SSEA-1⁺ cells are at the apex of the murine mesenchymal compartment. (A) Purified SSEA-1⁺ (top left plot) and SSEA-1⁻ (bottom left plot) cells were plated and analyzed a few days later. Analysis of SSEA-1 expression on SSEA-1⁺ cultures after attaining half-confluency (i, n = 2) or confluency (ii, n = 3) and on SSEA-1⁻ cultures at confluency (iii, n = 3). (B) Schematic representation of the hierarchical organization of SSEA-1⁺ cells inferred from our studies. (C) Both enriched SSEA-1⁺ (R1) and SSEA-1⁻ (R2) fractions were first purified from CD45/Ter119-depleted BMMNCs and then cultured independently in the presence of hematopoietic (R3) cells (i). (ii-iii) Overlay FACS plots show the purity of each enriched fraction (black dots) with the supporting hematopoietic cells (gray dots) as initiating culture populations. (iv) Representative FACS plots of adherent culture initiated by enriched SSEA-1⁻ cells (R2-derived cultures; n = 5) showing the lack of the putative mesenchymal fraction (CD45/CD11b⁻ cells) at passage 0. In contrast, cultures initiated with SSEA-1⁺ cells (R1-derived cultures; n = 3) were able to propagate and the presence of these cells was detected in different cell passages.