Search

Background: The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion., Methods: In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate. This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes)., Results: Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs. This method allowed the generation of numerous high-purity Sca-1 + CD44 + F4/80 - mBMSCs (> 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays., Conclusion: Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs., (© 2021. The Author(s).)

STAT5 is a transcription factor that is activated by various cytokines, hormones, and growth factors. Activated STAT5 is then translocated to the nucleus and regulates the transcription of target genes, affecting several biological processes. Several studies have investigated the role of STAT5 in adipogenesis, but unfortunately, its role in adipogenesis remains controversial. In the present study, we generated adipocyte-specific Stat5 conditional knockout (cKO) (Stat5 fl/fl ;Apn-cre) mice to investigate the role of STAT5 in the adipogenesis of bone marrow mesenchymal stem cells (BMSCs). BMSC adipogenesis was significantly inhibited upon overexpression of constitutively active STAT5A, while it was enhanced in the absence of Stat5 in vitro. In vivo adipose staining and histological analyses revealed increased adipose volume in the bone marrow of Stat5 cKO mice. ATF3 is the target of STAT5 during STAT5-mediated inhibition of adipogenesis, and its transcription is regulated by the binding of STAT5 to the Atf3 promoter. ATF3 overexpression was sufficient to suppress the enhanced adipogenesis of Stat5-deficient adipocytes, and Atf3 silencing abolished the STAT5-mediated inhibition of adipogenesis. Stat5 cKO mice exhibited reduced bone volume due to an increase in the osteoclast number, and coculture of bone marrow-derived macrophages with Stat5 cKO adipocytes resulted in enhanced osteoclastogenesis, suggesting that an increase in the adipocyte number may contribute to bone loss. In summary, this study shows that STAT5 is a negative regulator of BMSC adipogenesis and contributes to bone homeostasis via direct and indirect regulation of osteoclast differentiation; therefore, it may be a leading target for the treatment of both obesity and bone loss-related diseases.

The primary cilium acts as a sensory organelle with diverse receptors and ion channels to detect extracellular cues and regulate cellular functions, including cell migration. The migration of mesenchymal stem cells (MSCs) to bone remodeling sites is important for bone homeostasis. Recently, we have suggested that osteopontin (OPN) is a significant chemoattractant in MSC migration to bone remodeling sites. The objective of this study was to determine whether the primary cilium acts as a chemoattractant sensory unit to detect OPN cues and control MSC migration. We found that the loss of primary cilium induced by silencing of IFT88 reduced OPN-induced migration of MSCs. The effect of IFT88 silencing on cellular attachment, spreading, and proliferation was negligible. The loss of primary cilium did not affect the level of integrinβ1 or CD44, two known receptors for OPN. Interestingly, CD44 was localized to the primary cilium by OPN stimulus. Knockdown of IFT88 or CD44 dysregulated OPN-induced signaling activation and abolished OPN-induced Cdc42 activation. Our findings suggest that the primary cilium acts as a chemoattractant sensor for OPN to regulate MSC migration by controlling not only CD44-mediated OPN signaling, but also Cdc42-mediated actin cytoskeleton rearrangement., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Supplementation of mesenchymal stem cells (MSCs) at sites of bone resorption is required for bone homeostasis because of the non-proliferation and short lifespan properties of the osteoblasts. Calcium ions (Ca 2+ ) are released from the bone surfaces during osteoclast-mediated bone resorption. However, how elevated extracellular Ca 2+ concentrations would alter MSCs behavior in the proximal sites of bone resorption is largely unknown. In this study, we investigated the effect of extracellular Ca 2+ on MSCs phenotype depending on Ca 2+ concentrations. We found that the elevated extracellular Ca 2+ promoted cell proliferation and matrix mineralization of MSCs. In addition, MSCs induced the expression and secretion of osteopontin (OPN), which enhanced MSCs migration under the elevated extracellular Ca 2+ conditions. We developed in vitro osteoclast-mediated bone resorption conditions using mouse calvaria bone slices and demonstrated Ca 2+ is released from bone resorption surfaces. We also showed that the MSCs phenotype, including cell proliferation and migration, changed when the cells were treated with a bone resorption-conditioned medium. These findings suggest that the dynamic changes in Ca 2+ concentrations in the microenvironments of bone remodeling surfaces modulate MSCs phenotype and thereby contribute to bone regeneration.

Introduction: Because a root-end filling material comes into contact with the surrounding cells or tissues, understanding the cell-material interfacial activity is important. Thus, the purpose of this study was to assess the biocompatibility of 3 endodontic bioactive cements (MTA [Dentsply, Tulsa, OK], Bioaggregate [BA; Innovative Bioceramix, Vancouver, BC, Canada], and Biodentine [BD; Septodont, St Maur des Fosses, France]) and to investigate the effect of cements on the differentiation of mesenchymal stem cells., Methods: Cell viability, mineralization, and differentiation were evaluated using an 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay and alkaline phosphatase (ALP) staining. The expressions of ALP, osteocalcin, and bone sialoprotein at the gene level were detected by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction., Results: Cell viability of BD in concentrations of 1, 1/2, and 1/4 was significantly lower than MTA and BA (P < .05). There was no statistically significant difference in cell viability between materials in concentrations of 1/10 and 1/50 (P < .05). The messenger RNA level of osteogenic genes increased significantly in the MTA and BA groups compared with controls (P < .05). However, although the messenger RNA level of osteogenic genes increased in the BD group, there was no statistically significant difference compared with controls. MTA, BA, and BD led to an increase in ALP staining compared with controls., Conclusions: In conclusion, MTA, BA, and BD have effects on osteoblast differentiation in mesenchymal stem cells, suggesting that these cements may be useful for root-end filling material., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aims: MicroRNAs (miRNA) are involved in various biological processes including cellular differentiation. However, the role of miR-433 in osteoblast differentiation remains poorly understood. The objective of this study was to investigate the effect of miR-433 on BMP2-induced osteoblast differentiation., Main Methods: The expression of mature miR-433 in cells was detected by real-time PCR. RT-PCR or real-time PCR was used to confirm the expression of osteogenic genes. For the activation or inhibition of miR-433 expression, we used a precursor form of miR-433 or anti-miR-433. Functional activity of miR-433 and Runx2 was evaluated by promoter study. Osteoblast differentiation was evaluated by analyzing alkaline phosphatase (ALP) activity., Key Finding: ERRγ increased miR-433 expression in the mesenchymal stem cell lineage C3H10T1/2. During the BMP2-induction of osteoblastic differentiation of C3H10T1/2, ERRγ and miR433 expression decreased. In addition, during the osteoblastic differentiation, overexpression of ERRγ or miR-433 inhibited the expression of osteogenic marker genes such as Runx2 and ALP. A computer-based prediction algorithm led to the identification of three miR-433 binding sites [S1 (114-145 bp), S2 (3735-3766 bp) and S3 (3828-3860 bp)] on the 3'-UTR of Runx2 mRNA. Furthermore, miR-433 directly targeted S1 and S2, and decreased the level of Runx2 transcript. In addition, miR-433 inhibited BMP2-induced 6×OSE-Luc activities. Anti-miR-433 recovered ERRγ-suppressed Runx2 expression and ALP activity., Significance: These results demonstrated that miR-433 suppressed BMP2-indcued osteoblast differentiation by decreasing the level of Runx2 transcript., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background/aims: Cartilage oligomeric matrix protein (COMP)-angiopoietin 1 (Ang1) is a soluble and stable form of Ang1 which plays important roles in vessel formation and the survival of endothelial cells, neurons and cardiomyocytes. However, the effects of COMP-Ang1 on the survival of mesenchymal cells are unknown. Mesenchymal cells have been transplanted with some scaffolds for bone tissue regeneration, but they occasionally underwent cell death due to a lack of nutrient supply. This study examined the effects of COMP-Ang1 on the survival of mesenchymal cells under nutrient-deprived conditions., Methods: Primary and C3H10T1/2 mesenchymal cells were cultured under serum deprivation with or without COMP-Ang1. The effects of COMP-Ang1 on mesenchymal cell survival and its molecular mechanism were determined using a viability test, RT-PCR, Western blotting and fluorescence-activated cell sorting analysis., Results and Conclusion: COMP-Ang1 inhibited the nutrient-deprived apoptotic cell death of mesenchymal cells through the Akt, p38 and extracellular-signal-regulated kinase (ERK) pathways. In addition, COMP-Ang1 reversed the nutrient-deprived suppression of cyclin D1 mRNA expression. These results suggest that COMP-Ang1 has a protective role in the survival of nutrient-deprived mesenchymal cells. The use of COMP-Ang1 with some scaffolds might be useful for bone tissue engineering., (Copyright © 2010 S. Karger AG, Basel.)

Stabilin-1 is a transmembrane receptor that regulates molecule recycling and cell homeostasis by controlling the intracellular trafficking and participates in cell-cell adhesion and transmigration. Stabilin-1 expression is observed in various organs, including bones; however, its function and regulatory mechanisms in the bone remain unclear. In this study, we evaluated the physiological function of stabilin-1 in bone cells and tissue using a stabilin-1 knockout (Stab1 KO) mouse model. In wild-type (WT) mice, stabilin-1 was expressed in osteoblasts and osteoclasts, and its expression was maintained during osteoblast differentiation but significantly decreased after osteoclast differentiation. There was no difference in osteoblast differentiation and function, or the expression of osteoblast differentiation markers between mesenchymal stem cells isolated from Stab1 KO and WT mice. However, osteoclast differentiation marker levels demonstrated a non-significant increase and bone-resorbing activity was significantly increased in vitro in RANKL-induced osteoclasts from Stab1-deficient bone marrow macrophages (BMMs) compared with those of WT BMMs. Microcomputed tomography showed a negligible difference between WT and Stab1 KO mice in bone volume and trabecular thickness and number. Moreover, no in vivo functional defect in bone formation by osteoblasts was observed in the Stab1 KO mice. The osteoclast surface and number showed an increased tendency in Stab1 KO mice compared to WT mice in vivo, but this difference was not statistically significant. Overall, these results indicate that Stab1 does not play an essential role in in vivo bone development and bone cell function, but it does affect in vitro osteoclast maturation and function for bone resorption. [ABSTRACT FROM AUTHOR]