Whole bone marrow cells (WBMCs) were isolated from wild-type (WT), Fanca−/− and Fancc−/− mice by gently flushing out of tibias and femurs using DPBS + 10% FBS. Cells obtained from two tibias and two femurs were pooled and plated in 100mm culture dish (BD Falcon, Tewksbury, MA) in 10 ml of MSCs media (Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Gaithersburg, MD) supplemented with 15% fetal bovine serum (FBS, Gibco, Gaithersburg, MD) and 1% Penicillin-Streptomycin (Life Technologies # 15140-122 adapted and modified from previous report [27 (link)].
Plastic adherent cells were passaged 3 times followed by staining with fluorescently labelled antibodies for mesenchymal (CD29, CD44, CD73, SCA-1, CD106) and hematopoietic markers (CD45, CD11b) using the mouse multipotent mesenchymal stromal cell marker antibody panel (Cat # SC018; R&D systems; Minneapolis, MN) [28 –30 (link)]. We confirmed that at least 98% purity of MSCs was obtained with this culture method.
For Wnt5a treatment, MSCs at passages three were plated to obtain 95% confluence. Cells were pre-treated with different doses of Wnt5a (R&D System, Minneapolis, MN) for 16 hrs [31 (link)], followed by co-culture in fresh media with WT LSK (Lin−Sca1+c-kit+) cells.
Plastic adherent cells were passaged 3 times followed by staining with fluorescently labelled antibodies for mesenchymal (CD29, CD44, CD73, SCA-1, CD106) and hematopoietic markers (CD45, CD11b) using the mouse multipotent mesenchymal stromal cell marker antibody panel (Cat # SC018; R&D systems; Minneapolis, MN) [28 –30 (link)]. We confirmed that at least 98% purity of MSCs was obtained with this culture method.
For Wnt5a treatment, MSCs at passages three were plated to obtain 95% confluence. Cells were pre-treated with different doses of Wnt5a (R&D System, Minneapolis, MN) for 16 hrs [31 (link)], followed by co-culture in fresh media with WT LSK (Lin−Sca1+c-kit+) cells.
