Soleimani, M. & Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat. Protoc. 4, 102-106

Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, PO Box, 14115-111, Tehran, Iran.
Nature Protocol (Impact Factor: 9.67). 02/2009; 4(1):102-6. DOI: 10.1038/nprot.2008.221
Source: PubMed


We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees C-5% CO(2) incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 degrees C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

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    • "MSCs were isolated from the AT or BM of C57/BL6 mice according to three adapted protocols (Soleimani and Nadri, 2009; Sung et al., 2008; Yamamoto et al., 2007). Cells (2 3 10 5 ) at passage 3–5 were injected into the hindlimb of syngeneic mice the day following resection of the femoral artery. "
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    ABSTRACT: Mesenchymal stromal cells (MSCs) are defined as multipotent, self-renewing cells residing in several tissues, including the bone marrow, adipose tissue, umbilical cord blood, and placenta (Pittenger et al., 1999). These cells are defined as multipotent, as they are capable of generating different mesenchymal cell types, traditionally adipocytes, chondrocytes, and osteocytes, but also smooth muscle cells and cardiomyocytes (Makino et al., 1999 and Pittenger et al., 1999). MSCs have been at the forefront of clinical research for the therapy of cardiovascular disorders for many years. In particular, cardiac and peripheral ischemia is a leading cause of morbidity and mortality in our aging society and suffers from a lack of curative therapies (Tendera et al., 2011). In this setting, MSC transplantation has been proposed as an innovative therapy for no-option ischemic patients. Originally, the therapeutic potential of these cells was thought to arise through their putative capacity to transdifferentiate, thereby directly contributing to vasculogenesis and tissue regeneration (Quevedo et al., 2009). This attractive hypothesis led to the prompt, perhaps immature transition of the results obtained in animal models to the clinics, with the ambitious goal to regenerate ischemic tissues (Hare et al., 2009 and Tateishi-Yuyama et al., 2002). However, MSC plasticity has been later harshly questioned (Noiseux et al., 2006), and the therapeutic potential of these cells is currently considered to derive from the secretion of a variety of growth factors and cytokines exerting a paracrine, protective effect on ischemic cells (Gnecchi et al., 2012).
    Stem Cell Reports 02/2015; 110(3). DOI:10.1016/j.stemcr.2015.01.001 · 5.37 Impact Factor
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    • "The Ethical Review Board of Shanghai Sixth People's Hospital, Shanghai Jiaotong University School of Medicine approved the experimental procedures. Primary human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated and sub-cultured using a method described previously [38]. First, cell aggregates and bone debris were removed by filtering the bone marrow suspension through a 70 mm cell strainer. "
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    ABSTRACT: There is growing interest in the use of synthetic biomaterials to deliver inorganic ions that are known to stimulate angiogenesis and osteogenesis in vivo. In the present study, we investigated the effects of varying amounts of copper (Cu) in a bioactive glass on the response of human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro and on blood vessel formation and bone regeneration in rat calvarial defects in vivo. Porous scaffolds of a borosilicate bioactive glass (composition 6Na2O, 8K2O, 8MgO, 22CaO, 36B2O3, 18SiO2, 2P2O5; mol%) doped with 0.5, 1.0 and 3.0 wt% CuO were created using a foam replication method. When immersed in simulated body fluid, the scaffolds released Cu ions into the medium and converted to hydroxyapatite. At the concentrations used, the Cu in the glass was not toxic to the hBMSCs cultured on the scaffolds in vitro. The alkaline phosphatase activity of the hBMSCs and the expression levels of angiogenic-related genes (vascular endothelial growth factor and basic fibroblast growth factor) and osteogenic-related genes (runt-related transcription factor 2, bone morphogenetic protein-2 and osteopontin) increased significantly with increasing amount of Cu in the glass. When implanted in rat calvarial defects in vivo, the scaffolds (3 wt% CuO) significantly enhanced both blood vessel formation and bone regeneration in the defects at 8 weeks post-implantation. These results show that doping bioactive glass implants with Cu is a promising approach for enhancing angiogenesis and osteogenesis in the healing of osseous defects. Copyright © 2014. Published by Elsevier Ltd.
    Acta Biomaterialia 12/2014; 14. DOI:10.1016/j.actbio.2014.12.010 · 6.03 Impact Factor
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    • "Group 3 diabetic rats, 1 month after diabetic induction, were injected with 5× 10 6 dMSCs by the tail vein (i.v.). Briefly, mesenchymal cells (MSCs) from the bone marrow of 10 male albino rats were processed as per the standard protocol cited elsewhere (Soleimani and Nadri 2009). Phosphate-buffered saline (PBS) alone was also injected via tail vein in the control group (Group 2). "
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    ABSTRACT: Cardiovascular manifestations are one of the major complications of type 1 diabetes mellitus (T1DM) and supersede the slow progression of DM in most cases as the leading cause of mortality. There have been many studies and trials in regenerating the functional β-cells of islets from mesenchymal stem cells (MSCs) with varied success. The effect of MSCs ex vivo differentiated to mimic functional insulin-secreting β-cells of islets and their impact on restoration of diabetic complications and transplantation via systemic delivery have not been well studied. In the current study, bone marrow MSCs differentiated to insulin-secreting β-cells are used to treat STZ-induced diabetic rats. The post-homing effects of the differentiated MSCs (dMSCs) were endogenous with definite reversal of diabetic parameters. Consequently, the altered cardiac functions like heart beat rate, left ventricular performance, contractility index and physiological body weight gain due to hyperglycemia were amelorated into normacy. The primary onset cardiac perfomance and the endothelial activation were well evidenced by high fibrinogen levels and systolic blood pressure (SBP) being reversed on the treatment by dMSCs. Further high basal [Ca(2+)]c in isolated endothelial cells and thereby increased ROS confirmed the endothelial activation. The levels of pro-apoptotic makers p53 and Bax were highly expressed in the diabetic groups indicating oxidative stress through ROS induced by high cytosolic calcium skewing the cells towards apoptosis. The expression of the anti-apoptotic marker Bcl-2 was observed to be low in the diabetic group further augmenting the stress state of endothelial cells (ECs) in T1DM. Restoration of [Ca(2+)]c chelates ROS and the subsequent reversal of pro- and anti-apoptotic markers after the successful treatment of dMSCs proved that endogenous reconstitution of insulin secretion improves diabetic-induced cardiac manifestations.
    Cell and Tissue Research 11/2014; 359(2). DOI:10.1007/s00441-014-2034-2 · 3.57 Impact Factor
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