Figure 1.
PDGFR⍺ and PDGFRβ expression identifies 4 subpopulations within Sca-1– CD51+ cells that differ in their potential to form CFU-F, mineralized osteoblasts, and adipocytes in culture. (A) Representative FACS plots show viable CD45–Ter119–CD31– cells with gates for Sca-1+CD51+ and Sca-1–CD51+ bone-derived cells and the expression of PDGFR⍺ and PDGFRβ within these populations. (B) Proportions of the PDGFR⍺+PDGFRβ–, PDGFR⍺+PDGFRβ+, PDGFR⍺–PDGFRβ+, and PDGFR⍺–PDGFRβ– cells in Sca-1+CD51+ and Sca-1–CD51+ (termed A, AB, B, and DN, respectively) populations (n = 45 mice pooled from >10 separate experiments). (C) Messenger RNA (mRNA) expression of Pdgfra and Pdgfrb in A, AB, B, and DN sorted cells. (D) The percentages of EYFP+ cells within the A, AB, B, and DN populations in each Cre reporter. Data are mean ± SD. Mice were pooled from separate experiments (Prrx1:EYFP, n = 7; Nes:EYFP, n = 6; Col2a1:EYFP, n = 3; Osx1:EYFP, n = 12; Dmp1:EYFP, n = 4). (E) Representative digital interference contrast photos of the different cell populations at 7 days of culture; images are cropped from pictures taken at original magnification, ×20. (F) A limiting dilution assay using ELDA (n = 24) determined the CFU-F frequencies (solid line) of A, AB, B, and DN cells; 95% confidence intervals are indicated by dashed lines. The table shows CFU-F frequencies for each population and a pairwise test for differences in CFU-F frequencies (significant differences are shown in bold). (G-N) Freshly sorted A, AB, B, and DN cells were seeded at 1000 cells per well into a 96-well plate and allowed to expand for 1 week to reach confluence, then cultured in osteogenic or adipogenic media for 14 days. Staining for minerals was performed on populations at day 14: Alizarin Red, A, AB, B, and DN cells (G-J); and Oil Red O, adipocytes (K-N). Original magnification for all images. ×40; all photos are representative of 5 osteogenic and 4 adipogenic independent experiments with 3 to 6 wells per population. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test: **P < .01; ***P < .001; ****P < .001. APC, allophycocyanin; Cy7, cyanine; PE, phycoerythrin.

PDGFR⍺ and PDGFRβ expression identifies 4 subpopulations within Sca-1– CD51+ cells that differ in their potential to form CFU-F, mineralized osteoblasts, and adipocytes in culture. (A) Representative FACS plots show viable CD45–Ter119–CD31– cells with gates for Sca-1+CD51+ and Sca-1–CD51+ bone-derived cells and the expression of PDGFR⍺ and PDGFRβ within these populations. (B) Proportions of the PDGFR⍺+PDGFRβ–, PDGFR⍺+PDGFRβ+, PDGFR⍺–PDGFRβ+, and PDGFR⍺–PDGFRβ– cells in Sca-1+CD51+ and Sca-1–CD51+ (termed A, AB, B, and DN, respectively) populations (n = 45 mice pooled from >10 separate experiments). (C) Messenger RNA (mRNA) expression of Pdgfra and Pdgfrb in A, AB, B, and DN sorted cells. (D) The percentages of EYFP+ cells within the A, AB, B, and DN populations in each Cre reporter. Data are mean ± SD. Mice were pooled from separate experiments (Prrx1:EYFP, n = 7; Nes:EYFP, n = 6; Col2a1:EYFP, n = 3; Osx1:EYFP, n = 12; Dmp1:EYFP, n = 4). (E) Representative digital interference contrast photos of the different cell populations at 7 days of culture; images are cropped from pictures taken at original magnification, ×20. (F) A limiting dilution assay using ELDA (n = 24) determined the CFU-F frequencies (solid line) of A, AB, B, and DN cells; 95% confidence intervals are indicated by dashed lines. The table shows CFU-F frequencies for each population and a pairwise test for differences in CFU-F frequencies (significant differences are shown in bold). (G-N) Freshly sorted A, AB, B, and DN cells were seeded at 1000 cells per well into a 96-well plate and allowed to expand for 1 week to reach confluence, then cultured in osteogenic or adipogenic media for 14 days. Staining for minerals was performed on populations at day 14: Alizarin Red, A, AB, B, and DN cells (G-J); and Oil Red O, adipocytes (K-N). Original magnification for all images. ×40; all photos are representative of 5 osteogenic and 4 adipogenic independent experiments with 3 to 6 wells per population. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test: **P < .01; ***P < .001; ****P < .001. APC, allophycocyanin; Cy7, cyanine; PE, phycoerythrin.

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