American Society of Hematology

Fig. 12.

$Fig. 12. Single-cell origin of muscle and endothelial differentiated progeny. / MPCs obtained after 20 cell doublings were transduced with MFG-eGFP. (A) MFG-eGFP retroviral vector. Shown are EcoRI andNcoI digestion sites. Also shown are probes that span the eGFP gene only (a) or the complete retroviral vector (b). (B) FACS-selected eGFP+ MPCs were cultured at 10 cells per well until approximately 4 × 107 cells was obtained. Then, 5 × 106 MPCs were frozen, and groups of 5 × 106 MPCs each were induced to differentiate to osteoblasts, chondrocytes, adipocytes, stroma, skeletal muscle, and endothelium. Western blot analysis for the different lineages is shown for 3 individual clones in Figures 6, 7, 9, 10, and 11. DNA was extracted from 5 × 106 undifferentiated MPCs and 0.5 to 2 × 106 cells from osteoblast, chondrocyte, adipocyte, stroma, skeletal muscle, and endothelium differentiation cultures. Following digestion overnight with EcoRI, fragments were separated by gel electrophoresis, blotted to (+) nylon, and probed with a biotin-labeled eGFP-cDNA probe (a). Detection was by ECL. A single retroviral insert was detected in clone no. 3 (arrow), which was also present in cells recovered from osteoblast, chondrocyte, adipocyte, skeletal muscle, and endothelium differentiation cultures (representative of 3 clones). (C-D) Cultures were established in 96-well plates with 95 untransduced MPCs and 5 MFG-eGFP–transduced MPCs. Cultures were expanded to 2 × 107 cells; 5 × 106 MPCs were frozen; and groups of 5 × 106 MPCs were each induced to differentiate to skeletal muscle and endothelium. DNA was extracted from 5 × 106 undifferentiated MPCs and from 0.5 and 1 × 106 cells from myoblast and endothelium differentiation cultures. Following digestion overnight withNcoI, fragments were separated by gel electrophoresis, blotted to (+) nylon, and probed with a biotin-labeled MFG-eGFP-cDNA probe (b). Detection was by ECL. A single retroviral insert was detected (clone 2A3; 2 arrows indicate both retroviral fragments), which was also present in cells recovered from the myoblast and endothelium differentiation cultures (C). A fraction of the cells were fixed and stained with antibodies against fast-twitch myosin and vWF and examined by confocal microscopy for costaining of these markers with eGFP (D). A representative example of 2 clones is shown.$

Single-cell origin of muscle and endothelial differentiated progeny.

MPCs obtained after 20 cell doublings were transduced with MFG-eGFP. (A) MFG-eGFP retroviral vector. Shown are EcoRI andNcoI digestion sites. Also shown are probes that span the eGFP gene only (a) or the complete retroviral vector (b). (B) FACS-selected eGFP⁺ MPCs were cultured at 10 cells per well until approximately 4 × 10⁷ cells was obtained. Then, 5 × 10⁶ MPCs were frozen, and groups of 5 × 10⁶ MPCs each were induced to differentiate to osteoblasts, chondrocytes, adipocytes, stroma, skeletal muscle, and endothelium. Western blot analysis for the different lineages is shown for 3 individual clones in Figures 6, 7, 9, 10, and 11. DNA was extracted from 5 × 10⁶ undifferentiated MPCs and 0.5 to 2 × 10⁶ cells from osteoblast, chondrocyte, adipocyte, stroma, skeletal muscle, and endothelium differentiation cultures. Following digestion overnight with EcoRI, fragments were separated by gel electrophoresis, blotted to (+) nylon, and probed with a biotin-labeled eGFP-cDNA probe (a). Detection was by ECL. A single retroviral insert was detected in clone no. 3 (arrow), which was also present in cells recovered from osteoblast, chondrocyte, adipocyte, skeletal muscle, and endothelium differentiation cultures (representative of 3 clones). (C-D) Cultures were established in 96-well plates with 95 untransduced MPCs and 5 MFG-eGFP–transduced MPCs. Cultures were expanded to 2 × 10⁷ cells; 5 × 10⁶ MPCs were frozen; and groups of 5 × 10⁶ MPCs were each induced to differentiate to skeletal muscle and endothelium. DNA was extracted from 5 × 10⁶ undifferentiated MPCs and from 0.5 and 1 × 10⁶ cells from myoblast and endothelium differentiation cultures. Following digestion overnight withNcoI, fragments were separated by gel electrophoresis, blotted to (+) nylon, and probed with a biotin-labeled MFG-eGFP-cDNA probe (b). Detection was by ECL. A single retroviral insert was detected (clone 2A3; 2 arrows indicate both retroviral fragments), which was also present in cells recovered from the myoblast and endothelium differentiation cultures (C). A fraction of the cells were fixed and stained with antibodies against fast-twitch myosin and vWF and examined by confocal microscopy for costaining of these markers with eGFP (D). A representative example of 2 clones is shown.

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