Isolation of FGFR1/2⁺ skeletal cells in vitro. Plastic adherent skeletal cell preparation from 6- to 8-week-old C57Bl/6 whole-bone digest could be expanded greatly by FGF-2 stimulation in a dose-dependent manner (A). (B) Expansion capacity of whole bone–derived cells in the presence of FGF-2, EGF, TGF-β, and PDGF alone or in combination. Minimal expansion was observed when FGF-2 was absent. Data were calculated from growth curves generated for all conditions using the same initial cell number and harvest conditions. FGFR expression profile of whole bone–derived cells expanded with FGF-2 (98% CD11b⁻/CD31⁻/CD45⁻ by FACS, not shown) at passages 2-5 was analyzed by RT-qPCR, and PCR amplification products were loaded on agarose gel for visualization (passage 5 is shown; C). Liver, brain, and spleen extracts were used as positive controls for the primers (not shown). (D) Relative quantification of the expression of various growth factor receptors expressed by MSCs in vitro as analyzed by RT-qPCR. Cells isolated by whole-bone crush were seeded on coverslips at passage 2 and analyzed by immunofluorescence and confocal microscopy (E). Three-dimensional reconstructions (without deconvolution) of Z-stacks 10-15 μm in depth taken at 0.3-μm intervals are shown. Most cells expressed FGFR1 at various levels, but FGFR2c was limited to rare FGFR1⁺ cells. FGFR1/2⁺ cells typically expressed mesenchymal progenitor cell markers such as PN, PTHrP, CD90, CD73, and CD105. Only rare cells expressed osteoblast (osteocalcin, OC), hematopoietic (Lin), or endothelial (CD31) cell markers (see also supplemental Figures 2 and 3). Scale bars indicate 50 μm.