Figure 3. Mx1-Induced Cells Have MSC Characteristics and Maintain Long-Term Repopulation of Osteoblasts In Vivo.
(A) Mx1-induced osteogenic cells (YFP+) in calvaria (left) or femur sections (middle and right) of Mx1/YFP mice prepared 20 days after pIpC treatment (day 20) or 6 months after pIpC treatment (at 6–8 weeks after irradiation and wild-type bone marrow transplantation, +WT-BMT) were analyzed by undergoing intravital microscopy (left, blue, bone; green, Mx1-induced osteoblasts) or by being stained with anti-GFP/YFP (YFP), anti-osterix (Osx, for preosteoblasts), and anti-osteocalcin (Ocn, for mature osteoblasts) antibodies (right).
(B) Labeling efficiency of osteoblasts by Mx1. From collagenase-treated bones of Mx1/Tomato/Ocn-GFP trigenic mice at 30 days after pIpC treatment following WT-BMT, the percentage of Mx1-induced (Tomato+GFP+) or noninduced (GFP+) osteoblasts was analyzed by flow cytometry.
(C) Percentage of Mx1-induced osteoblasts (YFP+Ocn+) before (−pIpC) or after pIpC (+pIpC) treatment as analyzed by anti-GFP/YFP, anti-osteocalcin immunohistochemistry, and FACs of cells from femurs at the indicated time points (n = 5 per group, right panel).
(D) Enrichment of clonogenic stromal cells in the CD105+CD140a+ fraction. From collagenase-treated wild-type bone cells, the nonhematopoietic fraction (CD45−CD31−Ter119−) was further separated with CD105 and CD140a. Indicated fractions (A, B, and C) were sorted and their clonogenicity was analyzed by CFU-F assays (>50 cells/colony at 14 days) (right, n = 5).
(E) Mx1-induced labeling in Mx1/YFP mice was examined using the same antibodies and analyzed as in Figure 3D. The percentage of YFP− (box A) and YFP+ (box B) within CD105+CD140a+ cells was determined and these fractions were used for CFU-F assays (right, n = 4).
(F) Multilineage differentiation potential of Mx1-induced clones was examined using single-cell-driven, clonally expanded Mx1-induced progenitors (Figure 3E, box B) that were further incubated with indicated condition medium or MSC medium (Control) for 28 days (n = 8). Cells were stained with Oil-Red for adipocytes (Adipogenic), Alizarin-Red for osteoblasts (Osteogenic), or Toluidine-blue for chondrocytes (Chondrogenic). NIH 3T3 fibroblasts cultured with conditioned medium were stained as a control (inserted images).
(G) Eight weeks after lethal irradiation followed by wild-type marrow transplantation (WT-BMT), Mx1/mTmG mice were treated with five doses of pIpC every other day. After 90 days of further tracing, Mx1-induced (mGFP+, green) or Mx1-noninduced (mTomato+, red) osteoblasts from femur sections were analyzed by anti-osteocalcin (top right) or by anti-nestin immunohistochemistry (bottom). Red arrow indicates Mx1 and nestin double positive cells. Bone (B), bone marrow (BM), and osteoblasts (Ob) are indicated. Blue, DAPI (A and G).
Numbers in the histogram indicate the average of 5–10 different analyses; error bars, ± SD (D and E). Scale bars are 20 µm (G). Data represent more than three independent experiments with comparable results (A and G). See also Figures S3 and S4.