In this graphic, the %mtDNA copy number for 143B.TK- Rho-0 is also represented in relation to that of its parental cell line (143B.TK-). (c). This graph shows the % of mtDNA copy number for C-3 cultured in DMEM without d4t for 288 hours. The results show the stability of the mtDNA copy number during culture without the drug.
Fig 4.
The response of the KP cell line to different substances that deplete mitochondrial DNA.
All mtDNA copy numbers are expressed as percentages compared to those of untreated cells valued at 100%. (a). Treatment with ethidium bromide (EtBr) at three different concentrations, 1 mM, 500 nM and 100 nM, for 240 hours. (b). Treatment with Rhodamine6g (Rd6g) at 3 μg/ml for 24, 48, 72 and 96 hours; the positive response started at 48 hours. (c). Treatment with Zidovudine (AZT) at 10 μM and 0.5 mM for 168 and 240 hours. (d). Treatment with Stavudine (d4t) at three concentrations, 5 μM, 10 μM and 0.5 mM, for 240 hours. 8x104 cells were plated in each experiment for treatment with different agents. The figures represent at least two independent experiments.
Table 3.
Summary of the most effective reagent, in KP human mesenchymal stem cells (hMSCs).
Fig 5.
Characterization of 3a6 cells without treatment (3a6-wt) and 3a6 Rho-0 like cells.
(a). Phenotypic characterization is represented as the percentage of positivity for CD29, CD73, CD90, CD105, CD166 and SSEA4 for both cell types. (b). Cellular levels of reactive oxygen species (ROS) in both cell types and in 143B.TK-Rho-0 cells (used as a reference for a typical Rho-0 line). Total ROS production was measured with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA); data are expressed as mean fluorescence intensity. (c). Apoptosis measured with Annexin-V-FIT: data are expressed as percentage of positive cells for Annexin-V and propidium iodide (PI) in basal conditions and culture in presence of Staurosporine (Stau) at 2 μM for 2 hours. (d). Mitochondrial membrane potential (Δψm) measured with DilC1(5) [1,1´,3,3,3´-hexamethylindodicarbo-cyanine iodide], data are expressed as percentages of cells that were positive for DilC1(5) fluorescence. (e). Mitochondrial network in 3a6-wt and 3a6-Rho 0 cells incubated with 250 nM MitoTraker Red solution for 30 min in a 37°C incubator. The cells were fixed with 4% paraformaldehyde and counterstained with Hoechst-33258 nuclear dye. The cells were photographed with a confocal microscope Nikon AR-1. (f-g) The mitochondrial respiration [oxygen consumption (OCR)] pattern was obtained using a SeaHorse XFp for 3a6-wt and 3a6 Rho-0 cells (f) and for 143B.TK- and 143B.TK-Rho-0 cells (g). All data were obtained from three independent experiments, expressed as mean ±SD and analyzed by the unpaired t-test (*, p≤0.05; *** p<0.001).
Fig 6.
Osteogenic, adipogenic and chondrogenic differentiation in 3a6 without treatment (3a6-wt) and 3a6 Rho-0 cells.
(a) Gene expressions implicated in the non-differentiation stage: Nano-HomeoBox (Nano-g), POU Class 5 Homeobox 1(Oct 3/4) and SRY (Sex determining region)-Y-box 2 (Sox-2) and SRY (Sex determining region)-Y-box 9 (Sox-9) in 3a6-wt and 3a6 Rho-0 cells. (b). Gene expressions implicated in the osteogenic process: alkaline phosphatase-4 (ALP) and osteocalcin-1 (OC-1) in basal condition in 3a6-wt and 3a6 Rho-0 cells. (c). Fold increase in the gene levels of ALP and OC-1 during osteogenic induction for 9 days; data are expressed relative to the basal condition (basal condition equal to 1). (d). Basal gene expressions implicated in the adipogenic process: fatty acid synthase (FASN) and peroxisome proliferator-activated receptor gamma (PPAR-γ) in 3a6-wt and 3a6 Rho-0 cells. (e). Fold increase in the gene levels of FASN and PPAR-γ following 9 days under adipogenic differentiation; data are expressed relative to the basal condition (basal condition equal to 1). (f). LD540, which stains lipid droplets, was used to measure adipogenic differentiation in 3a6-wt and 3a6 Rho-0 cells. Cells were cultured in standard medium (control) and in adipogenic medium for 9 days. Cells were fixed and double-stained with LD540 (red) for lipid droplets and Hoechst 33258 (blue) for DNA. Fluorescence was visualized with Olympus BX61 fluorescence microscopy and photographed at 20X. (g). Fold increase in the gene levels of CoL-I and CoL-II in the micropellet model following 21 days under chondrogenic differentiation; data are expressed relative to the basal condition (basal condition equal to 1). All data were obtained from three independent experiments, expressed as mean ±SD and analyzed by the unpaired t-test (*, p≤0.05; ** p<0.005). Basal, without induction. Osteo (Osteogenic), Adipo (Adipogenic), Chondro (Chondrogenic) cells grown in the presence of indicated medium.
Fig 7.
mRNA expression of mitochondrial biogenesis genes in 3a6 without treatment (3a6-wt) and 3a6 Rho-0 cells.
(a). To analyze mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and transcription factor A, mitochondrial (TFAM) gene expression levels were determined under basal conditions. (b). Fold increase of PGC-1α gene levels during osteogenic and adipogenic induction. and (c). TFAM gene levels during osteogenic and adipogenic induction. Data are expressed relative to basal condition (basal condition equal to 1). All data were obtained from two independent experiments, expressed as mean ± SD and analyzed by the unpaired t-test (* p<0.05; ** p<0.005). Basal, in standard medium, Adipo (Adipogenic) and Osteo (Osteogenic); all experiments were developed during 9 days in each specific differentiation culture medium.