title: Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α.
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.
authors.affiliation: Centre for Liver Research, National Institute for Health Research Biomedical Research Unit, University of Birmingham, Birmingham, UK.
authors.forename: Diarmaid D
authors.initials: DD
authors.lastname: Houlihan
authors.forename: Yo
authors.initials: Y
authors.lastname: Mabuchi
authors.forename: Satoru
authors.initials: S
authors.lastname: Morikawa
authors.forename: Kunimichi
authors.initials: K
authors.lastname: Niibe
authors.forename: Daisuke
authors.initials: D
authors.lastname: Araki
authors.forename: Sadafumi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Okano
authors.forename: Yumi
authors.initials: Y
authors.lastname: Matsuzaki
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism*; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism*
chemical_list: D000950:Antigens, Ly; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D020796:Receptor, Platelet-Derived Growth Factor alpha