Differentiation Of Mouse Bone Marrow Research Articles

RANKL-dependent osteoclast differentiation and gene expression in bone marrow-derived cells from adult mice is sexually dimorphic

Sex-specific differences in bone integrity and properties are associated with age as well as the number and activity of cells involved in bone remodeling. The aim of this study was to investigate sex-specific differences in adhesion, proliferation, and differentiation of mouse bone marrow derived cells into osteoclasts. The adherent fraction of bone marrow- derived cells from 12-week-old male and female C57BL/6J mice were assessed for their adhesion, proliferation, and receptor activator of nuclear factor κB (RANKL)-induced differentiation into osteoclasts. Female bone marrow derived macrophages (BMDMs) displayed higher adhesion and proliferation ratio upon macrophage colony stimulating factor (M-CSF) (day 0) and M-CSF + RANKL (day 4) treatment, respectively. On the contrary, male BMDMs differentiated more efficiently into osteoclasts upon RANKL-treatment compared to females (day 5). To further understand these sex-specific differences at the gene expression level, BMDMs treated with M-CSF (day 0) and M-CSF + RANKL (day 4), were assessed for their differential expression of genes through RNA sequencing. M-CSF treatment resulted in 1106 differentially expressed genes, while RANKL-treatment gave 473 differentially expressed genes. Integrin, adhesion, and proliferation-associated genes were elevated in the M-CSF-treated female BMDMs. RANKL-treatment further enhanced the expression of the proliferation- associated genes, and of genes associated with inhibition of osteoclast differentiation in the females, while RANK-signaling-associated genes were upregulated in males. In conclusion, BMDM adhesion, proliferation and differentiation into osteoclasts are sex-specific and may be directed by the PI3K-Akt signaling pathway for proliferation, and the colony stimulating factor 1-receptor and the RANKLsignaling pathway for the differentiation.

Uncarboxylated osteocalcin alleviates the inhibitory effect of high glucose on osteogenic differentiation of mouse bone marrow\u2013derived mesenchymal stem cells by regulating TP63

BackgroundProgressive population aging has contributed to the increased global prevalence of diabetes and osteoporosis. Inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by hyperglycemia is a potential pathogenetic mechanism of osteoporosis in diabetic patients. Uncarboxylated osteocalcin (GluOC), a protein secreted by mature osteoblasts, regulates bone development as well as glucose and lipid metabolism. In our previous studies, GluOC was shown to promote osteoblastic differentiation of BMSCs; however, the underlying mechanisms are not well characterized. Tumor protein 63 (TP63), as a transcription factor, is closely related to bone development and glucose metabolism.ResultsIn this study, we verified that high glucose suppressed osteogenesis and upregulated adipogenesis in BMSCs, while GluOC alleviated this phenomenon. In addition, high glucose enhanced TP63 expression while GluOC diminished it. Knock-down of TP63 by siRNA transfection restored the inhibitory effect of high glucose on osteogenic differentiation. Furthermore, we detected the downstream signaling pathway PTEN/Akt/GSK3β. We found that diminishing TP63 decreased PTEN expression and promoted the phosphorylation of Akt and GSK3β. We then applied the activator and inhibitor of Akt, and concluded that PTEN/Akt/GSK3β participated in regulating the differentiation of BMSCs.ConclusionsOur results indicate that GluOC reduces the inhibitory effect of high glucose on osteoblast differentiation by regulating the TP63/PTEN/Akt/GSK3β pathway. TP63 is a potential novel target for the prevention and treatment of diabetic osteoporosis.

Early stage mechanical adaptability and osteogenic differentiation of mouse bone marrow derived mesenchymal stem cell under micro-vibration stimulation environment

This study investigated the early mechanical adaptability and osteogenic differentiation of mouse bone marrow mesenchymal stem cells (M-BMSCs) under micro-vibration stimulation (MVS). M-BMSCs were stimulated by MVS in vitro, cell proliferation, alkaline phosphatase (ALP) activity assay, and cytoskeleton were measured, and cell apoptosis was observed by flow cytometry. Early osteoblast-associated genes, runt-related transcription factor 2 (Runx2), Collagen Ⅰ (Col-Ⅰ) and ALP, were observed by RT-PCR and the activation of extracellular regulated protein kinases 1/2 (ERK1/2) was determined by Western blotting. The results showed that MVS had no significant effect on the proliferation of M-BMSCs. The early apoptosis was induced by mechanical stimulation (for one day), but the apoptosis was decreased after cyclic stimulation for 3 days. At the same time, MVS significantly accelerated the expression of F-actin protein in cytoskeleton, the synthesis of ALP and the ERK1/2 pathway, also up-regulated the expressions of Runx2, Col-Ⅰ and ALP genes. This study indicates that MVS could regulate cellular activity, alter early adaptive structure and finally promote the early osteogenic differentiation of M-BMSCs.

The Coumarin Derivative 5'-Hydroxy Auraptene Suppresses Osteoclast Differentiation via Inhibiting MAPK and c-Fos/NFATc1 Pathways.

The phytochemical substances, coumarin derivatives, have demonstrated antiresorptive bone effects by suppressing osteoclast differentiation in vitro and in vivo. Recently, we have identified 5′-hydroxy auraptene (5′-HA), a coumarin derivative isolated from Lotus lalambensis Schweinf, as a novel stimulator for osteoblast differentiation. In this study, we investigated the effect of 5′-HA on osteoclast differentiation of mouse bone marrow (BM) cells. The effect of 5′-HA on BM cell proliferation and osteoclast differentiation was determined by measuring cell viability and tartrate-resistant acid phosphatase (TRAP) enzyme activity, quantification of TRAP+ multinucleated cells (TRAP+MNCs), and quantitative real-time PCR (qPCR) of osteoclastic gene expression. Regulation of NF-κB, c-Fos/NFATc1, and MAPK signaling pathways by 5′-HA during osteoclastogenesis was measured by the NF-κB reporter assay and Western blot analysis. 5′-HA significantly suppresses the receptor activator of NF-κB ligand (RANKL) induced osteoclast differentiation of BM cells in a dose-dependent manner. Consistently, treatment of BM cells with 5′-HA significantly inhibited RANKL-induced activation of NF-κB and c-Fos/NFATc1 pathways in a dose-dependent manner. Furthermore, RANKL-induced phosphorylation of ERK1/2, p-38, and JNK was significantly inhibited by 5′-HA in BM cells. In conclusion, we identified 5′-HA as a novel coumarin derivative that suppresses RANKL-induced osteoclastogenesis via inhibiting c-Fos/NFATc1 and MAPK signaling pathways.

Effect of the saliva from different triatomine species on the biology and immunity of TLR-4 ligand and Trypanosoma cruzi-stimulated dendritic cells.

BackgroundTriatomines are blood-sucking vectors of Trypanosoma cruzi, the causative agent of Chagas disease. During feeding, triatomines surpass the skin host response through biomolecules present in their saliva. Dendritic cells (DCs) play a crucial role in the induction of the protection to aggressive agents, including blood-sucking arthropods. Here, we evaluated if salivary components of triatomines from different genera evade the host immunity by modulating the biology and the function of LPS- or T. cruzi-stimulated DCs.MethodsSaliva of Panstrongylus lignarius, Meccus pallidipennis, Triatoma lecticularia and Rhodnius prolixus were obtained by dissection of salivary glands and the DCs were obtained from the differentiation of mouse bone marrow precursors.ResultsThe differentiation of DCs was inhibited by saliva of all species tested. Saliva differentially inhibited the expression of MHC-II, CD40, CD80 and CD86 in LPS-matured DCs. Except for the saliva of R. prolixus, which induced IL-6 cytokine production, TNF-α, IL-12 and IL-6 were inhibited by the saliva of the other three tested species and IL-10 was increased in all of them. Saliva per se, also induced the production of IL-12, IL-6 and IL-10. Only the saliva of R. prolixus induced DCs apoptosis. The presence of PGE2 was not detected in the saliva of the four triatomines studied. Finally, T. cruzi invasion on DCs is enhanced by the presence of the triatomine saliva.ConclusionsThese results demonstrate that saliva from different triatomine species exhibit immunomodulatory effects on LPS and T. cruzi-stimulated DCs. These effects could be related to hematophagy and transmission of T. cruzi during feeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1890-x) contains supplementary material, which is available to authorized users.

Culture and Identification of Mouse Bone Marrow-Derived Dendritic Cells and Their Capability to Induce T Lymphocyte Proliferation.

BackgroundThe aim of this study was to establish a culture method for mouse dendritic cells (DCs) in vitro and observe their morphology at different growth stages and their ability to induce the proliferation of T lymphocytes.Material/MethodsGranulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were used in combination to induce differentiation of mouse bone marrow (BM) mononucleocytes into DCs. The derived DCs were then assessed for morphology, phenotype, and function.ResultsThe mouse BM-derived mononucleocytes had altered cell morphology 3 days after induction by GM-CSF and IL-4 and grew into colonies. Typical dendrites appeared 8 days after induction. Many mature DCs were generated, with typical dendritic morphology observed under scanning electron microscopy. Expression levels of CD11c, a specific marker of BM-derived DCs, and of co-stimulatory molecules such as CD40, CD80, CD86, and MHC-II were elevated in the mature DCs. Furthermore, the mature DCs displayed a strong potency in stimulating the proliferation of syngenic or allogenic T lymphocytes.ConclusionsMouse BM-derived mononucleocytes cultured in vitro can produce a large number of DCs, as well as immature DCs, in high purity. The described in vitro culture method lays a foundation for further investigations of anti-tumor vaccines.

Fibroblasts feeder niche and Flt3 Ligand as a novel inducer of plasmacytoid dendritic cells development in vitro.

Plasmacytoid dendritic cell (pDC), plays central role in antiviral immunity. The aim of this study was to assess the effect of Flt3 ligand (FL) alone or with L929 fibroblast feeder or L929 conditioned media on differentiation of mouse bone marrow (BM) cells into pDC in vitro. Murine BM cells were cultured with FL or with L929 or conditioned media for 9days. The differentiated cells were analyzed using flow cytometry for PDCA-1, B220 and CXCR4. The relative expression of Stat3, CXCR4, CXCR7, IFN-β, TGF-β and Runx2 in differentiated cells determined by real time PCR. The development of pDC showed up to 19% increase after co-culture of BM cells with fibroblast feeder. Upregulation of Stat3, Runx2 and CXCR4 due to the presence of fibroblast feeder with FL in culture results in improved pDC development. Furthermore, 30% L929 supernatant along with Flt3 ligand was able to derive pDC up to 8.9% in comparison with FL alone, which was 6.6% in vitro. Thus, for the first time we introduced L929 fibroblast feeder as a niche producer of M-CSF and probably other growth factors and chemokines, which promotes the development of pDC in vitro along with FL, similar to in vivo niche.

AB0145 Sodium Chloride Aggravates Arthritis by TH17 Polarization

BackgroundRecent studies demonstrated that sodium chloride (NaCl) can be a risk factor for autoimmune disease through the induction of pathogenic IL-17 producing T helper (Th17) cells.ObjectivesThis study was aimed to...

Differentiation of mouse bone marrow derived stem cells toward microglia-like cells

BackgroundMicroglia, the macrophages of the brain, have been implicated in the causes of neurodegenerative diseases and display a loss of function during aging. Throughout life, microglia are replenished by limited proliferation of resident microglial cells. Replenishment by bone marrow-derived progenitor cells is still under debate. In this context, we investigated the differentiation of mouse microglia from bone marrow (BM) stem cells. Furthermore, we looked at the effects of FMS-like tyrosine kinase 3 ligand (Flt3L), astrocyte-conditioned medium (ACM) and GM-CSF on the differentiation to microglia-like cells.MethodsWe assessed in vitro-derived microglia differentiation by marker expression (CD11b/CD45, F4/80), but also for the first time for functional performance (phagocytosis, oxidative burst) and in situ migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices.ResultsThe cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation.ConclusionWe conclude that in vitro-derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.

Effect of partial oxygen pressure on survival, proliferation, and differentiation of mouse bone marrow mesenchymal stem cells

Effects of partial oxygen pressure in a gas mixture surrounding the culture medium on survival, proliferation and differentiation of mesenchymal stem cells (MSC) isolated from mouse bone marrow was studied. It was found that 3% oxygen increased the survival of cells seeded at low density; the rate and duration of the MSC proliferation were also elevated. Effect of oxygen concentration on replicative activity of MSC was manifested as early as the first few days after the onset of cell growth. Studies of colony formation revealed that preincubation of cells with 21% oxygen had a negative effect on cell growth under 3% oxygen, while preincubation with 3% oxygen stimulated MSC proliferation in the presence of 21% oxygen. Notwithstanding, this effect of oxygen cannot be interpreted as unequivocally deleterious. Low partial oxygen pressure inhibited osteogenic differentiation of MSC, but adipogenic differentiation was insensitive to oxygen concentration. It is concluded that proliferation and differentiation of MSC depend critically on oxygen content in the culture medium.

Impact of stromal cell composition on BMP-induced chondrogenic differentiation of mouse bone marrow derived mesenchymal cells

Chondrogenic differentiation in mesenchymal stromal cells (MSCs) has been actively studied due to their potential use in mesenchymal tissue repair. Our goal was to develop a simple isolation protocol for adherent mouse MSCs to simultaneously clear off hematopoietic cells and expand to obtain enough starting material for differentiation studies. CD34 and CD45 expressing cells were rapidly removed by inhibiting growth of hematopoietic cells to yield short-term selected (STS) cells. Further passaging enriched more primitive, uniformly Sca-1 expressing, long-term selected (LTS) cells. The efficacy of several BMPs to induce chondrogenesis in pellet culture was compared in STS and LTS cells. In STS cells, chondrogenesis progressed rapidly to terminal differentiation while LTS cells differentiated at a slower rate with no hypertrophy. In LTS cells, rhBMP homodimers -2, -4, -6 and rhBMP2/7 heterodimer were effective enhancers of chondrogenesis over that of rhBMP-5 and -7. In STS cells, rhBMP-2 and rhBMP-7 supported rapid chondrogenesis and terminal differentiation over that of rhBMP-6. These data indicate the impact of stromal cell composition on the chondrogenic differentiation profile, which is an important aspect to be considered when standardizing differentiation assay conditions as well as developing MSC based cartilage repair technologies.

2,3,7,8-Tetrachlorodibenzo- p-dioxin modulates functional differentiation of mouse bone marrow-derived dendritic cells : Downregulation of RelB by 2,3,7,8-tetrachlorodibenzo- p-dioxin

We have previously shown that benzo( a)pyrene inhibits the growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DCs) [Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo( a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo( a)pyrene. Toxicol. Lett. 169, 82–90]. Since the toxic effects of benzo( a)pyrene are aryl hydrocarbon receptor (AhR)-dependent, we examined the effects of the very potent AhR agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the growth and functional differentiation of mouse BM-derived DCs. Ten nanomolars of TCDD had significant effects on functional differentiation of mouse DCs derived from BM cultured in the presence of GM-CSF and IL-4. The yields of DCs, flow-cytometrically analyzed for co-expression of CD11c/MHCII or CD11c/CD86, were reduced for TCDD-treated cultures, but TCDD itself had no effect on the growth of BM. DCs from TCDD-treated cultures expressed higher levels of MHCII and CD86, whereas expression of CD11c was reduced, compared with vehicle-treated cultures. Production of IL-10, but not IL-12, by the DCs from TCDD-treated cultures was decreased. Allogeneic T-cell stimulating ability of TCDD-treated DCs was increased compared to control DCs. The effects of TCDD were dependent on aryl hydrocarbon receptor (AhR), because α-naphthoflavone, an AhR antagonist, suppressed the effects of TCDD on IL-10 production and T-cell stimulating ability. RT-PCR revealed the downregulation of RelB, a transcription factor necessary for DCs differentiation and function. Taken together, although benzo( a)pyrene and TCDD exert their effects via binding to AhR, their effects on the growth and functional differentiation of bone marrow-derived DCs are different.

Benzo( a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells : Downregulation of RelB and eIF3 p170 by benzo( a)pyrene

In this study, we have investigated effects of benzo( a)pyrene (BP) on growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DC). 1 μM BP dramatically inhibited growth of BM cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although little alterations in surface expression of CD11c, major histocompatibility complex (MHC II), and CD86 molecules characteristic of mature DC were induced by BP, production of cytokines including IL-12, IL-10, and TNF-α, and allogeneic T cell stimulating ability were severely impaired. Some of the effects of BP were dependent on arylhydrocarbon receptor (AhR), because α-naphthoflavone, an AhR antagonist, suppressed the effects of BP on IL-12 production and T cell stimulating ability, but not on DC proliferation. Expression of RelB, a transcription factor necessary for DC differentiation and function, and eIF3 p170, a subunit of eukaryotic translation initiation factor (eIF)3, was reduced upon BP treatment.

Interleukin 10 inhibits transforming growth factor-beta (TGF-beta) synthesis required for osteogenic commitment of mouse bone marrow cells.

Interleukin 10 (IL-10) suppressed TGF-beta synthesis in mouse bone marrow cultures. Coincidingly, IL-10 down-regulated the production of bone proteins including alkaline phosphatase (ALP), collagen and osteocalcin, and the formation of mineralized extracellular matrix. The mAb 1D11.16 which neutralizes TGF-beta 1 and TGF-beta 2, induced suppressive effects comparable to IL-10 when administered before the increase of cell proliferation in the culture. It appears that mainly TGF-beta 1 plays a role in this system since (a) TGF-beta 2 levels were undetectable in supernatants from osteogenic cultures, (b) no effect was observed when the anti-TGF-beta 2 neutralizing mAb 4C7.11 was added and (c) the suppressive effect of IL-10 could be reversed by adding exogenous TGF-beta 1. It is unlikely that TGF-beta 1 modulates osteogenic differentiation by changing the proliferative potential of marrow cells since 1D11.16 did not affect [3H]thymidine ([3H]TdR) incorporation or the number of fibroblast colony forming cells (CFU-F) which harbor the osteoprogenitor cell population. Furthermore, 1D11.16 did not alter [3H]TdR uptake by the cloned osteoprogenitor cell lines MN7 and MC3T3. Light and scanning electron microscopy showed that IL-10 and 1D11.16 induced comparable morphological changes in the marrow cultures. Control cultures contained flat adherent cells embedded in a mineralized matrix. In contrast, IL-10 and 1D11.16 treated cultures were characterized by round non-adherent cells and the absence of a mineralized matrix. In this study, the mechanism by which IL-10 suppresses the osteogenic differentiation of mouse bone marrow was identified as inhibition of TGF-beta 1 production which is essential for osteogenic commitment of bone marrow cells.