An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Huang, Shuo; Xu, Liangliang; Sun, Yuxin; Wu, Tianyi; Wang, Kuixing; Li, Gang

doi:10.1016/j.jot.2014.07.005

Cited by 150 publications

(125 citation statements)

References 31 publications

(37 reference statements)

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“…They are main source for tissue repair, tissue engineering and vehicles of cell-based gene therapy. 5 Stem cells have been isolated from different types of tissues. Mesenchymal stem cells can differentiate into several tissue-forming cells such as bone, cartilage, fat, kidney, liver, tendon, muscle, heart and even brain cells.…”

Section: Resultsmentioning

confidence: 99%

Mesenchymal Stem Cells Derived from Rat Bone Marrow (rBM MSC): Techniques for Isolation, Expansion and Differentiation

Palakkara

Mohan

et al. 2017

JSRT

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Abbreviations: rBM-MSC, rat bone marrow derived mesenchymal stem cells; CFU-F, colony forming unit-fibroblast; DPBS, dulbecco's phosphate buffer saline; DMEM, dulbecco's modified eagles medium; EDTA, ethylene diamine tetra acetic acid IntroductionIn the past decade, the field of stem cells and cell-based therapies has undergone a remarkable evolution. The potential of stem cell to differentiate into various types of cells has revolutionized their use in clinics for the treatment of a variety of clinical conditions. Mesenchymal stem cells can be cultured and grown for many generations under appropriate conditions in the laboratory and still retain a stable morphology and chromosome complement.1 Bone marrow obtained mesenchymal stem cells are the most studied stem cell type that is capable of differentiating into variety of cell lineages. Differences in differentiation ability to osteogenic chondrogenic and adipogenic lineages of MSCs harvested from Murine species of various age groups and the number of passage of these cultured cells has been reported. Osteogenic and chondrogenic potential reduced with each and every age group and adipogenic differentiation ability reduced only in cells obtained from oldest donors. 2 The technique of bone marrow collection and stem cell culture vary for different species.3 Culturing of rodent bone marrow derived stem cell is a little bit difficult when compared to its human counterpart. 4 Here we describe a simple and easy technique of stem cells isolation and differentiation from adult Wistar rats. Materials and methods Bone marrow collectionTwo male Wistar rats of 14 weeks age were used for bone marrow collection. Prior permission was obtained from Institutional Animal Ethics Committee (IAEC) for conducting animal trials. Shortly after euthanizing the animal, back side extending from lumbar region to toes were shaved and aseptically prepared for dissection ( Figure 1A). Skin incision was given on the lateral aspect of the thigh and both tibia and femur were exteriorized after removing the muscular and tendinous attachment ( Figure 1B). Femur and tibia of both the legs were placed separately in a 50ml centrifuge tube containing DPBS (Dulbecco's Phosphate Buffered Saline) and antibiotics. It was then placed under laminar hood in petridish containing DMEM (Dulbecco's Modified Eagles Media) with antibiotics. With a scissor the metaphyseal region of both bones were cut. After inserting a needle into the medullary cavity the bone marrow was flushed out using DMEM into a 15ml centrifuge tube. Flushed out bone marrow was centrifuged for 5minutes at 1000rpm in order to concentrate the cells. The supernatant was decanted and 5ml of complete media was added to the tube and mixed well by pipetting. Cell suspension was layered over equal quantity of histopaque and again subjected for centrifugation at 2500rpm for half an hour. After this density gradient centrifugation mononuclear cells were collected from the interface and washed with Ca ++ and Mg ++ free DPBS ( Figure 1C). Obtained cell ...

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Section: Resultsmentioning

confidence: 99%

Mesenchymal Stem Cells Derived from Rat Bone Marrow (rBM MSC): Techniques for Isolation, Expansion and Differentiation

Palakkara

Mohan

et al. 2017

JSRT

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“…MSCs were prepared according to the method of Huang et al (61). Briefly, femurs were excised from two 8-wk-old female CD1 mice, and the tissue was removed from the outside of the bone.…”

Section: Methodsmentioning

confidence: 99%

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

McKinlay

Vargas

Blake

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

212

264

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Functional delivery of mRNA to tissues in the body is key to implementing fundamentally new and potentially transformative strategies for vaccination, protein replacement therapy, and genome editing, collectively affecting approaches for the prevention, detection, and treatment of disease. Broadly applicable tools for the efficient delivery of mRNA into cultured cells would advance many areas of research, and effective and safe in vivo mRNA delivery could fundamentally transform clinical practice. Here we report the stepeconomical synthesis and evaluation of a tunable and effective class of synthetic biodegradable materials: charge-altering releasable transporters (CARTs) for mRNA delivery into cells. CARTs are structurally unique and operate through an unprecedented mechanism, serving initially as oligo(α-amino ester) cations that complex, protect, and deliver mRNA and then change physical properties through a degradative, charge-neutralizing intramolecular rearrangement, leading to intracellular release of functional mRNA and highly efficient protein translation. With demonstrated utility in both cultured cells and animals, this mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.

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“…The medium was slowly changed after 3 days to remove non‐adherent cells. Meantime, the adherent marrow cells were subcultured until obtaining purify population of mesenchymal stem cells with spindle‐shaped morphology …”

Section: Methodsmentioning

confidence: 99%

Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF‐1α signalling

et al. 2019

J Cellular Molecular Medi

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Mesenchymal stem cells (MSCs) have drawn great attention because of their therapeutic potential. It has been suggested that intra‐venous infused MSCs could migrate the site of injury to help repair the damaged tissue. However, the mechanism for MSC migration is still not clear so far. In this study, we reported that hypoxia increased chemotaxis migration of MSCs. At 4 and 6 hours after culturing in hypoxic (1% oxygen) conditions, the number of migrated MSCs was significantly increased. Meanwhile, hypoxia also increased the expression of HIF‐1α and SDF‐1. Using small interference RNA, we knocked down the expression of HIF‐1α in MSCs to study the role of HIF‐1α in hypoxia induced migration. Our data indicated that knocking down the expression of HIF‐1α not only abolished the migration of MSCs, but also reduced the expression of SDF‐1. Combining the results of migration assay and expression at RNA and protein level, we demonstrated a novel mechanism that controls the increase of MSCs migration. This mechanism involved HIF‐1α mediated SDF‐1 expression. These findings provide new insight into the role of HIF‐1α in the hypoxia induced MSC migration and can be a benefit for the development of MSC‐based therapeutics for wound healing.

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An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Cited by 150 publications

References 31 publications

Mesenchymal Stem Cells Derived from Rat Bone Marrow (rBM MSC): Techniques for Isolation, Expansion and Differentiation

Mesenchymal Stem Cells Derived from Rat Bone Marrow (rBM MSC): Techniques for Isolation, Expansion and Differentiation

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF‐1α signalling

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