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

Diarmaid D. Houlihan, Yo Mabuchi, Satoru Morikawa, Kunimichi Niibe, Daisuke Araki, Sadafumi Suzuki, Hideyuki Okano, Yumi Matsuzaki

Research output: Contribution to journalArticlepeer-review

149 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2103-2111
Number of pages9
JournalNature Protocols
Volume7
Issue number12
DOIs
Publication statusPublished - 2012 Dec

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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