American Society of Hematology

Figure 1

$Figure 1. Revealing the hierarchical organization of the murine mesenchymal compartment. (A-C) Representative flow-cytometry plots of mesenchymal cell (MC) cultures from passage 5 (P5). (A) Bulk mesenchymal cultures contain RS1-like cells. (B) SSEA-1 is expressed by some mesenchymal cells and this population is still heterogeneous for KDR and Trk expressions. (C) Hst/PY staining showing that the quiescent (G0) cell subpopulation is highly enriched for SSEA-1–expressing cells. (D) Identification of SSEA-1–expressing cells directly in bone marrow cells. Lineage-depleted BMMNCs were stained for Lin/CD45/CD31, SSEA-1, and Sca-1. Representative FACS-staining profile of cells derived from 2-week-old mice. The negative fraction for the expression of Lin/CD45/CD31 was gated (R1) and plotted for SSEA-1 and Sca-1 expressions. (E) Frequency variation of SSEA-1+ cells during mouse ontogeny. The frequency was calculated by multiplying the percentage of mesenchymal cells in BMMNCs (R1) with that of SSEA-1+ cells in the mesenchymal population (R2) obtained for each group of mice. The analysis was performed on pooled samples of BMMNCs from 8 (2 weeks old), 10 (4 weeks old), 6 (6 weeks old), and 3 (> 12 weeks old; n = 2) mice, respectively, for each group. (F) Expression of Oct-3/4 and Nanog determined by QRT-PCR in SSEA-1+ (S+) and SSEA-1− (S−) cells isolated from MC cultures and directly from the bone marrow (from R1 fraction); representative gel electrophoresis with GAPDH as the internal PCR control is shown. The relative mRNA expressions were obtained after normalizing the cycle threshold (CT) values from each gene with the internal PCR control, then using the ΔCT values from ES cells as a reference. All quantitative results shown are the mean of fold change (x) from 2 independent experiments, each performed in duplicate.$

Revealing the hierarchical organization of the murine mesenchymal compartment. (A-C) Representative flow-cytometry plots of mesenchymal cell (MC) cultures from passage 5 (P5). (A) Bulk mesenchymal cultures contain RS1-like cells. (B) SSEA-1 is expressed by some mesenchymal cells and this population is still heterogeneous for KDR and Trk expressions. (C) Hst/PY staining showing that the quiescent (G0) cell subpopulation is highly enriched for SSEA-1–expressing cells. (D) Identification of SSEA-1–expressing cells directly in bone marrow cells. Lineage-depleted BMMNCs were stained for Lin/CD45/CD31, SSEA-1, and Sca-1. Representative FACS-staining profile of cells derived from 2-week-old mice. The negative fraction for the expression of Lin/CD45/CD31 was gated (R1) and plotted for SSEA-1 and Sca-1 expressions. (E) Frequency variation of SSEA-1⁺ cells during mouse ontogeny. The frequency was calculated by multiplying the percentage of mesenchymal cells in BMMNCs (R1) with that of SSEA-1⁺ cells in the mesenchymal population (R2) obtained for each group of mice. The analysis was performed on pooled samples of BMMNCs from 8 (2 weeks old), 10 (4 weeks old), 6 (6 weeks old), and 3 (> 12 weeks old; n = 2) mice, respectively, for each group. (F) Expression of Oct-3/4 and Nanog determined by QRT-PCR in SSEA-1⁺ (S⁺) and SSEA-1⁻ (S⁻) cells isolated from MC cultures and directly from the bone marrow (from R1 fraction); representative gel electrophoresis with GAPDH as the internal PCR control is shown. The relative mRNA expressions were obtained after normalizing the cycle threshold (CT) values from each gene with the internal PCR control, then using the ΔCT values from ES cells as a reference. All quantitative results shown are the mean of fold change (x) from 2 independent experiments, each performed in duplicate.

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