Tissue-type plasminogen activator expands CD45⁻Ter119⁻Sca-1⁺PDGFRα⁺ MSCs in mice. C57BL/6 (WT B6) mice were injected daily intraperitoneally with a serpine-resistant recombinant tissue tPA on day 0 and day 1. MSCs were identified using the following marker profile: CD45⁻Ter119⁻Sca-1⁺PDGFRα⁺ (PαS-MSCs) by FACS. (A) Absolute numbers of PαS-MSCs per femur in WT B6 and tPA^−/−, Plg^−/−, and PAI-1^−/− BM cells under steady state as determined by FACS (n = 3-4/group). (B) Absolute numbers of MSCs per femur from tPA-treated or PBS-treated WT B6 mice following the indicated treatment schedule as determined by FACS analysis (n = 5/group; left). FACS gating schedule shown (right). (C) Immunohistologic staining for Nestin in ex vivo cultured MSCs derived from tPA and vehicle-treated mice. Nuclei were counterstained using DAPI (n = 3/group). Scale bars, 20 µm. (D) The percentage of CD45⁻Ter119⁻CD31⁻LeptinR⁺ MSCs in BM cells of tPA-treated or PBS-treated WT B6 mice as determined by FACS (n = 3/group). (E) Cell cycle analysis from freshly isolated PαS-MSCs derived from tPA and control BM cells (n = 3-4/group). (F) BrdU cell proliferation assay was performed using PαS-MSCs derived from mice treated with/without tPA (n = 4 /group). (G) Using the colorimetric cell counting kit-8, the relative absorbance at 450 nm as a measure for cell proliferation is given for PαS-MSCs derived from control or tPA-treated mice (n = 3/group). (H) Freshly isolated PαS-MSCs from tPA or PBS-treated WT B6 mice were grown as single cell cultures for 14 days. The number of CFU-F was counted (n = 2000/group). (I) The percentage of PαS-MSCs in femoral muscle and subcutaneous white fat isolated from tPA- or PBS-treated WT B6 mice on day 2 is indicated as determined by FACS (n = 4/group). Mean ± SEM. *P < .05; **P < .01; ***P < .001 by Student t test. Data are representative of 2 independent experiments.