During osteoporosis, the shift of mesenchymal stem cell (MSC) lineage commitment to adipocyte leads to the imbalance between bone mass and fat, which increases the risk of fracture. The Enhancer of Zeste homology 2 (EZH2), which methylates histone H3 on lysine 27 (H3K27me3), controls MSC cell lineage commitment. However, whether EZH2 is related to osteoporosis remains elusive. In our study, we found EZH2 expression was significantly increased in osteoporotic MSCs. EZH2 directly increased H3K27me3 levels on promoters of Wnt1, Wnt6, and Wnt10a to silence Wnt gene transcription. The inhibition of Wnt/β-catenin signaling shifted MSC cell lineage commitment to adipocyte. Knockdown of EZH2 by lentivirus-expressing shRNA rescued the abnormal fate of osteoporotic MSC. By employing the H3K27me3 inhibitor DZNep, we effectively derepressed Wnt signaling and improved osteogenic differentiation of osteoporotic MSCs in vitro. Furthermore, in vivo administration of DZNep successfully increased bone formation and repressed excessive bone marrow fat formation in osteoporotic mice. Noteworthy, DZNep treatment persistently enhanced osteogenic differentiation of endogenous MSCs. In conclusion, our study demonstrated that redundant EZH2 shifted MSC cell lineage commitment to adipocyte, which contributed to the development of osteoporosis. We also provided EZH2 as a novel therapeutic target for improving bone formation during osteoporosis.

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of MSCs, resulting in failure of MSCs therapy. Here, we report a melatonin-based strategy to improve cell therapy of in vitro cultured MSCs. Among four small molecules with anti-aging and stem cell-protection properties (rapamycin, resveratrol, quercetin and melatonin), colony forming, proliferation, and osteogenic differentiation assay showed that melatonin was the most efficient to preserve self-renewal and differentiation properties of rat bone marrow MSCs (BMMSCs) after long-term passaging. Functional assays confirmed melatonin treatment did not affect the colony forming, proliferation and osteogenic differentiation of BMMSCs cultured for 1 or 4 passages, but largely prevented the decline of self-renew and differentiation capacity of BMMSCs cultured for 15 passages in vitro. Furthermore, heterotopic osteogenesis assay, critical size calvarial defects repair assay, osteoporosis treatment and experimental colitis therapy assay strongly certified that melatonin preserved the therapeutic effect of long-term passaged BMMSCs on bone regeneration and immunotherapy in vivo. Mechanistically, melatonin functioned by activating antioxidant defense system, inhibiting the pathway of cell senescence, and preserving the expression of gene governing the stemness. Taken together, our findings showed that melatonin treatment efficiently prevented the dysfunction and therapeutic failure of BMMSCs after long-term passaging, providing a practical strategy to improve the application of BMMSCs in tissue engineering and cytotherapy.

Decline of antioxidant defense after estrogen deficiency leads to oxidative damage in bone marrow-derived mesenchymal stem cells (BMMSCs), resulting a defect of bone formation in osteoporosis. Forkhead box O1 (FoxO1) protein is crucial for defending physiological oxidative damage in bone. But whether FoxO1 is involved in the oxidative damage during osteoporosis is largely unknown. In this study, we found that FoxO1 protein accumulation was decreased in BMMSCs of ovariectomized mice. The decrease of FoxO1 resulted in the suppression of manganese superoxide dismutase (Sod2) and catalase (Cat) expression and accumulation of reactive oxygen species (ROS), inhibiting the osteogenic differentiation of BMMSCs. The decline of FoxO1 protein was caused by tumor necrosis factor-alpha (TNF-a) accumulated after estrogen deficiency. Mechanistically, TNF-a activated NF-jB pathway to promote microRNA-705 expression, which function as a repressor of FoxO1 through post-transcriptional regulation. Inhibition of NFjB pathway or knockdown of miR-705 largely prevented the decline of FoxO1-mediated antioxidant defense caused by TNF-a and ameliorated the oxidative damage in osteoporotic BMMSCs. Moreover, the accumulated ROS further activated NF-jB pathway with TNF-a, which formed a feed-forward loop to persistently inhibiting FoxO1 protein accumulation in BMMSCs. In conclusion, our study revealed that the decline of FoxO1 is an important etiology factor of osteoporosis and unclosed a novel mechanism of FoxO1 regulation by TNF-a. These findings suggested a close correlation between inflammation and oxidative stress in stem cell dysfunction during degenerative bone diseases. STEM CELLS 2016;34:1054-1067 SIGNIFICANCE STATEMENTThis study revealed two novel findings. (a) The decline of FOXO1 protein after estrogen deficiency is a crucial etiological factor of osteoporosis. Previous study confirmed that FOXO1 is an important mediator of skeleton resistance to physiologic oxidative stress. Our study further showed that oxidative damage in osteoporosis is, at least partly, caused by FOXO1 dysfunction, suggesting FOXO1 is a potential target for osteoporosis prevention and treatment. (b) TNF-a decreased FOXO1-mediated antioxidant defense through post-transcriptional regulation by miR-705. This study uncovered that TNF-a directly inhibits antioxidant defense to aggravate oxidative stress damage, suggesting a central role of TNF-a in oxidative damage regulation. Since oxidative stress could induce inflammatory events, these results unclosed a novel network between inflammation and oxidative stress, suggesting intervention of inflammation signaling is a potential strategy for recovery of mesenchymal stem cell dysfunction and treatment of degenerative bone diseases.

MicroRNAs (miRNAs) emerge as important regulators of stem cell lineage commitment and bone development. MiRNA-26a (miR-26a) is one of the important miRNAs regulating osteogenic differentiation of both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs). However, miR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs, suggesting distinct post-transcriptional regulation of tissue-specific MSC differentiation. However, the molecular basis is largely unknown. Here, we report that the function of miR-26a is largely depended on the intrinsic signaling regulation network of MSCs. Using bioinformatics and functional assay, we confirmed that miR-26a potentially targeted on GSK3β and Smad1 to regulate Wnt and BMP signaling pathway. Overall comparative analysis revealed that Wnt signaling was enhanced more potently and played a more important role than BMP signaling in osteogenic differentiation of BMSCs, whereas BMP pathway was more essential for promoting osteogenic differentiation of ADSCs. The distinct activation pattern and role of signaling pathways determined that miR-26a majorly targeted on GSK3β to activate Wnt signaling for promoting osteogenic differentiation of BMSCs, whereas it inhibited Smad1 to suppress BMP signaling for interfering with the osteogenic differentiation of ADSCs. Taken together, our study demonstrated that BMSCs and ADSCs applied different signaling pathway to facilitate their osteogenic differentiation, which determined the inverse function of miR-26a. The distinct transcriptional regulation and post-transcriptional regulation network suggested the intrinsic molecular differences between tissue-specific MSCs and the complexity in MSC research and MSC-based cell therapy.

Disrupted Wnt signaling in osteoblastic-lineage cells leads to bone formation defect in osteoporosis. However, the factors repressing Wnt signaling are unclear. In our study, we found that Wnt signaling was suppressed persistently in bone marrow-derived mesenchymal stem cells (BMSCs) during osteoporosis. Accordingly, histone acetylation levels on Wnt genes (Wnt1, Wnt6, Wnt10a, and Wnt10b) were declined in BMSCs from OVX mice. By screening the family of histone acetyltransferase, we identified that GCN5 expression increased during osteogenic differentiation of BMSCs, whereas decreased after osteoporosis. Further analysis revealed that GCN5 promoted osteogenic differentiation of BMSCs by increasing acetylation on histone 3 lysine 9 loci on the promoters of Wnt genes. Reduced GCN5 expression suppressed Wnt signaling, resulting in osteogenic defect of BMSCs from OVX mice. Moreover, restoring GCN5 levels recovered BMSC osteogenic differentiation, and attenuated bone loss in OVX mice. Taken together, our study demonstrated that disrupted histone acetylation modification in BMSCs lead to bone formation defect during osteoporosis. The findings also introduced a novel therapeutic target for osteoporosis.

Angiogenesis is disrupted in age-related and postmenopausal osteoporosis. However, the mechanisms of the disorder remain elusive. We confirmed in this study that, in accordance with the decrease of H-type vessels, the proangiogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs) declined during osteoporosis. Screening of the histone acetyltransferase family revealed that GCN5 decreased in BMSCs derived from osteoporotic femur. Further analysis identified that GCN5 plays important roles in regulating the proangiogenic potential of BMSCs. GCN5 promoted BMSC-mediated angiogenesis by enhancing H3K9ac levels on the promoter of The decrease of GCN5 in osteoporotic BMSCs led to the decline of proangiogenic capacity. Accordingly, overexpression of GCN5 enhanced the proangiogenic potency of osteoporotic BMSCs. Furthermore, recovering GCN5 expression by lentiviral expression vector significantly attenuated the loss of angiogenesis in ovariectomized mouse femurs. Our study results revealed an epigenetic mechanism controlling BMSC-mediated angiogenesis and provided a novel therapeutic target for osteoporosis treatment.-Jing, H., Liao, L., Su, X., Shuai, Y. Zhang, X., Deng, Z., Jin, Y. Declining histone acetyltransferase GCN5 represses BMSC-mediated angiogenesis during osteoporosis.

Cutaneous wounds are among the most common soft tissue injuries. Wounds involving dermis suffer more from outside influence and higher risk of chronic inflammation. Therefore the appearance and function restoration has become an imperative in tissue engineering research. In this study, cell-aggregates constructed with green fluorescent protein-expressing (GFP+) rat bone marrow mesenchymal stem cells (BMMSCs) were applied to rat acute full-layer cutaneous wound model to confirm its pro-regeneration ability and compare its regenerative efficacy with the currently thriving subcutaneous and intravenous stem cell administration strategy, with a view to sensing the advantages, disadvantages and the mechanism behind. According to results, cell-aggregates cultured in vitro enjoyed higher expression of several pro-healing genes than adherent cultured cells. Animal experiments showed better vascularization along with more regular dermal collagen deposition for cell-aggregate transplanted models. Immunofluorescence staining on inflammatory cells indicated a shorter inflammatory phase for cell-aggregate group, which was backed up by further RT-PCR. The in situ immunofluorescence staining manifested a higher GFP+-cell engraftment for cell-aggregate transplanted models versus cell administered ones. Thus it is safe to say the BMMSCs aggregate could bring superior cutaneous regeneration for full layer cutaneous wound to BMMSCs administration, both intravenous and subcutaneous.

Programmed cell death-1 (PD-1) belongs to an inhibitory signaling pathway capable of maintaining central and peripheral immune tolerance. Blockage of PD-1 has been identified as a promising immunotherapeutic approach for cancer and chronic infectious diseases. However, it is unknown whether PD-1 pathway regulates stem cell function. It is generally believed that mesenchymal stem cells (MSCs) produce PD-1 ligand, but fail to express PD-1. In this study, we show that neural crest-derived MSCs from dental pulp (MSC-DP), but not MSCs from bone marrow, expressed PD-1. Knocking down PD-1 expression in MSC-DP results in a significantly reduced capacity for cell proliferation and accelerated multipotential differentiation. Mechanistically, we show that PD-1 regulates a SHP2/ERK/Notch cascade to maintain proliferation and a SHP2/ERK/β-catenin cascade to inhibit osteo-/odontogenic differentiation. This study indicates that PD-1 is a key surface molecule controlling cell proliferation and multipotential differentiation of MSC-DP. Through regulating PD-1/SHP2/ERK signaling, we can significantly improve the quality and quantity of culture-expanded MSC-DP for potential clinical therapies.