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. 2017 Summer;10(3):208-213.

Isolation, differentiation, and characterization of mesenchymal stem cells from human bone marrow

Kaveh Baghaei  1 , Seyed Mohmoud Hashemi  2 , Samaneh Tokhanbigli  1 , Ali Asadi Rad  2 , Hamid Assadzadeh-Aghdaei  1 , Abdolhamid Sharifian  3 , Mohammad Reza Zali  1   4
Affiliations

Isolation, differentiation, and characterization of mesenchymal stem cells from human bone marrow

Kaveh Baghaei et al. Gastroenterol Hepatol Bed Bench. 2017 Summer.

Abstract

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: Differentiation; Flow cytometry; Mesenchymal Stromal Cell.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Microscopic images of human mesenchymal stem cells isolated from bone marrow. (a) hMSCs spindle-shaped fibroblast- like appearance, extended in opposite directions from a small cell body (20X) (passage number 2). (b) adipogenic-induced hMSC, intracellular staining using oil red-O. (c) Osteogenic differentiation assay, Alizarin staining specifically shows calcium oxalates in differentiated hMSCs (5 days in differentiation medium, passage number 2-3
Figure 2
Figure 2
Flow cytometry analysis of cell surface markers present on hMSCs derived bone marrow. (a) positive markers CD73, CD90, and CD105 (b) while being negative for CD14, CD34and CD45
See this image and copyright information in PMC

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