Abstract
Platelet-derived growth factor receptor α (PDGFR-α) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-α+Sca-1+ (PαS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PαS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PαS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 °C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45+Ter-119+)-positive cells. Approximately 10,000 PαS MSCs may then be isolated per mouse. The total protocol takes ∼7 h to complete.
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Acknowledgements
This work was supported by the Project for Realization of Regenerative Medicine and Support for the Core Institutes for Induced Pluripotent Stem (iPS) Cell Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT; to H.O. and Y. Matsuzaki). This study was also supported, in part, by a grant-in-aid for Encouragement of Young Medical Scientists from Keio University (to Y. Mabuchi), a grant-in-aid for Scientific Research (KAKENHI; to Y. Mabuchi) and a grant-in-aid from the Global Century COE program of the MEXT, Japan, to Keio University. D.D.H. is funded by the Medical Research Council, UK.
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Y. Mabuchi, D.D.H. and Y. Matsuzaki designed the study. Y. Mabuchi and D.D.H. performed the experiments. H.O. and Y. Matsuzaki supervised all the experiments. Y. Mabuchi, D.D.H., S.M., K.N., D.A. and S.S. analyzed the data. Y. Mabuchi, D.D.H. and Y. Matsuzaki contributed to writing the manuscript.
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Houlihan, D., Mabuchi, Y., Morikawa, S. et al. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α. Nat Protoc 7, 2103–2111 (2012). https://doi.org/10.1038/nprot.2012.125
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DOI: https://doi.org/10.1038/nprot.2012.125
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