Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells

We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs) and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5), which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine

Transplanted neural progenitor cells expressing mutant NT3 promote myelination and partial hindlimb recovery in the chronic phase after spinal cord injury

Neutrotrophin-3 (NT3) plays a protective role in injured central nervous system tissues through interaction with trk receptors. To enhance the regeneration of damaged tissue, a combination therapy with cell transplantation and neurotrophins has been under development. We examined whether the transplantation of neural progenitor cells (NPCs) secreting NT3/D15A, a multi-neurotrophin with the capacity to bind both trkB and trkC, would enhance the repair of damaged tissues and the functional recovery in a chronic phase of spinal cord injury. The cultured NPCs with lentiviral vector containing either GFP or NT3/D15A were transplanted into the contused spinal cord at 6 weeks after the initial thoracic injury. Eight weeks after the transplantation, the NT3/D15A transplants displayed better survival than the GFP transplants, and they exhibited enhanced myelin formation and partial improvement of hindlimb function. Our study revealed that NT3/D15A produced positive effects in injured spinal cords even in the chronic phase. These effects suggest an enhanced neurotrophin-trk signaling by NT3/D15A

Dexamethasone enhances osteogenic differentiation of bone marrow- and muscle-derived stromal cells and augments ectopic bone formation induced by bone morphogenetic protein-2.

UNLABELLED:We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. HIGHLIGHTS:1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2

Ectopic bone formation analyses.

<p>A: Bone formation capability of muscle-derived cells. Representative histological sections of a scaffold loaded with BMSCs or MuSCs cultured with both dexamethasone and BMP-2. Scale bar: 1 mm (left panels), 200 μm (middle panels) and 50 μm (right panels). Black arrows indicate new bone formation in the scaffold. Black arrow heads indicate osteocytes and green arrow heads indicate bone lining cells. B: Newly formed bone, T: β-TCP, P: Porous area. B: Recruitment of cells residing in muscle tissue to participate in BMP-2-induced ectopic bone formation. Cells labeled prior to local BMP-2 administration were detected in the newly formed woven bone area. Representative histological sections were stained with H&E and evaluated for i-QD fluorescence. The black arrows in the H&E image show the locations of fluorescently labeled cells indicated with white arrows in the fluorescent image. Scale bar: 200 μm. B: Newly formed bone, T: β-TCP, P: Porous area. C: Augmentation of ectopic bone formation by dexamethasone. C-1, 2: Representative histological sections of an excised scaffold that had been loaded with BMP-2 alone and a scaffold that had been loaded with dexamethasone and BMP-2. The sections were stained with H&E and immunostained for osteocalcin. Black arrows indicate new bone formation in the scaffold. Red arrows indicate osteocalcin positive staining area. Scale bar: 1 mm (top panels of C-1), 200 μm (bottom panels of C-1) and 50 μm (C-2). B: Newly formed bone, T: β-TCP, P: Porous area. C-3: Quantification of bone formation at 3 weeks after transplantation. The Y axis indicates the bone formation ratio calculated as total bone area/total scaffold area. Each bar represents the mean with the standard deviation (SD). *denotes <i>P</i> < 0.05.</p

Dexamethasone affects cell proliferation during osteogenic differentiation.

<p>The absorbance at 585 nm was measured for dye extracted from the wells, and ratios relative to the standard are presented in the graphs. A: BM, B: BM-Dex, C: Mu, and D: Mu-Dex</p

Dexamethasone pretreatment and osteogenic induction with combined dexamethasone and BMP-2 treatment enhance the osteogenic differentiation of BMSCs and MuSCs.

<p>A: Schematic representation of the cell culture protocol. Gross images of ALP staining (ALP) and Von Kossa staining (VK) of BMSCs (B) and MuSCs (D). Quantitative analysis of the mRNA expression of ALP and osteocalcin in BMSCs (C) and MuSCs (E). The fold change in gene expression was normalized to that of BM-Dex-AG or Mu-Dex-AG. Bars show the mean and SEM. Statistical significance was confirmed between the BM and BM-Dex groups (ALP: p = 0.015, OCN: p = 0.023) and between the Mu and Mu-DEX groups (ALP: p = 0.019, OCN: p = 0.015). Effects of combinations of differentiation reagents were significant for ALP in BM-Dex (p = 0.032) and Mu-DEX (p = 0.019) and for OCN in Mu-DEX (p = 0.024).</p

RT-PCR primers used in this study.

<p>RT-PCR primers used in this study.</p