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

Platelet-derived growth factor receptor alpha (PDGFR-alpha) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-alpha(+)Sca-1(+) (P alpha S) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of P alpha S MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate P alpha S 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 degrees 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-alpha, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter-119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Approximately 10,000 P alpha S MSCs may then be isolated per mouse. The total protocol takes similar to 7 h to complete.