Novel SCRG1/BST1 axis regulates self-renewal, migration, and osteogenic differentiation potential in mesenchymal stem cells.
ABSTRACT: Human mesenchymal stem cells (hMSCs) remodel or regenerate various tissues through several mechanisms. Here, we identified the hMSC-secreted protein SCRG1 and its receptor BST1 as a positive regulator of self-renewal, migration, and osteogenic differentiation. SCRG1 and BST1 gene expression decreased during osteogenic differentiation of hMSCs. Intriguingly, SCRG1 maintained stem cell marker expression (Oct-4 and CD271/LNGFR) and the potentials of self-renewal, migration, and osteogenic differentiation, even at high passage numbers. Thus, the novel SCRG1/BST1 axis determines the fate of hMSCs by regulating their kinetic and differentiation potentials. Our findings provide a new perspective on methods for ex vivo expansion of hMSCs that maintain native stem cell potentials for bone-forming cell therapy.
Project description:OBJECTIVES:The aim of this study was to investigate whether sclerostin (SOST) regulates the osteogenic differentiation of rat ectomesenchymal stem cells (EMSCs) and whether SOST and low-affinity nerve growth factor receptor (LNGFR) regulate the osteogenic differentiation of EMSCs. MATERIALS AND METHODS:EMSCs were isolated from embryonic facial processes from an embryonic 12.5-day (E12.5d) pregnant Sprague-Dawley rat. LNGFR+ EMSCs and LNGFR- EMSCs were obtained by fluorescence-activated cell sorting and were subsequently induced to undergo osteogenic differentiation in vitro. SOST/LNGFR small-interfering RNAs and SOST/LNGFR overexpression plasmids were used to transfect EMSCs. RESULTS:LNGFR+ EMSCs displayed a higher osteogenic capacity and lower SOST levels compared with LNGFR- EMSCs. SOST silencing enhanced the osteogenic differentiation of LNGFR- EMSCs, while SOST overexpression attenuated the osteogenic differentiation of LNGFR+ EMSCs. Moreover, LNGFR was present upstream of SOST and strengthened the osteogenic differentiation of EMSCs by decreasing SOST. CONCLUSIONS:SOST alleviated the osteogenic differentiation of EMSCs, and LNGFR enhanced the osteogenic differentiation of EMSCs by decreasing SOST, suggesting that the LNGFR/SOST pathway may be a novel target for promoting dental tissue regeneration and engineering.
Project description:Considerable evidence has shown that the Wnt/?-catenin pathway is involved in osteogenic differentiation in various stem cells. However, the role of Wnt/?-catenin pathway in regulating the osteogenic differentiation of rat ectomesenchymal stem cells (EMSCs), which are considered to be the progenitors of dental mesenchymal stem cells, remains unknown. In this study, we demonstrated that nuclear ?-catenin was upregulated during EMSC osteogenic differentiation. The Wnt signalling inhibitor IWR-1-endo inhibited EMSC osteogenic differentiation, while the Wnt signalling agonist SKL2001 promoted it. Moreover, nuclear ?-catenin was further upregulated by the overexpression of low-affinity nerve growth factor receptor (LNGFR) during EMSC osteogenic differentiation. Further experiments demonstrated that LNGFR overexpression enhanced EMSC osteogenic differentiation, while LNGFR silencing decreased it. Additionally, IWR-1-endo attenuated LNGFR-enhanced EMSC osteogenic differentiation. Collectively, our data reveal that LNGFR targets the Wnt/?-catenin pathway and positively regulates EMSC osteogenic differentiation, suggesting that Wnt/?-catenin pathway may be involved in the development of teeth and that the targeting Wnt/?-catenin pathway may have great potential for applications in dental tissue engineering regeneration.
Project description:Human mesenchymal stem cells (hMSCs), which conventionally are isolated based on their adherence to plastic, are heterogeneous and have poor growth and differentiation, limiting our ability to investigate their intrinsic characteristics. We report an improved prospective clonal isolation technique and reveal that the combination of three cell-surface markers (LNGFR, THY-1, and VCAM-1) allows for the selection of highly enriched clonogenic cells (one out of three isolated cells). Clonal characterization of LNGFR(+)THY-1(+) cells demonstrated cellular heterogeneity among the clones. Rapidly expanding clones (RECs) exhibited robust multilineage differentiation and self-renewal potency, whereas the other clones tended to acquire cellular senescence via P16INK4a and exhibited frequent genomic errors. Furthermore, RECs exhibited unique expression of VCAM-1 and higher cellular motility compared with the other clones. The combination marker LNGFR(+)THY-1(+)VCAM-1(hi+) (LTV) can be used selectively to isolate the most potent and genetically stable MSCs.
Project description:Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days' culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days' culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-beta signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.
Project description:Lineage specification is an essential process in stem cell fate, tissue homeostasis and development. Microenvironmental cues provide direct and selective extrinsic signals to regulate lineage specification of stem cells. Microenvironmental milieu consists of two essential components, one being extracellular matrix (ECM) as the substratum, while the other being cell secreted exosomes and growth factors. ECM of differentiated cells modulates phenotypic expression of stem cells, while their exosomes contain phenotype specific instructive factors (miRNA, RNA and proteins) that control stem cell differentiation. This study demonstrates that osteoblasts-derived (Os-Exo) and adipocytes-derived (Ad-Exo) exosomes contain instructive factors that regulate the lineage specification of human mesenchymal stem cells (hMSCs). Analyses of exosomes revealed the presence of transcription factors in the form of RNA and protein for osteoblasts (RUNX2 and OSX) and adipocytes (C/EBP? and PPAR?). In addition, several miRNAs reported to have osteogenic and adipogenic differentiation potentials are also identified in these exosomes. Kinetic and differentiation analyses indicate that both osteoblast and adipocyte exosomes augment ECM-mediated differentiation of hMSCs into the respective lineage. The combination of osteoblast/adipocyte ECM and exosomes turned-on the lineage specific gene expressions at earlier time points of differentiation compared to the respective ECM or exosomes administered individually. Interestingly, the hMSCs differentiated on osteoblast ECM with adipogenic exosomes showed expression of adipogenic lineage genes, while hMSCs differentiated on adipocyte ECM with osteoblast exosomes showed osteogenic lineage genes. Based on these observations, we conclude that exosomes might override the ECM mediated instructive signals during lineage specification of hMSC.
Project description:Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3.
Project description:Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However, these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking, which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study, we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs), a type of adult stem cells that reside in a number of tissues, at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM, resveratrol promotes cell self-renewal by inhibiting cellular senescence, whereas at 5 µM or above, resveratrol inhibits cell self-renewal by increasing senescence rate, cell doubling time and S-phase cell cycle arrest. At 1 µM, its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect, accompanied with increased senescence rate. At all concentrations, resveratrol promotes osteogenic differentiation in a dosage dependent manner, which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary, resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs.
Project description:Abstract Implantation of stem cells for tissue regeneration faces significant challenges such as immune rejection and teratoma formation. Cell?free tissue regeneration thus has a potential to avoid these problems. Stem cell derived exosomes do not cause immune rejection or generate malignant tumors. Here, exosomes that can induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) are identified and used to decorate 3D?printed titanium alloy scaffolds to achieve cell?free bone regeneration. Specifically, the exosomes secreted by hMSCs osteogenically pre?differentiated for different times are used to induce the osteogenesis of hMSCs. It is discovered that pre?differentiation for 10 and 15 days leads to the production of osteogenic exosomes. The purified exosomes are then loaded into the scaffolds. It is found that the cell?free exosome?coated scaffolds regenerate bone tissue as efficiently as hMSC?seeded exosome?free scaffolds within 12 weeks. RNA?sequencing suggests that the osteogenic exosomes induce the osteogenic differentiation by using their cargos, including upregulated osteogenic miRNAs (Hsa?miR?146a?5p, Hsa?miR?503?5p, Hsa?miR?483?3p, and Hsa?miR?129?5p) or downregulated anti?osteogenic miRNAs (Hsa?miR?32?5p, Hsa?miR?133a?3p, and Hsa?miR?204?5p), to activate the PI3K/Akt and MAPK signaling pathways. Consequently, identification of osteogenic exosomes secreted by pre?differentiated stem cells and the use of them to replace stem cells represent a novel cell?free bone regeneration strategy. Exosomes secreted from human mesenchymal stem cells (hMSCs) pre?differentiated for a certain period of time can serve as inducers to induce osteogenic differentiation of hMSCs in vitro. They can decorate 3D printed titanium alloy scaffolds, which are further implanted into radial bone defect. They are found to enable the scaffolds to achieve efficient cell?free bone regeneration in vivo.
Project description:The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.
Project description:In this study we developed a dual therapeutic metal ion-releasing nanoparticle for advanced osteogenic differentiation of stem cells. In order to enhance the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and induce angiogenesis, zinc (Zn) and iron (Fe) were synthesized together into a nanoparticle with a pH-sensitive degradation property. Zn and Fe were loaded within the nanoparticles to promote early osteogenic gene expression and to induce angiogenic paracrine factor secretion for hMSCs. In vitro studies revealed that treating an optimized concentration of our zinc-based iron oxide nanoparticles to hMSCs delivered Zn and Fe ion in a controlled release manner and supported osteogenic gene expression (RUNX2 and alkaline phosphatase) with improved vascular endothelial growth factor secretion. Simultaneous intracellular release of Zn and Fe ions through the endocytosis of the nanoparticles further modulated the mild reactive oxygen species generation level in hMSCs without cytotoxicity and thus improved the osteogenic capacity of the stem cells. Current results suggest that our dual ion releasing nanoparticles might provide a promising platform for future biomedical applications.