Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α

Nat Protoc. 2012 Dec;7(12):2103-11. doi: 10.1038/nprot.2012.125. Epub 2012 Nov 15.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Ly / metabolism*
  • Cell Culture Techniques
  • Cell Separation / methods*
  • Flow Cytometry / methods*
  • Membrane Proteins / metabolism*
  • Mesenchymal Stem Cells / cytology*
  • Mesenchymal Stem Cells / metabolism*
  • Mice
  • Receptor, Platelet-Derived Growth Factor alpha / metabolism*

Substances

  • Antigens, Ly
  • Ly6a protein, mouse
  • Membrane Proteins
  • Receptor, Platelet-Derived Growth Factor alpha