Liangliang Xu

H19 activates Wnt signaling and promotes osteoblast differentiation by functioning as a competing endogenous RNA

Bone homeostasis is tightly orchestrated and maintained by the balance between osteoblasts and osteoclasts. Recent studies have greatly expanded our understanding of the molecular mechanisms of cellular differentiation. However, the functional roles of non-coding RNAs particularly lncRNAs in remodeling bone architecture remain elusive. In our study, lncRNA H19 was found to be upregulated during osteogenesis in hMSCs. Stable expression of H19 significantly accelerated in vivo and in vitro osteoblast differentiation. Meanwhile, by using bioinformatic investigations and RIP assays combined with luciferase reporter assays, we demonstrated that H19 functioned as an miRNA sponge for miR-141 and miR-22, both of which were negative regulators of osteogenesis and Wnt/β-catenin pathway. Further investigations revealed that H19 antagonized the functions of these two miRNAs and led to de-repression of their shared target gene β-catenin, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. In addition, we also identified a novel regulatory feedback loop between H19 and its encoded miR-675-5p. And miR-675-5p was found to directly target H19 and counteracted osteoblast differentiation. To sum up, these observations indicate that the lncRNA H19 modulates Wnt/β-catenin pathway by acting as a competing endogenous RNA, which may shed light on the functional role of lncRNAs in coordinating osteogenesis.

Salvianolic acid B promotes osteogenesis of human mesenchymal stem cells through activating ERK signaling pathway

Salvianolic acid B, a major bioactive component of Chinese medicine herb, Salvia miltiorrhiza, is widely used for treatment of cardiovascular diseases. Our recent studies have shown that Salvianolic acid B can prevent development of osteoporosis. However, the underlying mechanisms are still not clarified clearly. In the present study, we aim to investigate the effects of Salvianolic acid B on viability and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The results showed Salvianolic acid B (Sal B) had no obvious toxic effects on hMSCs, whereas Sal B supplementation (5μM) increased the alkaline phosphatase activity, osteopontin, Runx2 and osterix expression in hMSCs. Under osteogenic induction condition, Sal B (5μM) significantly promoted mineralization; and when the extracellular-signal-regulated kinases signaling (ERK) pathway was blocked, the anabolic effects of Sal B were diminished, indicating that Sal B promoted osteogenesis of hMSCs through activating ERK signaling pathway. The current study confirms that Sal B promotes osteogenesis of hMSCs with no cytotoxicity, and it may be used as a potential therapeutic agent for the management of osteoporosis.

Aqp1 Enhances Migration of Bone Marrow Mesenchymal Stem Cells Through Regulation of FAK and β-Catenin

Bone marrow mesenchymal stem cells (MSCs) have the potential to migrate to the site of injury and regulate the repair process. Aquaporin 1 (Aqp1) is a water channel molecule and a regulator of endothelial cell migration. To study the role of Apq1 in MSC migration, we manipulated the expression of the Aqp1 gene in MSCs and explored its effects on MSC migration both in vitro and in vivo. Overexpression of Aqp1 promoted MSC migration, while depletion of Aqp1 impaired MSC migration in vitro. When the green fluorescent protein (GFP) labeled Aqp1 overexpressing MSCs were systemically injected into rats with a femoral fracture, there were significantly more GFP-MSCs found at the fracture gap in the Aqp1-GFP-MSC-treated group compared to the GFP-MSC group. To elucidate the underlying mechanism, we screened several migration-related regulators. The results showed that β-catenin and focal adhesion kinase (FAK) were upregulated in the Aqp1-MSCs and downregulated in the Aqp1-depleted MSCs, while C-X-C chemokine receptor type 4 had no change. Furthermore, β-catenin and FAK were co-immunoprecipitated with Aqp1, and depletion of FAK abolished the Aqp1 effects on MSC migration. This study demonstrates that Aqp1 enhances MSC migration ability mainly through the FAK pathway and partially through the β-catenin pathway. Our finding suggests a novel function of Aqp1 in governing MSC migration, and this may aid MSC therapeutic applications.

mir-21 Overexpressing Mesenchymal Stem Cells Accelerate Fracture Healing in a Rat Closed Femur Fracture Model

MicroRNAs are small noncoding RNAs involved in numerous biological processes. Emerging pieces of evidence suggest that microRNAs play important roles in osteogenesis and skeletal homeostasis. Recent studies indicated the significant regulation function of mir-21 in osteogenesis in vitro, but little information is known about its veritable functions in vivo. In the present study, we aimed to investigate the effect of mir-21 intervention on osteogenic differentiation of rats bone marrow derived mesenchymal stem cells (rBMSCs) and repair capacity in rats closed femur fracture model with internal fixation. The results showed that the upregulation of mir-21 not only increased the expression of osteopontin and alkaline phosphatase in rBMSCs but also promoted mineralization in the condition of osteogenic induction. Furthermore, the bone healing properties were also improved in fracture healing model according to the results of micro-CT, mechanical test, and histological analysis. The current study confirms that the overexpression of mir-21 could promote osteogenesis and accelerate bone fracture healing, which may contribute to a new therapeutic way for fracture repair.

Cellular retinol-binding protein 1 (CRBP-1) regulates osteogenenesis and adipogenesis of mesenchymal stem cells through inhibiting RXRα-induced β-catenin degradation.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can differentiate into osteoblasts, chondrocytes and adipocytes, providing a potential source for musculoskeletal tissue engineering. Retinoid signaling plays very important roles in skeletal development. CRBP1 (cellular retinol binding protein 1), a key component of retinoid signaling pathway, is known to take part in vitamin A metabolism and intracellular transporting of retinoids. However, the role of CRBP1 in MSCs remains still obscure. In this study, we investigated the cellular effects of CRBP1 on osteogenic and adipogenic differentiation of bone marrow derived MSCs in vitro and in vivo. Our results showed that CRBP1 overexpression promoted osteogenic differentiation of bone marrow derived MSCs, while inhibited their adipogenic differentiation. We also demonstrated that the possible underlying mechanism for CRBP1 promoting osteogenic differentiation of MSCs was by inhibiting RXRα-induced β-catenin degradation, maintaining β-catenin and pERK1/2 at higher levels. These findings reveal a potential role of CRBP1 in the regulation of β-catenin turnover which can greatly affect the process of osteogenesis and adipogenesis of MSCs.

Sox11-modified mesenchymal stem cells (MSCs) accelerate bone fracture healing: Sox11 regulates differentiation and migration of MSCs

Mesenchymal stem cells (MSCs) are a promising cell resource for tissue engineering. Sry‐related high‐mobility group box 11 (Sox11) plays critical roles in neural development and organogenesis. In the present study, we investigated the role of Sox11 in regulating trilineage differentiation (osteogenesis, adipogenesis, and chondrogenesis) and migration of MSCs, and explored the effect of systemically administrated Sox11‐modified MSCs on bone fracture healing using the rat model of open femur fracture. Our results demonstrated that Sox11 overexpression increased the trilineage differentiation and migration of MSCs, as well as cell viability under oxidative stress. The effect of Sox11 on osteogenesis was confirmed by ectopic bone formation assay conducted in nude mice. In addition, we found that Sox11 could activate the bone morphogenetic protein (BMP)/Smad signaling pathway in MSCs. By dual‐luciferase reporter assay, we also demonstrated that Sox11 could transcriptionally activate runt‐related transcription factor 2 (Runx2) and CXC chemokine receptor‐4 (CXCR4) expression. The activation of the BMP/Smad signaling pathway and Runx2, CXCR4 expression may have a synergic effect, which largely contributed to the effect of Sox11 on MSC fate determination and migration. Finally, using an open femur fracture model in rats, we found that a larger number of MSCs stably expressing Sox11 migrated to the fracture site and improved bone fracture healing. Taken together, our study shows that Sox11 is an important regulator of MSC differentiation and migration, and Sox11‐modified MSCs may have clinical implication for accelerating bone fracture healing, which can reduce the delayed unions or nonunions.—Xu, L., Huang, S., Hou, Y., Liu, Y., Ni, M., Meng, F., Wang, K., Rui, Y., Jiang, X., Li, G. Sox11‐modified mesenchymal stem cells (MSCs) accelerate bone fracture healing: Sox11 regulates differentiation and migration of MSCs. FASEB J. 29, 1143‐1152 (2015). www.fasebj.org

An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Summary Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2) Our culture medium is not supplemented with any additional growth factor. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

Epigenetic memory gained by priming with osteogenic induction medium improves osteogenesis and other properties of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

The Effects of Secretion Factors from Umbilical Cord Derived Mesenchymal Stem Cells on Osteogenic Differentiation of Mesenchymal Stem Cells

Factors synthesized by mesenchymal stem cells (MSCs) contain various growth factors, cytokines, exosomes and microRNAs, which may affect the differentiation abilities of MSCs. In the present study, we investigated the effects of secretion factors of human umbilical cord derived mesenchymal stem cells (hUCMSCs) on osteogenesis of human bone marrow derived MSCs (hBMSCs). The results showed that 20 μg/ml hUCMSCs secretion factors could initiate osteogenic differentiation of hBMSCs without osteogenic induction medium (OIM), and the amount of calcium deposit (stained by Alizarin Red) was significantly increased after the hUCMSCs secretion factors treatment. Real time quantitative reverse transcription-polymerase chain reaction (real time qRT-PCR) demonstrated that the expression of osteogenesis-related genes including ALP, BMP2, OCN, Osterix, Col1α and Runx2 were significantly up-regulated following hUCMSCs secretion factors treatment. In addition, we found that 10 μg hUCMSCs secretion factors together with 2×105 hBMSCs in the HA/TCP scaffolds promoted ectopic bone formation in nude mice. Local application of 10 μg hUCMSCs secretion factors with 50 μl 2% hyaluronic acid hydrogel and 1×105 rat bone marrow derived MSCs (rBMSCs) also significantly enhanced the bone repair of rat calvarial bone critical defect model at both 4 weeks and 8 weeks. Moreover, the group that received the hUCMSCs secretion factors treatment had more cartilage and bone regeneration in the defect areas than those in the control group. Taken together, these findings suggested that hUCMSCs secretion factors can initiate osteogenesis of bone marrow MSCs and promote bone repair. Our study indicates that hUCMSCs secretion factors may be potential sources for promoting bone regeneration.

Stepwise Differentiation of Mesenchymal Stem Cells Augments Tendon-Like Tissue Formation and Defect Repair In Vivo

Tendon injuries are common and present a clinical challenge, as they often respond poorly to treatment and result in long‐term functional impairment. Inferior tendon healing responses are mainly attributed to insufficient or failed tenogenesis. The main objective of this study was to establish an efficient approach to induce tenogenesis of bone marrow‐derived mesenchymal stem cells (BMSCs), which are the most common seed cells in tendon tissue engineering. First, representative reported tenogenic growth factors were used as media supplementation to induce BMSC differentiation, and the expression of teno‐lineage transcription factors and matrix proteins was compared. We found that transforming growth factor (TGF)‐β1 significantly induced teno‐lineage‐specific gene scleraxis expression and collagen production. TGF‐β1 combined with connective tissue growth factor (CTGF) elevated tenomodulin and Egr1 expression at day 7. Hence, a stepwise tenogenic differentiation approach was established by first using TGF‐β1 stimulation, followed by combination with CTGF for another 7 days. Gene expression analysis showed that this stepwise protocol initiated and maintained highly efficient tenogenesis of BMSCs. Finally, regarding in situ rat patellar tendon repair, tendons treated with induced tenogenic BMSCs had better structural and mechanical properties than those of the control group, as evidenced by histological scoring, collagen I and tenomodulin immunohistochemical staining, and tendon mechanical testing. Collectively, these findings demonstrate a reliable and practical strategy of inducing tenogenesis of BMSCs for tendon regeneration and may enhance the effectiveness of cell therapy in treating tendon disorders.