Isolation, differentiation, and Characterization of Mesenchymal Stem Cells from Human bone marrow

Kaveh Baghaei, Seyed Mahmoud Hashemi, Samaneh Tokhanbigli, Ali Asadi Rad, Hamid Asadzadeh-Aghdaie, Abdolhamid Sharifian, Mohammad Reza Zali

Abstract


68

Aim: We describe the minimum requirements and a simplified method for isolation and characterization of mesenchymal stem cells (MSCs) from human bone marrow.

Background: MSCs are well known adult stem cells present in many tissues such as adipocytes, chondrocytes, osteoblasts, and neurons. Many isolations and characterization methods have emerged to apply MSCs in the clinical applications, which many of them are expensive and time-consuming.

Methods: MSC isolation was carried out from human bone marrow, and cultured in defined medium. Cultures were maintained at 370C in a humidified atmosphere containing 5% CO2 for 48h. The medium was exchanged every 3-4 days. Adherent cells were characterized according to main criteria defined by ISCT, such as differentiation capability to adipocyte and osteoblast using specific differentiation mediums; also, flow cytometry verified MSC specific markers.

Results: Isolated MSCs had a fibroblastic-like appearance with adherent property to the culture plate. Differentiation function was proved with the formation of lipid drops and calcium oxalates on the differentiated MSCs and finally, purified MSCs from bone marrow were positive for cell surface markers, CD73, CD90, and CD105 while being negative for CD34 and CD45.

Conclusion: These findings confirm that the represented method is capable of isolating MSCs from bone marrow with proven results according to all minimum criteria defined by the International Society for Cellular Therapy (ISCT).

Keywords: Mesenchymal Stromal Cell, Flow cytometry, Differentiation


Keywords


Mesenchymal stem cells (MScs), Flow cytometery, differentiation

Full Text:

PDF - View Count=30

References


Ullah I, Baregundi Subbarao R, Rho G-J. Human Mesenchymal Stem Cells - Current trends and future prospective. Biosci Rep [Internet]. 2015;35(2):e00191. Available from: http://www.bioscirep.org/content/35/2/e00191.abstract

Schüring AN, Schulte N, Kelsch R, Röpke A, Kiesel L, Götte M. Characterization of endometrial mesenchymal stem-like cells obtained by endometrial biopsy during routine diagnostics. Fertil Steril. 2011;95(1):423–6.

Hadjizadeh A, Doillon CJ. Directional migration of endothelial cells towards angiogenesis using polymer fibres in a 3D co-culture system. J Tissue Eng Regen Med. 2010;4(7):524–31.

Eriksson M, Taskinen M, Leppä S. Mitogen Activated Protein Kinase-Dependent Activation of c-Jun and c-Fos is required for Neuronal differentiation but not for Growth and Stress Reposne in PC12 cells. J Cell Physiol. 2006;207(1):12–22.

Nauta AJ, Fibbe WE. Review in translational hematology Immunomodulatory properties of mesenchymal stromal cells. Library (Lond). 2008;110(10):3499–506.

Le Blanc K, Ringdén O. Immunomodulation by mesenchymal stem cells and clinical experience. J Intern Med. 2007;262(5):509–25.

Nicola M Di, Carlo-stella C, Magni M, Milanesi M, Longoni PD, Grisanti S, et al. induced by cellular or nonspecific mitogenic stimuli Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. 2013;99(10):3838–43.

Bieback K, Schallmoser K, Klüter H, Strunk D. Clinical protocols for the isolation and expansion of mesenchymal stromal cells. Transfus Med Hemotherapy. 2008;35(4):286–94.

Meirelles Lda S, Nardi NB, Meirelles L da S, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol [Internet]. 2003;123(4):702–11. Available from: http://www.ncbi.nlm.nih.gov/pubmed/14616976nhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14616976

Expanded C, Mesenchymal C, Possess SC, Vivo PIN. Stem Cells Possess. Science (80- ). 2001;6(2):125–34.

Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol. 2006;34(11):1606–7.

Ringe J, Kaps C, Schmitt B, Büscher K, Bartel J, Smolian H, et al. Porcine mesenchymal stem cells: Induction of distinct mesenchymal cell lineages. Cell Tissue Res. 2002;307(3):321–7.

Caterson EJ, Nesti LJ, Danielson KG, Tuan RS. Human marrow-derived mesenchymal progenitor cells. Mol Biotechnol. Springer; 2002;20(3):245–56.

Méndez-Ferrer S, Michurina T V, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature [Internet]. 2010;466(7308):829–34. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3146551&tool=pmcentrez&rendertype=abstract

Tondreau T, Lagneaux L, Dejeneffe M, Delforge a, Massy M, Mortier C, et al. Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential. Cytotherapy [Internet]. 2004;6(4):372–9. Available from: http://dx.doi.org/10.1080/14653240410004943

Van Vlasselaer P, Falla N, Snoeck H, Mathieu E. Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin. Blood. 1994;84(3):753–63.

Eslaminejad MB, Nadri S. Murine mesenchymal stem cell isolated and expanded in low and high density culture system: Surface antigen expression and osteogenic culture mineralization. Vitr Cell Dev Biol - Anim. 2009;45(8):451–9.

Eslaminejad MB, Nikmahzar a, Taghiyar L, Nadri S, Massumi M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev Growth Differ [Internet]. 2006;48(6):361–70. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16872449

Baddoo M, Hill K, Wilkinson R, Gaupp D, Hughes C, Kopen GC, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem. 2003;89(6):1235–49.

Soleimani M, Nadri S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc [Internet]. 2009;4(1):102–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19131962

Siclari VA, Zhu J, Akiyama K, Liu F, Zhang X, Chandra A, et al. Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow. Bone [Internet]. Elsevier Inc.; 2013;53(2):575–86. Available from: http://dx.doi.org/10.1016/j.bone.2012.12.013

Pittenger M, Mackay M. Multilineage potential of adult human mesenchymal stem cells. Science (80- ). 1999;284(5411):143–7.

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy [Internet]. Elsevier; 2006;8(4):315–7. Available from: http://www.sciencedirect.com/science/article/pii/S1465324906708817

Neagu M, Suciu E, Ordodi V, Unescu VP. Human Mesenchymal Stem Cells As Basic Tools for Tissue Engineering : Isolation and Culture. October. 2005;15(October):29–34.

Kaiser S, Hackanson B, Follo M, Mehlhorn A, Geiger K, Ihorst G, et al. BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype. Cytotherapy [Internet]. Elsevier; 2007;9(5):439–50. Available from: http://dx.doi.org/10.1080/14653240701358445




DOI: http://dx.doi.org/10.22037/ghfbb.v0i0.1089

Creative Commons License
GHFBB by Gastroenterology and Liver Diseases Research Institute is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

 

PISSN: 2008-2258

EISSN: 2008-4234


•  

Privacy Policy | For Author | Online Submission | About | Contact

Copyright © 2016 Shahid Beheshti University of Medical Sciences. All rights reserved.