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. 2012 Jan 6;417(1):211-6.
doi: 10.1016/j.bbrc.2011.11.087. Epub 2011 Nov 28.

Role of Nanog in the Maintenance of Marrow Stromal Stem Cells During Post Natal Bone Regeneration

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Free PMC article

Role of Nanog in the Maintenance of Marrow Stromal Stem Cells During Post Natal Bone Regeneration

Manish V Bais et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ∼50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ∼80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ∼3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ∼50% decrease was seen in the expression of terminal osteogenic gene expression and a ∼50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ∼2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.

Figures

Figure 1
Figure 1. Characterization of Nanog Expression in Bone Marrow Stromal Cells
(A)The relative mRNA expression in adherent mouse marrow stromal cells isolated one day after plating in tissue culture, 6 days after plating but before switching to osteoinductive media and at 21 days in culture (15 days in osteoinductive media) when they are fully differentiated. B) Comparison of undifferentiated marrow stromal cells (MSCs) to W20-17 marrow stromal cell line. Levels are expressed relative to murine embryonic fibroblasts that have been used as a null expressing reference to which ESC are compared. C) Evaluation of relative 2.5 Kb Nanog promoter activities in MEFs and W20-17 cell line. D) BMP-7 down regulation of Nanog promoter activity with exogenous addition of BMP7 protein at 10, 100 and 300 ng concentration compared to control in W20-17 cell line E) Effect of lentiviral Nanog shRNA particle transduction on endogenous Nanog mRNA within W20-17 cells. F) Functional effect of Nanog shRNA mediated knockdown on Osteocalcin mRNA expression. NT= transduced with non target virus. Error bars represent standard deviation from replicate measurements from three experiments.
Figure 2
Figure 2. Effect of Nanog Knockdown and Overexpression on Bone Marrow Stromal Cell Proliferation
(Top panel)Effect of nanog shRNA and Nanog OE lentiviral particles transduction on proliferation. The temporal effect of nanog overexpression on cell proliferation was assessed by MTT assay. Horizontal line indicates control level. Mean values are measurements made from three separate preparations of cells. (Bottom Panel) Effect of lentivirus shRNA or Nanog OE particle transduction on expression of Nanog mRNA expression on days 10, 14 and 21. Error bars represent standard deviation from replicate measurements from three experiments. P-value reflects the comparison of different treatment groups calculated by t-test and denoted as * p<0.05. NT= transduced with non target virus. shRNA= lentivirus shRNA to Nanog. OE = overexpression lentivirus for Nanog. NT= non target lentiviral vector. Separate NT viral constructs were used for the shRNA and OE experiments so that the backbone vector that was used for each control was different between experimental groups
Figure 3
Figure 3. Time Course of Nanog Expression during Bone Regeneration after Surgical Marrow Ablation
(A)Histological assessment of endosteal bone formation in response to surgical marrow ablation on days 5, 7, 14 and 21. All images are orientated with the proximal end of the bone at the top (B) Steady state mRNA expression of Nanog, Runx2, Osteocalcin, RankL and TRAP over a 21 day time course of primary bone formation and coupled remodeling following surgical bone marrow ablation. Error bars represent standard deviation from replicates measurements from three different mice.
Figure 4
Figure 4. Functional Effect of Nanog shRNA Knockdown on Bone Regeneration Following Surgical Bone Marrow Ablation
(A)Graphical assessment of percent bone tissue volume (BV/TV) as determined from quantitative μCT analysis MicroCT. B) Representative μCT 3D renderings of bone formation from control mice and mice injected with NT and Nanog shRNA lentiviral particles are shown in the bottom panels. All images are orientated with the proximal end of the bone at the top and are from 7 and 21 days after the time of surgery. The Comparisons for all pairs using one way ANOVA showed statistical significant difference in mean in shNanog group. P<.0001 denoted as ***showing significant differences in each group (n=10/ group). C) Histological analysis of bone formation in response to surgical marrow ablation in NT shRNA and Nanog shRNA injected group on day 7 and day 21. Sections were cut in longitudinal orientations and selected sections from the central region of the bone are presented in the space occupied by the regenerating marrow. Sections were stained with Goldners trichome stain. D) Effect of nanog shRNA lentiviral treatment on the expression of various genes involved in regenerative response in skeletal and adipose tissue lineages within marrow tissues formed after surgical ablation. Effect of nanog shRNA treatment relative to NT shRNA on expression of genes involved in skeletal lineage shown in panel on left and major genes involved in adipogenesis are shown in right panel. Levels of expression are relative to unoperated control tibia bones. Error bars represent standard deviation from replicates measurements from pooled three experiments.

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