Mouse Bone Marrow-derived Mesenchymal Stem Research Articles

Zinc finger protein-36 deficiency inhibits osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells and preosteoblasts by activating the ERK/MAPK pathway

To explore the role of zinc finger protein 36(ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7(P < 0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P < 0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P < 0.05). ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Aucubin Promotes Osteogenic Differentiation and Facilitates Bone Formation through the lncRNA-H19 Driven Wnt/β-Catenin Signaling Regulatory Axis.

Traditional Chinese medicine Cortex Eucommiae has been used to treat bone fracture for hundreds of years, which exerts a significant improvement in fracture healing. Aucubin, a derivative isolated from Cortex Eucommiae, has been demonstrated to possess anti-inflammatory, immunoregulatory, and antioxidative potential. In the present study, our aim was to explore its function in bone regeneration and elucidate the underlying mechanism. The effects of Aucubin on osteoblast and osteoclast were examined in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) and RAW 264.7 cells, respectively. Moreover, the lncRNA H19 and Wnt/β-catenin signaling were detected by qPCR examination, western blotting, and luciferase activity assays. Using the femur fracture mice model, the in vivo effect of Aucubin on bone formation was monitored by X-ray, micro-CT, histomorphometry, and immunohistochemistry staining. In the present study, Aucubin was found to significantly promote osteogenic differentiation in vitro and stimulated bone formation in vivo. Regarding to the underlying mechanism, H19 was found to be obviously upregulated by Aucubin in MSCs and thus induced the activation of Wnt/β-catenin signaling. Moreover, H19 knockdown partially reversed the Aucubin-induced osteogenic differentiation and successfully suppressed the activation of Wnt/β-catenin signaling. We therefore suggested that Aucubin induced the activation of Wnt/β-catenin signaling through promoting H19 expression. Our results demonstrated that Aucubin promoted osteogenesis in vitro and facilitated fracture healing in vivo through the H19-Wnt/β-catenin regulatory axis.

Bioactive mesoporous silica nanoparticle-functionalized titanium implants with controllable antimicrobial peptide release potentiate the regulation of inflammation and osseointegration

Bacterial infection and delayed osseointegration are two major challenges for titanium-based orthopedic implants. In the present study, we developed a functionalized titanium implant Ti-M@A by immobilizing antimicrobial peptide (AMP) HHC36-loaded diselenide-bridged mesoporous silica nanoparticles (MSNs) on the surface, which showed good long-term and mechanical stability. The functionalized implants can realize the sustained release of AMP over 30 days and exhibit over 95.71 % antimicrobial activity against four types of clinical bacteria (S. aureus, E. coli, P. aeruginosa and MRSA), which arose from the capability to destroy the bacterial membranes. Moreover, Ti-M@A can efficiently inhibit the biofilm formation of the bacteria. The functionalized implants can also significantly promote the osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) because of the Se in MSNs. Notably, it can trigger macrophages toward M2 polarization in vitro by scavenging ROS in LPS-activated macrophages. Consequently, in vivo assays with infection and non-infection bone defect models demonstrated that such bioactive implants can not only kill over 98.82 % of S. aureus, but also promote osseointegration. Hence, this study provides a combined strategy to resolve bacterial infection and delayed osseointegration for titanium implants.

In Vitro Characterization of an Anodized Surface of a Dental Implant Collar and Dental Abutment on Peri-Implant Cellular Response.

The purpose of this paper was to determine the effect of anodization on the in vitro proliferation and adhesion of immortalized human keratinocytes (HaCats) and mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) in Titanium Grade 23 (Ti6Al4V ELI) discs and to describe the surface topography, roughness, and composition of dental implants (body and collar) and abutments submitted to an area-specific anodization process. HaCat cells and BM-MSCs were seeded onto discs with three different surface treatments: machined, area-specific anodization for abutments, and area-specific anodization for implant collars. Cell proliferation was assessed using a resazurin-based fluorescent dye on days 1, 3, and 7, while cell adhesion was examined using scanning electron microscopy (SEM). Surface topography, roughness, and composition were evaluated for six implant bodies with an anodized rough surface, six anodized implant smooth collars, and six anodized prosthetic abutments. Both HaCats and BM-MSCs showed increased viability over time (p < 0.001) with no statistically significant differences among the different surfaces (p = 0.447 HaCats and p = 0.631 BM-MSCs). SEM analysis revealed an enhanced presence and adhesion of HaCat cells on the anodized surface for the implant collars and an increased adhesion of BM-MSCs on both the anodized and machined surface abutments. The topography characteristics of the treated implants and abutments varied depending on the specific implant region. Chemical analysis confirmed the presence of oxygen, calcium, phosphorus, and sodium on the anodized surfaces. The area-specific anodization process can be utilized to create variable topography, increase the specific surface area, and introduce oxygen, calcium, phosphorus, and sodium to dental implants and abutments. While BM-MSCs and HaCat cells showed similar adhesion and proliferation on anodized and machined surfaces, a positive interaction between anodized Ti6Al4V ELI surfaces and these two cell lines present in the peri-implant mucosa was observed. Due to the limitations of the present study, further research is necessary to confirm these findings.

Optimization of decellularization methods using human small intestinal submucosa for scaffold generation in regenerative medicine.

Porcine small intestinal submucosa, despite its successful use as a scaffold in regenerative medicine, has innate biomechanical heterogeneity. In this study, we hypothesized that human small intestinal submucosa could be a viable alternative bio-scaffold. For the first time, we characterize submucosal extraction from human small intestine and examine appropriate decellularization methods. In total, 16 human small intestinal submucosal samples were obtained and decellularized using three reported methods of porcine decellularization: Abraham, Badylak, and Luo. For each method, four specimens were decellularized. The remaining four specimens were designated as non-decellularized. We measured the amount of residual DNA and growth factors in decellularized human intestinal samples. Additionally, decellularized human small intestinal submucosa was co-cultured with mouse bone marrow-derived mesenchymal stem cells to examine mesenchymal stem cell survival and proliferation. The reference value for the amount of residual DNA deemed appropriate in decellularized tissue was established as 50 ng/mg of extracellular matrix dry weight or less. Abraham's method most successfully met this criterion. Measurement of residual growth factors revealed low levels observed in samples decellularized using the Abraham and Badylak methods. Co-culture of each small intestinal submucosal sample with mouse bone marrow-derived mesenchymal stem cells confirmed viable cell survival and proliferation in samples derived using protocols by Abraham and Badylak. Abraham's method most successfully met the criteria for efficient tissue decellularization and cell viability and proliferation. Thus, we consider this method most suitable for decellularization of human small intestinal submucosa.

Early depletion of M1 macrophages retards the progression of glucocorticoid-associated osteonecrosis of the femoral head

Inflammation stands as a pivotal factor in the pathogenesis of glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH). However, the vital role played by M1 macrophages, the principal constituents of the inflammatory process, remains largely underexplored. In this study, we employed reverse transcription-quantitative polymerase chain Reaction (RT-PCR), western blot, and flow cytometry to assess the impact of M1-conditioned medium on cultures of mouse bone marrow-derived mesenchymal stem cells (BMSCs) and Murine Long bone Osteocyte-Y4 (MLO-Y4) in vitro. Moreover, we quantified the levels of inflammatory cytokines in the M1-conditioned medium through the employment of an enzyme-linked immunosorbent assay (ELISA). For in vivo analysis, we examined M1 macrophages and investigated the NF-kB signaling pathway in specimens obtained from the femoral heads of animals and humans. We found that the number of M1 macrophages in the femoral head of GA-ONFH patients grew significantly, and in the mice remarkably increase, maintaining high levels in the intramedullary. In vitro, the M1 macrophage-conditioned medium elicited apoptosis in BMSCs and MLO-Y4 cells, shedding light on the intricate interplay between macrophages and these cell types. The presence of TNF-α within the M1-conditioned medium activated the NF-κB pathway, providing mechanistic insight into the apoptotic induction. Moreover, employing a robust rat macrophage clearance model and GA-ONFH model, we demonstrated a remarkable attenuation in TNF-α expression and NF-kB signaling subsequent to macrophage clearance. This pronounced reduction engenders diminished cellular apoptosis and engenders a decelerated trajectory of GA-ONFH progression. In conclusion, our study reveals the crucial involvement of M1 macrophages in the pathogenesis of GA-ONFH, highlighting their indispensable role in disease progression. Furthermore, early clearance emerges as a promising strategy for impeding the development of GA-ONFH.

Galangin mitigates glucocorticoid-induced osteoporosis by activating autophagy of BMSCs via triggering the PKA/CREB signaling pathway.

Glucocorticoid-induced osteoporosis (GIOP), one of the most common and serious adverse effects associated with glucocorticoid administration, manifests as decreased bone formation and increased bone resorption, eventually culminating in bone loss. Galangin (GAL) is a flavonoid extracted from the medicinal herbal galangal that possesses a variety of pharmacological activities and can inhibit osteoclastogenesis. However, the effects of GAL on GIOP remain unclear. Our study aims to explore the effects of GAL on GIOP in mice and the underlying mechanism. Our results show that GAL markedly mitigates the severity of dexamethasone (Dex)-induced osteoporosis in mice and potentiates osteogenic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). Furthermore, GAL also significantly counteracts Dex-mediated suppression of osteogenic differentiation and autophagy in human BMSCs. GAL augments PKA/CREB-mediated autophagic flux in BMSCs and the bones of osteoporotic mice. GAL-mediated osteogenic differentiation in Dex-treated BMSCs is significantly decreased by the PKA inhibitor H89 and autophagy inhibitor 3-methyladenine. Collectively, our data indicate that GAL can ameliorate GIOP, partly by augmenting the mineralization of BMSCs by potentiating PKA/CREB-mediated autophagic flux, highlighting its potential therapeutic use in treating glucocorticoid-related osteoporosis.

Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis.

Eccrine sweat gland (SG) plays a crucial role in thermoregulation but exhibits very limited regenerative potential. Although SG lineage-restricted niches dominate SG morphogenesis and benefit SG regeneration, rebuilding niches in vivo is challenging for stem cell therapeutic applications. Hence, we attempted to screen and tune the critical niche-responding genes that dually respond to both biochemical and structural cues, which might be a promising strategy for SG regeneration. An artificial SG lineage-restricted niche consisting of mouse plantar dermis homogenates (i.e. biochemical cues) and 3D architecture (i.e. structural cues) was built in vitro by using an extrusion-based 3D bioprinting approach. Mouse bone marrow-derived mesenchymal stem cells (MSCs) were then differentiated into the induced SG cells in the artificial SG lineage-restricted niche. To decouple biochemical cues from structural cues, the transcriptional changes aroused by pure biochemical cues, pure structural cues and synergistic effects of both cues were analyzed pairwise, respectively. Notably, only niche-dual-responding genes that are differentially expressed in response to both biochemical and structural cues and participate in switching MSC fates towards SG lineage were screened out. Validations in vitro and in vivo were respectively conducted by inhibiting or activating the candidate niche-dual-responding gene(s) to explore the consequent effects on SG differentiation. Notch4 is one of the niche-dual-responding genes that enhanced MSC stemness and promoted SG differentiation in 3D-printed matrix in vitro. Furthermore, inhibiting Notch4 specifically reduced keratin 19-positive epidermal stem cells and keratin 14-positive SG progenitor cells, thus further delaying embryonic SG morphogenesis in vivo. Notch4 not only participates in mouse MSC-induced SG differentiation in vitro but is also implicated in mouse eccrine SG morphogenesis in vivo.

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves diabetic wound healing by promoting angiogenesis and lymphangiogenesis.

Some clinical trials have shown the usefulness of stem cell therapy for diabetic foot ulcers. However, the donor supply is limited, and the process is time consuming and expensive. This study assessed the therapeutic effects of neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSC) xenotransplantation using diabetic wound model mice. All layers of back skin were removed from streptozotocin-induced diabetic mice. In the npBM-MSCs group, npBM-MSCs were transplanted to the wound, and syngeneic mouse bone marrow-derived mesenchymal stem cells (mBM-MSCs) were transplanted to the wound in the mBM-MSCs group. The control group comprised diabetic mice that did not receive cellular therapy. The therapeutic effects of the transplantation were evaluated according to the rate of wound closure and the promotion of neovascularization in the wound. The wound closure rate was significantly improved in the npBM-MSCs group compared with the control group (p<.001 at postoperative day [POD] 4 and p<.01 at POD 7) and mBM-MSCs groups (p<.05 at POD 4). Prominent promotion of both angiogenesis and lymphangiogenesis was observed in the npBM-MSCs group. Furthermore, the expression of murine Prox1 and both porcine and murine Vegfs and Tgfb1 in the wounds was enhanced until POD 4 by npBM-MSCs transplantation. The amounts of vascular endothelial growth factor (VEGF) A, VEGFC, and transforming growth factor β1 secreted from npBM-MSCs were higher than those from mBM-MSCs (p<.05). Xenotransplantation of npBM-MSCs improved diabetic wound healing by promoting both angiogenesis and lymphangiogenesis.

Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs.

Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (i) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (ii) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (75–150 μg/ml) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C2S extract (150 μg/ml) induced TNF-α, IL-1β and IL-6 production in macrophages. C2S extract (150 μg/ml) enhanced reactive oxygen species level and intracellular calcium level but reduced mitochondrial membrane potential and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C2S (150 μg/ml)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1 and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.

3D Printed Gelatin/Sodium Alginate Hydrogel Scaffolds Doped with Nano-Attapulgite for Bone Tissue Repair.

IntroductionBone tissue engineering (BTE) is a new strategy for bone defect repair, but the difficulties in the fabrication of scaffolds with personalized structures still limited their clinical applications. The rapid development in three-dimensional (3D) printing endows it capable of controlling the porous structures of scaffolds with high structural complexity and provides flexibility to meet specific needs of bone repair.MethodsIn this study, sodium alginate (SA)/gelatin (Gel) hydrogel scaffolds doped with different contents of nano-attapulgite were fabricated via 3D printing. The surface microstructure, hydrophilicity and mechanical properties were fully evaluated. Furthermore, mouse bone marrow-derived mesenchymal stem cells (BMSCs) were cultured with the composite hydrogels in vitro, and proliferation and osteoblastic differentiation were assessed. A rabbit tibia plateau defect model was used to evaluate the osteogenic potential of the composite hydrogel in vivo.ResultsWhen increasing nano-ATP content, the Gel/SA/nano-ATP composite hydrogels showed better mechanical property and printability. Moreover, Gel/SA/nano-ATP composite hydrogels showed excellent bioactivity, and a significant mineralization effect was observed on the surface after being incubated in simulated body fluid (SBF) for 14 days. The Gel/SA/nano-ATP composite hydrogel also showed good biocompatibility and promoted the osteogenesis of BMSCs. Finally, histological analysis demonstrates that the Gel/SA/nano-ATP composite hydrogels could effectively enhance bone regeneration in vivo.ConclusionThese properties render the Gel/SA/nano-ATP composite hydrogel scaffolds an ideal bone tissue engineering material for the repair of bone defects.

Irradiation Haematopoiesis Recovery Orchestrated by IL-12/IL-12Rβ1/TYK2/STAT3-Initiated Osteogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells.

PurposeRepairing the irradiation-induced osteogenic differentiation injury of bone marrow mesenchymal stem cells (BM-MSCs) is beneficial to recovering haematopoiesis injury in radiotherapy; however, its mechanism is elusive. Our study aimed to help meet the needs of understanding the effects of radiotherapy on BM-MSC osteogenic potential.Methods and MaterialsBalb/c mice and the BM-MSCs were used to evaluate the irradiation-induced osteogenic differentiation injury in vivo. The cellular and molecular characterization were applied to determine the mechanism for recovery of irradiation-derived haematopoiesis injuries.ResultsWe report a functional role of IL-12 in acute irradiation hematopoietic injury recovery and intend to dissect the possible mechanisms through BM-MSC, other than the direct effect of IL-12 on hematopoietic stem and progenitor cells (HSPCs). Specifically, we show that early use of IL-12 enhanced the osteogenic differentiation of BM-MSCs through IL-12Rβ1/TYK2/STAT3 signaling; furthermore, IL-12 induced osteogenesis facilitated bone formation and irradiation hematopoiesis recovery when transplanted BM-MSCs in the femur of Balb/c mice. For the mechanism of action, we found that IL-12 receptor beta 1 (IL-12Rβ1) expression of irradiated BM-MSCs was upregulated rapidly, coincidentally consistent with early use of IL-12 induced osteogenic differentiation enhancement. IL-12Rβ1 and tyrosine kinase 2 gene (Tyk2) silencing experiments and phosphotyrosine of signal transducer and activator of transcription 3 (p-STAT3) suppression experiments indicated the IL-12Rβ1/TYK2/STAT3 signaling was essential in IL-12-induced osteogenic differentiation enhancement of BM-MSCs.ConclusionThese findings suggested that IL-12 may exert BM-MSCs-based hematopoietic recovery by repairing osteogenic differentiation abilities damages through IL-12Rβ1/TYK2/STAT3 signaling pathway post-irradiation.

Anti-adipogenic peptides from ark shell protein hydrolysate: Purification, identification and anti-adipogenic effect

Ark shell protein hydrolysates-derived peptides showing anti-adipogenic effect were purified and identified using consecutive chromatographic techniques and tandem mass spectrometry. Two anti-adipogenic peptides LLRLTDL (843.02 Da) and GYALPCDCL (954.13 Da) were found to be highly effective for lipid accumulation inhibition in mouse bone marrow-derived mesenchymal stem cells differentiation into adipocyte. The inhibitory activity of LLRLTDL and GYALPCDCL for lipid accumulation was 48.53 % and 46.22 % at 100 μM, respectively. During adipocyte differentiation, two anti-adipogenic peptides LLRLTDL and GYALPCDCL significantly inhibited intracellular reactive oxygen species (ROS) generation by 47.39 % and 49.77 % at 100 μM. In addition, LLRLTDL and GYALPCDCL treatment increased cellular antioxidant enzyme activities, including superoxide dismutase, catalase, and glutathione peroxidase. Moreover, LLRLTDL and GYALPCDCL suppressed proinflammatory cytokine productions, including tumor necrosis factor-α, interleukin-1β and -6. These results suggest that LLRLTDL and GYALPCDCL could modulate adipocyte differentiation and be useful as anti-obesity ingredients or functional foods.

Studying the induction effect of different mouse neck scaffolds on the behavior of Mouse Bone marrow-derived mesenchymal stem cell

Objective: Studying the behavior of mesenchymal stem cells is important for understanding some physiological and pathological processes as well as long-term preservation of these cells in vitro. The neck area of mouse contains a wide variety of different organs and tissues and extracellular matrices. In the present study, the interaction of mouse bone marrow derived mesenchymal stem cells (BM-MSCs) with mouse neck scaffolds was performed in order to have a better understanding of the behavior of mesenchymal stem cells. Materials and methods: The neck areas of 9 two-week-old Syrian mice were cut into pieces with 4 mm thickness. Decellularization process was carried through snap freeze-thaw cycles followed by sodium dodecyl sulfate (SDS) treatments. After confirmation of acellularization through histological studies and DNA extraction process, scaffolds were co-cultured with mouse (BM-MSCs) up to 21 days, in vitro. Results: As indicated by histological studies and scanning of electron microscopy, stem cells migrate into the cartilage scaffold on third, seventh and fourteenth days of culture. Stem cells were divided on these days. On the fourteenth day of culture, migration to epidermal and blood vascular scaffolds was also observed. On the twenty-first day of culture, cells were observed only in the cartilage scaffold, while showing morphological differentiation. Conclusion: As indicated by the results of the present study, mouse neck scaffold, due to its diverse extracellular matrices, is a suitable structure for studying some cellular behaviors such as migration, cell division and cell differentiation. However, more molecular studies are needed to determine the extent of this differentiation. Keywords: Natural scaffold, Cell behaviors, Cell-matrix interaction, Experimental model scaffolding, mouse neck area

Uncarboxylated osteocalcin promotes osteogenesis and inhibits adipogenesis of mouse bone marrow-derived mesenchymal stem cells via the PKA-AMPK-SIRT1 axis.

Osteoporosis is a bone disease characterized by reduced bone density, thin cortical bone and large gaps in the bone's honeycomb structure, which increases the risk of bone fragility. Uncarboxylated osteocalcin (unOC), a vitamin K-dependent bone protein, is known to regulate carbohydrate and energy metabolism. A previous study demonstrated that unOC promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts, but inhibits their differentiation into adipocytes. However, the underlying mechanism remains unknown. The present study showed that unOC regulated the differentiation potential of BMSCs via protein kinase A (PKA)/AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signaling. SIRT1, a member of the sirtuin family with deacetylation functions, was upregulated by unOC in BMSCs. Transfection analyses with SIRT1 small interfering RNA indicated that the unOC-induced differentiation shift in BMSCs required SIRT1. Examination of SIRT1 downstream targets revealed that unOC regulated the acetylation levels of runt-related transcription factor (RUNX) 2 and peroxisome proliferator-activated receptor γ (PPARγ). Therefore, unOC inhibited adipogenic differentiation by PPARγ acetylation and promoted osteogenic differentiation by RUNX2 deacetylation. Moreover, phosphorylated PKA and AMPK protein levels increased after unOC treatment, which led to the upregulation of SIRT1. Western blot analysis with PKA and AMPK inhibitors indicated that the PKA-AMPK signaling pathway functioned upstream of SIRT1 and positively regulated SIRT1 expression. These findings led us to propose a model in which unOC regulated BMSC osteogenic differentiation through the PKA-AMPK-SIRT1 axis, giving evidence towards the therapeutic potential of unOC in osteoporosis treatment.

Carnosol induces the osteogenic differentiation of bone marrow-derived mesenchymal stem cells via activating BMP-signaling pathway.

Carnosol is a phenolic diterpene phytochemical found in rosemary and sage with reported anti-microbial, anti-oxidant, anti-inflammatory, and anti-carcinogenic activities. This study aimed to investigate the effect of carnosol on the lineage commitment of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) into osteoblasts and adipocytes. Interestingly, carnosol stimulated the early commitment of mBMSCs into osteoblasts in dose-dependent manner as demonstrated by increased levels of alkaline phosphatase activity and Alizarin red staining for matrix mineralization. On the other hand, carnosol significantly suppressed adipogenesis of mBMSCs and downregulated both early and late markers of adipogenesis. Carnosol showed to induce osteogenesis in a mechanism mediated by activating BMP signaling pathway and subsequently upregulating the expression of BMPs downstream osteogenic target genes. In this context, treatment of mBMSCs with LDN-193189, BMPR1 selective inhibitor showed to abolish the stimulatory effect of carnosol on BMP2-induced osteogenesis. In conclusion, our data identified carnosol as a novel osteoanabolic phytochemical that can promote the differentiation of mBMSCs into osteoblasts versus adipocytes by activating BMP-signaling.

RETRACTED: Mouse bone marrow derived mesenchymal stem cells-secreted exosomal microRNA-125b-5p suppresses atherosclerotic plaque formation via inhibiting Map4k4

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concern was raised about the reliability of the Western blot results in Figs. 2D and 4E, which appear to have the same eyebrow shaped phenotype as many other publications tabulated here (https://docs.google.com/spreadsheets/d/149EjFXVxpwkBXYJOnOHb6RhAqT4a2llhj9LM60MBffM/edit#gid=0). The journal requested the corresponding author comment on these concerns and provide the raw data. However the authors were not able to satisfactorily fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Secretome Analysis of Inductive Signals for BM-MSC Transdifferentiation into Salivary Gland Progenitors.

Severe dry mouth in patients with Sjögren’s Syndrome, or radiation therapy for patients with head and neck cancer, significantly compromises their oral health and quality of life. The current clinical management of xerostomia is limited to palliative care as there are no clinically-proven treatments available. Previously, our studies demonstrated that mouse bone marrow-derived mesenchymal stem cells (mMSCs) can differentiate into salivary progenitors when co-cultured with primary salivary epithelial cells. Transcription factors that were upregulated in co-cultured mMSCs were identified concomitantly with morphological changes and the expression of acinar cell markers, such as α-amylase (AMY1), muscarinic-type-3-receptor(M3R), aquaporin-5(AQP5), and a ductal cell marker known as cytokeratin 19(CK19). In the present study, we further explored inductive molecules in the conditioned media that led to mMSC reprogramming by high-throughput liquid chromatography with tandem mass spectrometry and systems biology. Our approach identified ten differentially expressed proteins based on their putative roles in salivary gland embryogenesis and development. Additionally, systems biology analysis revealed six candidate proteins, namely insulin-like growth factor binding protein-7 (IGFBP7), cysteine-rich, angiogenetic inducer, 61(CYR61), agrin(AGRN), laminin, beta 2 (LAMB2), follistatin-like 1(FSTL1), and fibronectin 1(FN1), for their potential contribution to mMSC transdifferentiation during co-culture. To our knowledge, our study is the first in the field to identify soluble inductive molecules that drive mMSC into salivary progenitors, which crosses lineage boundaries.

Coordinated Regulation of Mesenchymal Stem Cell Migration by Various Chemotactic Stimuli.

Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-β), and stromal cell-derived factor-1 (SDF-1). SP, TGF-β, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-β, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-β while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-β. TGF-β pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-β noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-β, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.

Long-isoform NRF1 protects against arsenic cytotoxicity in mouse bone marrow-derived mesenchymal stem cells by suppressing mitochondrial ROS and facilitating arsenic efflux

Acute exposure to arsenic is known to cause bone marrow depression and result in anemia, in which the dusfunction of cells in the bone marrow niche such as mesenchymal stem cells (MSCs) is vital. However, the mechanism underlying response of MSCs to arsenic challange is not fully understood. In the present study, we investigated the role of nuclear factor erythroid 2-related factor (NRF) 1 (NRF1), a sister member of the well-known master regulator in antioxidative response NRF2, in arsenite-induced cytotoxicity in mouse bone marrow-derived MSCs (mBM-MSCs). We found that arsenite exposure induced significant increase in the protein level of long-isoform NRF1 (L-NRF1). Though short-isoform NRF1 (S-NRF1) was induced by arsenite at mRNA level, its protein level was not obviously altered. Silencing L-Nrf1 sensitized the cells to arsenite-induced cytotoxicity. L-Nrf1-silenced mBM-MSCs showed decreased arsenic efflux with reduced expression of arsenic transporter ATP-binding cassette subfamily C member 4 (ABCC4), as well as compromised NRF2-mediated antioxidative defense with elevated level of mitochondrial reactive oxygen species (mtROS) under arsenite-exposed conditions. A specific mtROS scavenger (Mito-quinone) alleviated cell apoptosis induced by arsenite in L-Nrf1-silenced mBM-MSCs. Taken together, these findings suggest that L-NRF1 protects mBM-MSCs from arsenite-induced cytotoxicity via suppressing mtROS in addition to facilitating cellular arsenic efflux.