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Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential

Alexandra Peister, Jason A. Mellad, Benjamin L. Larson, Brett M. Hall, Laura F. Gibson and Darwin J. Prockop

Article Figures & Data

Figures

  • Figure 1.

    Schematic for the isolation of mMSCs. (A) The ends of each tibia or femur were removed, and the bones were placed in a pipet tip within a centrifuge tube. The cells were pelleted by centrifugation for 1 to 2 minutes at 400g. (B) Representative t-CFU and sc-CFU analysis. (C) The culturing steps for isolation and expansion of mMSCs.

  • Figure 2.

    Morphologies of mMSCs. mMSCs from Bl/6 mice: (A) passage 0, (B) passage 1, and (C) passage 5. Low-density plating to show the morphology of passage 7 mMSCs from the mouse strains: (D) Bl/6, (E) BALB/c, (F) FVB/N, (G) DBA1, and passage 4 GFPtg, phase contrast (H) and epifluorescence (I). Original magnification, × 10.

  • Figure 3.

    Dependence of expansion on initial plating density. (A) Expansion as a function of plating density for passage 3 mMSCs from Bl/6 mice. Cultures were counted every 3 days. Fold increase of cells was determined by total cell number recovered divided by the initial cell number plated. (B) The fold increase in cell number after 12 days of culture for mMSC from 5 different mouse strains. (C) t-CFU assays of the 12-day cultures after initial plating at increasing densities. (D) t-CFU and sc-CFU assays of Bl/6 mMSCs. Data are expressed as the mean ± SE, n = 3to5.

  • Figure 4.

    Medium requirements for mMSCs. (A) mMSCs were plated at 5 or 50 cells per cm2 (5 c or 50 c, respectively) and counted after 12 days of culture. (B) t-CFU assays were performed after the 12 days' incubation in the medium indicated. (C) t-CFU assays and sc-CFU assays were performed on Bl/6 mMSCs expanded in the different media. (D) Serum requirements (HS:FCS = 1:1) were determined for the 5 strains of mMSCs. The mMSCs were plated at 5 or 50 cells per cm2 (5 c or 50 c, respectively) and counted after 12 days. (E) t-CFU assays after the 12-day expansion in varying serum concentrations. (F) t-CFU and sc-CFU assays performed with mMSCs from Bl/6 mice. All strains were passage 7, except GFPtg, which was passage 4. Data are expressed as the mean ± SE, n = 3 to 5.

  • Figure 5.

    Differentiation of mMSCs in culture. mMSCs were incubated to confluency in CEM and then transferred to adipogenic or osteogenic medium for 21 days. Chondrogenesis was produced by incubating 200 000 mMSCs as a micromass pellet in chondrogenic media for 21 days. All mMSCs were passage 7, except GFPtg cells, which were passage 4. Original magnification, × 10.

  • Figure 6.

    Assays of CD34 and Sca-1 epitopes on mMSCs. Passage 7 mMSCs were incubated with antibodies for CD34 or Sca-1 and assayed by FACS. Each antibody was tested individually, and isotype controls were gated to less than 1%. Representative plots from 3 samples from each strain are shown.

Tables

  • Table 1.

    Summary of cell epitopes on mMSCs and human MSCs

    B1/6FVB/NBALB/cDBA1Human MSCs14
    Hematopoietic markers
        CD11b - - - - -
        CD34 +++ ++ + + -
        CD45 - - - - -
        Sca-1 (ly-6A/E) +++ +++ - + NA
        CD106 (VCAM-1) ++ ++ + + ++
    Endothelial markers
        Flk1 (VEGF-R2) - - - - ++
        CD31 (PECAM) - - - - -
        CD90 (Thy1)* - - - - +++
        CD117 (C-kit) - - - - -
        Lineage panel (CD11b, Ter-119, CD45R/B220, Ly-6G and Ly-6C, CD3e) - - - - NA
    • mMSCs were analyzed by FACS, as in Figure 6. Each antibody was tested individually, and isotype controls were gated to less than 1%. Samples were scored as - if less than 2% of the cells were positive, + if more than 2% and less than 20% were positive, ++ if more than 20% and less than 75% were positive, and +++ if more than 75% were positive. NA indicates not available.

    • * For B1/6, antibody CD90.2 (Thy1.2) was used. For all other strains, antibody CD90.1 (Thy1.1) was used.