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Updated: Jul 11, 2026
Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs
Published on: March 22, 2010
Isolation murine mesenchymal stem cells by positive selection
Samad Nadri1, Masoud Soleimani
1Stem Cells Department, Stem Cell Technology, Co, Tehran, Iran.
Abstract:
Isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures is arduous because of the unwanted growth of hematopoietic cells and non-MSCs. In this work, homogenous populations of CD34(+) MSCs from mouse bone marrow were isolated via positive selection. For this purpose, C57Bl/6 mice were killed and bone marrow cells were aspirated before incubation with magnetic bead conjugated to anti-CD34 antibody. A sample of positively selected CD34(+) cells were prepared for flow cytometry to examine the expression of CD34 antigen and others were subcultured in a 25-cm(2) culture flask. To investigate the mesenchymal nature, the plastic adherent cultivated cells were induced to differentiate along osteoblastic and adipogenic lineages. Furthermore, the expression of some surface markers was investigated by flow cytometry. According to the result, purified populations of fibroblast-like CD34(+) cells were achieved in the first passage (1 wk after culture initiation). The cells expressed CD34, CD44, Sca-1, and Vcam-1 antigens (markers) but not CD11b and CD45. They were capable of differentiating into osteocytes and adipocytes. This study indicated that our protocol can result in the efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We have shown that murine bone marrow-derived CD34(+) cells with plastic adherent properties and capability of differentiating into skeletal lineages in vitro are MSCs.
Insights
Researchers efficiently isolated homogenous mesenchymal stem cells (MSCs) from mouse bone marrow using CD34(+) positive selection. These purified MSCs exhibit plastic adherence and differentiate into bone and fat cells in vitro.
Area of Science:
- Stem Cell Biology
- Hematology
- Cellular and Molecular Medicine
Background:
- Isolating mesenchymal stem cells (MSCs) from mouse bone marrow using traditional plastic adherence is challenging due to contamination by hematopoietic and non-MSC populations.
- The CD34 antigen is a marker often associated with hematopoietic stem cells, but its role in MSC isolation requires further investigation.
Purpose of the Study:
- To develop an efficient protocol for isolating homogenous populations of mesenchymal stem cells (MSCs) from C57BL/6 mouse bone marrow.
- To characterize the isolated CD34(+) cells for their mesenchymal stem cell properties, including surface marker expression and differentiation potential.
Main Methods:
- Positive selection of CD34(+) cells from mouse bone marrow using magnetic beads conjugated to anti-CD34 antibodies.
- Culture of positively selected cells in plastic adherent flasks.
- Flow cytometry analysis to assess surface marker expression (CD34, CD44, Sca-1, Vcam-1, CD11b, CD45).
- In vitro induction of differentiation into osteoblastic and adipogenic lineages.
Main Results:
- Homogenous populations of fibroblast-like CD34(+) cells were successfully isolated within the first passage (1 week).
- The purified cells expressed MSC markers CD34, CD44, Sca-1, and Vcam-1, while lacking hematopoietic markers CD11b and CD45.
- Isolated cells demonstrated the capacity for osteogenic and adipogenic differentiation in vitro.
Conclusions:
- The described protocol enables efficient isolation of homogenous mesenchymal stem cells (MSCs) from C57BL/6 mouse bone marrow.
- Murine bone marrow-derived CD34(+) cells with plastic adherence and trilineage differentiation potential are identified as MSCs.
