A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone

Zhu, Heng; Guo, Zi-Kuan; Jiang, Xiaoxia; Li, Hong; Wang, Xiaoyan; Yao, Hong; Zhang, Yi; Mao, Ning

doi:10.1038/nprot.2009.238

Cited by 461 publications

(439 citation statements)

References 60 publications

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“…15 For the adipogenesis assay, cells were incubated in a-MEM supplemented with 10% FBS, 1 mM dexamethasone, 0.5 mM IBMX, 2 mg/mL insulin, and 2 mM rosiglitazone for 2 weeks. The results were analyzed by Oil Red O staining at day 14.…”

Section: Adipogenesis and Chondrogenesis Assaymentioning

confidence: 99%

NF-κB Decoy Oligodeoxynucleotide Enhanced Osteogenesis in Mesenchymal Stem Cells Exposed to Polyethylene Particle

Lin

Sato

Barcay

et al. 2015

Tissue Engineering Part A

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Excessive generation of wear particles after total joint replacement may lead to local inflammation and periprosthetic osteolysis. Modulation of the key transcription factor NF-kB in immune cells could potentially mitigate the osteolytic process. We previously showed that local delivery of ultrahigh-molecular-weight polyethylene (UHMWPE) particles recruited osteoprogenitor cells and reduced osteolysis. However, the biological effects of modulating the NF-kB signaling pathway on osteoprogenitor/mesenchymal stem cells (MSCs) remain unclear. Here we showed that decoy oligodeoxynucleotide (ODN) increased cell viability when primary murine MSCs were exposed to UHMWPE particles, but had no effects on cellular apoptosis. Decoy ODN increased transforming growth factor-beta 1 (TGF-b1) and osteoprotegerin (OPG) in MSCs exposed to UHMWPE particles. Mechanistic studies showed that decoy ODN upregulated OPG expression through a TGFb1-dependent pathway. By measuring the alkaline phosphatase activity, osteocalcin levels, Runx2 and osteopontin expression, and performing a bone mineralization assay, we found that decoy ODN increased MSC osteogenic ability when the cells were exposed to UHMWPE particles. Furthermore, the cellular response to decoy ODN and UHMWPE particles with regard to cell phenotype, cell viability, and osteogenic ability was confirmed using primary human MSCs. Our results suggest that modulation of wear particle-induced inflammation by NF-kB decoy ODN had no adverse effects on MSCs and may potentially further mitigate periprosthetic osteolysis by protecting MSC viability and osteogenic ability.

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Section: Adipogenesis and Chondrogenesis Assaymentioning

confidence: 99%

NF-κB Decoy Oligodeoxynucleotide Enhanced Osteogenesis in Mesenchymal Stem Cells Exposed to Polyethylene Particle

Lin

Sato

Barcay

et al. 2015

Tissue Engineering Part A

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“…We analyzed these MSC cultures by flow cytometry to determine whether the increased cell number reflected a greater degree of proliferation and survival by OSXCreSHIP flox/flox MSC. We quantified the frequency and absolute numbers of MSC in these cultures using the CD29 + Lin -MSC phenotype recently described by Zhu et al [50] (Fig. 4A-C).…”

Section: Ship1 Expression Represses Id2 To Facilitate Osteolineage Comentioning

confidence: 99%

SHIP1 Regulates MSC Numbers and Their Osteolineage Commitment by Limiting Induction of the PI3K/Akt/β-Catenin/Id2 Axis

Iyer

Viernes

Chisholm³

et al. 2014

Stem Cells and Development

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Here, we show that Src homology 2-domain-containing inositol 5¢-phosphatase 1 (SHIP1) is required for the efficient development of osteoblasts from mesenchymal stem cells (MSCs) such that bone growth and density are reduced in mice that lack SHIP1 expression in MSCs. We find that SHIP1 promotes the osteogenic output of MSCs by limiting activation of the PI3K/Akt/b-catenin pathway required for induction of the MSC stemness factor Id2. In parallel, we demonstrate that mice with myeloid-restricted ablation of SHIP1, including osteoclasts (OCs), show no reduction in bone mass or density. Hence, diminished bone mass and density in the SHIP1-deficient mice results from SHIP deficiency in MSC and osteolineage progenitors. Intriguingly, mice with a SHIP-deficient MSC compartment also exhibit decreased OC numbers. In agreement with our genetic findings we also show that treatment of mice with an SHIP1 inhibitor (SHIPi) significantly reduces bone mass. These findings demonstrate a novel role for SHIP1 in MSC fate determination and bone growth. Further, SHIPi may represent a novel therapeutic approach to limit bone development in osteopetrotic and sclerotic bone diseases.

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“…Primary osteoblasts were isolated from long bone metaphyses of 12-week-old control and osteoblast-specific p38a knockout mice as recently described [19]. Mice were sacrificed by cervical dislocation and rinsed in 70 % ethanol for 5 min.…”

Section: Primary Osteoblast Culturesmentioning

confidence: 99%

The p38α MAPK positively regulates osteoblast function and postnatal bone acquisition

Thouverey

Caverzasio

2012

Cell. Mol. Life Sci.

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Bone continuously remodels throughout life by coordinated actions of osteoclasts and osteoblasts. Abnormalities in either osteoclast or osteoblast functions lead to bone disorders. The p38 MAPK pathway has been shown to be essential in controlling osteoblast differentiation and skeletogenesis. Although p38a is the most abundant p38 member in osteoblasts, its specific individual contribution in regulating postnatal osteoblast activity and bone metabolism is unknown. To elucidate the specific role of p38a in regulating osteoblast function and bone homeostasis, we generated mice lacking p38a in differentiated osteoblasts. Osteoblast-specific p38a knockout mice were of normal weight and size. Despite non-significant bone alterations until 5 weeks of age, mutant mice demonstrated significant and progressive decrease in bone mineral density from that age. Adult mice deficient in p38a in osteoblasts displayed a striking reduction in cancellous bone volume at both axial and appendicular skeletal sites. At 6 months of age, trabecular bone volume was reduced by 62 % in those mice. Mutant mice also exhibited progressive decrease in cortical thickness of long bones. These abnormalities correlated with decreased endocortical and trabecular bone formation rate and reduced expressions of type 1 collagen, alkaline phosphatase, osteopontin and osteocalcin whereas bone resorption and osteoclasts remained unaffected. Finally, osteoblasts lacking p38a showed impaired marker gene expressions and defective mineralization in vitro. These findings indicate that p38a is an essential positive regulator of osteoblast function and postnatal bone formation in vivo.

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A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone

Cited by 461 publications

References 60 publications

NF-κB Decoy Oligodeoxynucleotide Enhanced Osteogenesis in Mesenchymal Stem Cells Exposed to Polyethylene Particle

NF-κB Decoy Oligodeoxynucleotide Enhanced Osteogenesis in Mesenchymal Stem Cells Exposed to Polyethylene Particle

SHIP1 Regulates MSC Numbers and Their Osteolineage Commitment by Limiting Induction of the PI3K/Akt/β-Catenin/Id2 Axis

The p38α MAPK positively regulates osteoblast function and postnatal bone acquisition

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