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. 2012 Dec;30(12):2746-59.
doi: 10.1002/stem.1223.

Nanog Reverses the Effects of Organismal Aging on Mesenchymal Stem Cell Proliferation and Myogenic Differentiation Potential

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Free PMC article

Nanog Reverses the Effects of Organismal Aging on Mesenchymal Stem Cell Proliferation and Myogenic Differentiation Potential

Juhee Han et al. Stem Cells. .
Free PMC article

Abstract

Although the therapeutic potential of mesenchymal stem cells (MSCs) is widely accepted, loss of cell function due to donor aging or culture senescence are major limiting factors hampering their clinical application. Our laboratory recently showed that MSCs originating from older donors suffer from limited proliferative capacity and significantly reduced myogenic differentiation potential. This is a major concern, as the patients most likely to suffer from cardiovascular disease are elderly. Here we tested the hypothesis that a single pluripotency-associated transcription factor, namely Nanog, may reverse the proliferation and differentiation potential of bone marrow-derived MSC (BM-MSC) from adult donors. Microarray analysis showed that adult (a)BM-MSC expressing Nanog clustered close to Nanog-expressing neonatal cells. Nanog markedly upregulated genes involved in cell cycle, DNA replication, and DNA damage repair and enhanced the proliferation rate and clonogenic capacity of aBM-MSC. Notably, Nanog reversed the myogenic differentiation potential and restored the contractile function of aBM-MSC to a similar level as that of neonatal (n)BM-MSC. The effect of Nanog on contractility was mediated--at least in part--through activation of the TGF-β pathway by diffusible factors secreted in the conditioned medium of Nanog-expressing BM-MSC. Overall, our results suggest that Nanog may be used to overcome the effects of organismal aging on aBM-MSC, thereby increasing the potential of MSC from aged donors for cellular therapy and tissue regeneration.

Figures

Figure 1
Figure 1. Ectopic expression of Nanog in neonatal and adult BM-MSC
(A) The presence of Nanog in aBM-MSC and nBM-MSC was measured by Western blot; β-actin served as a loading control. (B) Immunostaining showed that Nanog was present in the nucleus of aBM-MSC (green). Cell nuclei were counterstained with Hoechst 33342 (blue). (C) Reverse transcription–polymerase chain reaction (RT–PCR) analysis of endogenous (endo) and exogenous (exo) Nanog as well as endogenous Oct4 or Sox2. Genomic DNA (gDNA) from BM-MSC was used as a positive control template for PCR of endogenous genes; GAPDH served as a loading control.
Figure 2
Figure 2. Gene expression analysis by microarray
Gene expression profiles of control and Nanog-expressing nBM-MSC and aBM-MSC using Affymetrix GeneChip Bovine Genome Arrays (n=3 each group). A total of 1,670 differentially expressed genes (DEGs) were obtained that were at least 2-fold up-regulated (p<0.01) or 2-fold down-regulated (p<0.01) in at least one of the three groups (aBM.N vs. aBM.C; nBM.N vs. nBM.C; nBM.C vs. aBM.C). (A) Heat map for hierarchical cluster analysis of the total DEGs. Red represents upregulation and green represents downregulation. (B) Venn diagrams for total DEGs or up- or down-regulated DEGs in the three comparisons. (C) KEGG pathway analysis of genes that are differentially regulated upon Nanog expression in aBM-MSC. The numbers in the parentheses denote the number of genes that were up- or down-regulated (up, down) in the corresponding pathway. (D) Verification of microarray results by real-time qRT-PCR for the cell cycle related genes that were differentially expressed upon Nanog overexpression in aBM-MSC. GAPDH served as a loading control. Triplicate independent mRNA samples were used in qRT-PCR experiments.
Figure 3
Figure 3. Ectopic expression of Nanog significantly improved clonogenic and proliferative potential in BM-MSC
Cells were plated onto 10-cm dishes at clonal density (500 cells/dish) and cultured for 9 days (nBM-MSC, n=3) or 18 days (aBM-MSC, n=3). (A) Photographs of representative cell culture dishes and colonies. Bar=200 µm. (B) The number of colonies larger than 2mm in diameter was quantified for control and Nanog-expressing cells. The symbol (*) denotes p<0.05 between the indicated samples and their corresponding control. (C) To measure proliferation rate cells were seeded at 10,000 cells/cm2. After 6 days (nBM-MSC, n=3) or 9 days (aBM-MSC, n=3) the number of cells was counted and doubling time was calculated assuming exponential cell growth. (D) Representative images of SA-β-gal staining for control aBM-MSC (passage 8) or Nanog+ aBM-MSC (passage 20). Scale bar: 400 µm. (E) Quantitation of the percentage of SA-β-gal positive cells. (*) p<0.05 between the indicated sample and corresponding control. (F) Real-time qRT-PCR for p16INK4a. The results were normalized to GAPDH and plotted as average ± SE (n=3 independent experiments). (*) p<0.05 between the indicated samples.
Figure 4
Figure 4. Effects of Nanog on myogenic differentiation of BM-MSC
Control and Nanog expressing (A) nBM-MSC or (B) aBM-MSC were cultured under growth (DMEM/10% FBS, 2ng/mL bFGF) or myogenic differentiation medium (DMEM/10%FBS, 2ng/mL TGF-β1, 30µg/mL heparin). On day 3, the cells were fixed and immunostained for αSMA or calponin (red). Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 100µm. (C, D) Control and Nanog expressing nBM-MSCs or aBM-MSCs were cultured in the presence of TGF-β1 (2 ng/ml) and heparin (30 µg/ml) for 3 days. (C) Western blot for αSMA and calponin. (D) Real-time qRT-PCR for SM22, caldesmon and smoothelin; (*) p<0.05 (n=3).
Figure 5
Figure 5. Nanog increased the ability of BM-MSC to generate force
BM-MSCs were embedded in fibrin gels, which were allowed to polymerize for 1hr before they were released from the walls and allowed to compact. At the indicated times, the gels were photographed and their area was measured by using Image J software. The ratio of each gel area (A) at the indicated times over its initial area (A0) was plotted as a function of time. (A) Gel compaction profiles of control or Nanog expressing nBM-MSC and aBM-MSC in the presence of TGF-β1 (2ng/mL). (B) Representative gel pictures at the last time point (t = 67hr).
Figure 6
Figure 6. Nanog increased receptor-dependent and independent contractility of nBM-MSC and aBM-MSC
BM-MSCs were embedded in fibrin hydrogels and cultured around 6mm mandrels for two weeks in the presence of myogenic medium (2ng/mL TGF-β1, 2µg/mL insulin, and 300 µM vitamin C). Vascular reactivity was measured using an isolated tissue bath system. (A) Vascular reactivity (kPa) in response to Endothelin-1 (20 nM), U46619 (10−6 M) or KCl (118 mM). (B) Representative graph of isometric contraction of control or Nanog expressing nBM-MSC and aBM-MSC in response to Endothelin-1. (C–E) Vascular reactivity (kPa) of vascular tissue constructs prepared from human MSC. (C) Male aBM-MSC; (D) female aBM-MSC; (E) male aHF-MSCs. All values are the mean±SD of triplicate samples in a representative experiment. (*) p<0.05 between the indicated samples and corresponding control.
Figure 7
Figure 7. Nanog induced gel compaction by activating the TGF-β/Smad signaling pathway
(A) Cells were incubated in low serum medium (DMEM/2%FBS) and then next day they were treated with TGF-β1 (2ng/mL) or the TGF-β type I receptor inhibitor SB431542 (10µM) for 1 hr. The presence of p-Smad2 in cells was determined by Western blotting; GAPDH served as loading control. (B) The band intensity of p-Smad2 was determined using Image J software and normalized to GAPDH. All values are the mean±SE of three independent experiments (n=3). The symbol (*) denotes p<0.05 between the indicated samples. (C) Control and Nanog expressing neonatal or adult BM-MSCs were cultured in low serum medium (DMEM/2%FBS) and 24hr later the conditioned medium was harvested and filtered. Cells were incubated in low serum medium (DMEM/2%FBS) for 24hr and exposed to conditioned media from control or Nanog+ nBM-MSC or aBM-MSC in the presence or absence of SB431542 (10µM). After 1hr cells were lysed and the presence of p-Smad2 was measured by Western blot; GAPDH served as loading control. (D) Gel compaction assay was performed with conditioned media in the presence or absence of SB431542 (10µM). All values are the mean±SD of triplicate samples in a representative experiment (n=3). The symbol (*) denotes p<0.05 between the indicated samples, n=3.

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