Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy

Abstract

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use.

Figure 1

Characterization of hMSCs replicative senescence. (a) Growth curves of five independent primary hMSC samples (black) and four hMSC samples transduced with hTERT lentiviral vector (hTERT-MSC) at passage 5 (gray). Neither the proliferation rate nor the morphology of hMSCs was significantly changed after 15 passages. No evidence of spontaneous immortalization was observed in any primary cell sample over 23 passages. (b) Percentage of SA-β-gal-positive cells in non-transduced hMSCs at different passages and in transduced hMSCs at passage 20. Upper panels show representative images of SA-β-gal-positive cells. (c) P53, P21 and P16 mRNA gene expression by Taqman Assays at early (P≤5), middle (P>5–P≤10) and late (P≥15) passages in hMSCs, and passage >20 in hTERT-MSCs. (d) Representative images of western blot for P53, P21 and P16 protein levels of one hMSC sample (FT34hMSC) at early, middle and late passages, and one hTERT-MSC line at passage >20. β-actin was detected as a loading control. All independent hMSC cultures followed the same protein profile expression. All above experiments were performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (*P<0.050)

Figure 2

Replicative senescence in hMSCs is associated with aneuploidy. (a) Histogram of DNA content indicating the percentages of cells in apoptosis, G0/G1, S and G2/M phases of the cell cycle. Data were obtained by staining the DNA at various passages in hMSCs and passage >20 in hTERT-MSCs. Right panels are representative histograms of one independent hMSC sample and their respective transduced hTERT-MSC counterpart. Note the increase in the coefficient of variation of G0/G1 and G2/M peaks over time in culture in non-transduced cells. Experiment was performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (*P<0.050; **P<0.010). (b) Percentage of aneuploid cells for any of chromosomes 8, 11 and 17 classified according to type of aneusomy: (0) nulisomy, (1) monosomy, (3) trisomy, (4) tetrasomy and (>4) polisomy. Right panels show example images of hMSCs hybridized with CEP probes for chromosomes 8 (red), 11 (green) and 17 (light blue). Cultures preferentially accumulated trisomic cells with passages. A total of 100–200 nuclei were analyzed per hMSC culture. Four independent hMSC samples were used for this experiment, and data are means±S.E.M.

Figure 3

hTERT overexpression in hMSCs elongates telomeres and reduces chromosome abnormalities. (a) Telomere length analysis by Q-FISH in metaphases from four primary hMSC samples at passages P≤5 and passages P≥10, and in hTERT-MSCs at passage P>18. Upper panels show representative images of metaphases from hMSC and hTERT-MSC cultures, stained with PNA telomere probe (red) and centromere probe (green). (b) Cytogenetic analysis of structural chromosomal aberrations in metaphase cells from hMSC and hTERT-hMSC cultures. Right panels show examples of chromosomal aberrations in cells processed by FISH with centromere probes (green) and telomere PNA probes (red). All above experiments were performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (*P<0.050; **P<0.010) (c) Chromosome number analysis in metaphase of four hMSCs at passages P<5 and passage 14, and in four hTERT-MSCs at passage 20. Histogram represents the percentage of metaphases with a specific number of chromosomes in four independent hMSC samples and their transduced counterparts

Figure 4

The senescence-prone cell subpopulation is significantly aneuploid and has reduced proliferation rate. (a) Representative FSC-A/SSC-A plot diagrams at assorted passages in hMSCs and at passage P>20 in hTERT-MSCs. Black circles indicate the most common FSC-A/SSC-A subpopulation (alpha) at initial passages. The percentage of the alpha subpopulation is indicated for each passage. The above experiment was performed with four hMSC samples and their respective transduced hTERT-MSC counterparts, and all of them followed the same kinetics. (b) Representative scatter plot from a FACS assay in hMSCs at passages 5–7, separating cells of large size (forward scatter) and high complexity (side scatter) (subpopulation β) from cells of small size and low complexity (subpopulation α). (c) Percentage of SA-β-gal-positive hMSCs in subpopulations α and β at the third passage after sorting. (d) Percentage of aneuploid cells for any of the chromosomes 8, 11 and 17 in subpopulations α and β at the third passage after sorting. (e) Growth curves of subpopulations α and β over five passages after sorting, revealing impaired proliferation of the more senescent hMSC subpopulation (α). Cell sorter experiments were performed in triplicate with one independent hMSC sample (ft34hMSC). Data are means±S.E.M. (*P<0.050)

Figure 5

Replicative senescence in hMSCs promotes oxygen consumption and oxidative stress, and is decreased by hTERT overexpression. (a) Relative levels of total and mitochondrial ROS (O₂⁻) detected by flow cytometry using DHE (di-hydroethidium) and MitoSox red fluorescence in four hMSC lines at early, middle and late passage, and in derived hTERT-MSC cultures at late passage. (b) Levels of protein carbonyls and MDA in four hMSC samples (white) and derived hTERT-MSC samples (gray) at passage P=5 and passage P>20, respectively. All above experiments were performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M (*P<0.050; **P<0.010). (c) Basal oxygen consumption rate (pmoles/min) in hMSCs at early, 16.74±0.68; middle, 22.68±2.24 and late passages, 36.79±6.01 pMoles/min, and in hTERT-MSCs at late passage, 13±0.90 pMoles/min. Seahorse experiment was performed in eight replicates with four hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (*P<0.050; **P<0.010). (d) Relative differences of the mitochondrial content in four hMSC and their derived hTERT-MSCs using Mitotracker green staining and quantifying the fluorescence by flow cytometry. (e) Western blot of the mitochondrial protein Porin and the ROS scavenger MnSOD (SOD2) in three hMSC cultures at early and late passages, and their transduced counterparts at late passage. The lower histogram shows protein quantification by pixel density analysis with ImageJ software, normalized to the β-actin signal. Data are shown as means±S.E.M. (*P<0.050; **P<0.010)

Figure 6

Replicative senescence in hMSCs alters the expression of ploidy-controlling genes. (a) Taqman qRT-PCR quantification of mRNA transcripts for SCIN, EDN1, AKAP9, CD70, CXCL1 and CXCL12 in hMSCs at passage 22 versus passage 2 (control). SCIN, EDN1 and AKAP9 were significantly upregulated at passage 22 compared with passage 2 (10.46±3.65, 3.90±0.72 and 2.64±0.39-fold increases, respectively), whereas CXCL1 and CD70 were significantly downregulated (−6.22±2.99 and −7.31±2.68, respectively) (P<0.05); and CXCL12 expression was a downward trend (−3.26±0.97; P=0.09). (b) Expression of ploidy-controlling mRNA transcripts in hTERT-MSC cultures versus non-transduced hMSCs (control) at passage 22. hTERT transduction reverses the gene expression phenotype of high-passage hMSCs. The above experiments were performed in triplicate with four independent hMSC and four hTERT-MSC preparations. Data are means±S.E.M. (*P<0.050). All above experiments were performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (n=4) (*P<0.050; **P<0.010). (c) SA-β-gal-positive cells (%) at passage 4 in ft40hMSCs transduced with pRRL-SCIN and pLVX-Sh203 lentiviral vectors or their corresponding controls. Experiments were performed in triplicate for each condition. Data are means±S.E.M. (*P<0.050). (d) Aneuploid cells (%) at passage 4 in ft40hMSCs transduced with pRRL-SCIN and pLVX-Sh203 lentiviral vectors or their corresponding controls. A total of 100–200 nuclei were analyzed per condition. The fraction of aneuploid cells was calculated for each condition, and data were analyzed with Fisher's exact test for two binomials. Data are means±S.D. (χ² test *χ²>3.84)