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RB maintains quiescence and prevents premature senescence through upregulation of DNMT1 in mesenchymal stromal cells.

Lin SP, Chiu FY, Wang Y, Yen ML, Kao SY, Hung SC - Stem Cell Reports (2014)

Bottom Line: RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs.Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect.These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan, ROC.

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Overexpression of RB in Late-Passage MSCs Increased the Proliferation Rate and In Vitro Osteogenic and Adipogenic Differentiation Potential, and Reduced Senescence MarkersLate-passage MSCs were transfected with control (CTR) or RB overexpression vectors (OE RB).(A) MSCs without or with RB overexpression were seeded at 4.5 × 103 /cm2 and cultured for 7 days. MTT assay was performed during the indicated period, and data are shown as the relative fold increase.(B) MSCs without or with RB overexpression were treated with OIM. Upper: representative pictures of alizarin red S staining at 2 weeks. Lower: optical density measurements of extracted alizarin red S staining.(C) MSCs without or with RB overexpression were treated with AIM. Upper panel: representative pictures of oil red O staining at 2 weeks. Lower panel: optical density measurements of extracted oil red O staining.(D–F) Western blot analysis (D), quantitative RT-PCR (E), and β-gal staining (F) of MSCs without or with RB overexpression.The results are expressed as mean ± SD of three independent experiments. Asterisks indicate significant differences (∗∗p < 0.01). Scale bar, 50 μm. See also Figure S3.
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fig3: Overexpression of RB in Late-Passage MSCs Increased the Proliferation Rate and In Vitro Osteogenic and Adipogenic Differentiation Potential, and Reduced Senescence MarkersLate-passage MSCs were transfected with control (CTR) or RB overexpression vectors (OE RB).(A) MSCs without or with RB overexpression were seeded at 4.5 × 103 /cm2 and cultured for 7 days. MTT assay was performed during the indicated period, and data are shown as the relative fold increase.(B) MSCs without or with RB overexpression were treated with OIM. Upper: representative pictures of alizarin red S staining at 2 weeks. Lower: optical density measurements of extracted alizarin red S staining.(C) MSCs without or with RB overexpression were treated with AIM. Upper panel: representative pictures of oil red O staining at 2 weeks. Lower panel: optical density measurements of extracted oil red O staining.(D–F) Western blot analysis (D), quantitative RT-PCR (E), and β-gal staining (F) of MSCs without or with RB overexpression.The results are expressed as mean ± SD of three independent experiments. Asterisks indicate significant differences (∗∗p < 0.01). Scale bar, 50 μm. See also Figure S3.

Mentions: To further confirm the roles of RB in maintaining stem cell properties and preventing senescence, we overexpressed RB in late-passage MSCs and evaluated its effects on proliferation, differentiation potentials, and senescence marker expression. Overexpression of RB in late-passage MSCs increased the proliferation rate (Figure 3A) and differentiation potentials (Figures 3B and 3C); decreased the expression of senescence-associated markers such as p21, p16 (Figure 3D), and APO-1 (Figure 3E); and reduced β-gal staining (Figure 3F). Similarly, RB overexpression via retroviral transduction in late-passage MSCs also increased the proliferation rate (Figure S3A), decreased the expression of p21 and p16 (Figure S3B), and reduced β-gal staining (Figure S3C). However, overexpression of RB in IMR90 fibroblasts decreased the proliferation rate (Figure S3D) and increased β-gal staining (Figure S3E), consistent with a previous report that showed RB’s role in replicative senescence of normal fibroblasts (Li et al., 1994). Together, these data suggest that RB plays an essential role in maintaining stem cell properties and prevents senescence in MSCs, but induces replicative senescence in fibroblasts.


RB maintains quiescence and prevents premature senescence through upregulation of DNMT1 in mesenchymal stromal cells.

Lin SP, Chiu FY, Wang Y, Yen ML, Kao SY, Hung SC - Stem Cell Reports (2014)

Overexpression of RB in Late-Passage MSCs Increased the Proliferation Rate and In Vitro Osteogenic and Adipogenic Differentiation Potential, and Reduced Senescence MarkersLate-passage MSCs were transfected with control (CTR) or RB overexpression vectors (OE RB).(A) MSCs without or with RB overexpression were seeded at 4.5 × 103 /cm2 and cultured for 7 days. MTT assay was performed during the indicated period, and data are shown as the relative fold increase.(B) MSCs without or with RB overexpression were treated with OIM. Upper: representative pictures of alizarin red S staining at 2 weeks. Lower: optical density measurements of extracted alizarin red S staining.(C) MSCs without or with RB overexpression were treated with AIM. Upper panel: representative pictures of oil red O staining at 2 weeks. Lower panel: optical density measurements of extracted oil red O staining.(D–F) Western blot analysis (D), quantitative RT-PCR (E), and β-gal staining (F) of MSCs without or with RB overexpression.The results are expressed as mean ± SD of three independent experiments. Asterisks indicate significant differences (∗∗p < 0.01). Scale bar, 50 μm. See also Figure S3.
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fig3: Overexpression of RB in Late-Passage MSCs Increased the Proliferation Rate and In Vitro Osteogenic and Adipogenic Differentiation Potential, and Reduced Senescence MarkersLate-passage MSCs were transfected with control (CTR) or RB overexpression vectors (OE RB).(A) MSCs without or with RB overexpression were seeded at 4.5 × 103 /cm2 and cultured for 7 days. MTT assay was performed during the indicated period, and data are shown as the relative fold increase.(B) MSCs without or with RB overexpression were treated with OIM. Upper: representative pictures of alizarin red S staining at 2 weeks. Lower: optical density measurements of extracted alizarin red S staining.(C) MSCs without or with RB overexpression were treated with AIM. Upper panel: representative pictures of oil red O staining at 2 weeks. Lower panel: optical density measurements of extracted oil red O staining.(D–F) Western blot analysis (D), quantitative RT-PCR (E), and β-gal staining (F) of MSCs without or with RB overexpression.The results are expressed as mean ± SD of three independent experiments. Asterisks indicate significant differences (∗∗p < 0.01). Scale bar, 50 μm. See also Figure S3.
Mentions: To further confirm the roles of RB in maintaining stem cell properties and preventing senescence, we overexpressed RB in late-passage MSCs and evaluated its effects on proliferation, differentiation potentials, and senescence marker expression. Overexpression of RB in late-passage MSCs increased the proliferation rate (Figure 3A) and differentiation potentials (Figures 3B and 3C); decreased the expression of senescence-associated markers such as p21, p16 (Figure 3D), and APO-1 (Figure 3E); and reduced β-gal staining (Figure 3F). Similarly, RB overexpression via retroviral transduction in late-passage MSCs also increased the proliferation rate (Figure S3A), decreased the expression of p21 and p16 (Figure S3B), and reduced β-gal staining (Figure S3C). However, overexpression of RB in IMR90 fibroblasts decreased the proliferation rate (Figure S3D) and increased β-gal staining (Figure S3E), consistent with a previous report that showed RB’s role in replicative senescence of normal fibroblasts (Li et al., 1994). Together, these data suggest that RB plays an essential role in maintaining stem cell properties and prevents senescence in MSCs, but induces replicative senescence in fibroblasts.

Bottom Line: RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs.Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect.These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan, ROC.

Show MeSH
Related in: MedlinePlus