AML-MSCs characterization in vitro in 2D culture. (A) Alizarin red S staining (i) and quantification by ImageJ (ii) in h-MSCs (n = 3) and AML-MSCs (n = 8) after 7, 14, and 21 days of culture with or without (namely control medium) osteogenic differentiation medium; ×10 magnification; scale bar, 200 µm. (B) HUVEC tube formation assay. h-MSCs (n = 7) or AML-MSCs (n = 10) were stimulated (st) with a proinflammatory cytokine cocktail (hIL-1β, hIL-6, and hTNF-α) or not (unst) for 24 hours; after medium washout, new fresh medium was added for 18 hours, becoming conditioned medium (CM). CM or basal medium was used to culture HUVECs, and tube formation was evaluated after 4 hours; ×10 magnification; scale bar, 150 µm. (C) Quantification (%) of PHA-stimulated CD3⁺ T cells expressing CD69 and CD25 after 72 hours of coculture with h-MSCs (n = 15) and AML-MSCs (n = 10), relative to stroma-free conditions (SF, without MSCs, n = 6). (D) Cell proliferation in 2D was measured in AML-MSCs (n = 11) and h-MSCs (n = 5) by ATP cell proliferation assay. (E) Telomere length, expressed as base pair (bp), evaluated on h-MSCs (n = 3) and AML-MSCs (n = 10). (F-H) Percentage of CD34⁺ progenitor cells (gated on CD45⁺ and side scatter [SS] low) (F), CD34⁺CD33⁻ (G), and CD34⁺CD33⁺ (H) cells when cocultured with h-MSCs (n = 8) or AML-MSCs (n = 9) after 10 days of cocultures in 2D or without MSCs (stroma free [SF], n = 5). (I) Serial replating of CD34⁺ cells cocultured with h-MSCs or AML-MSCs (n = 8) or without MSCs (stroma free [SF], n = 6) for 10 days. (J) Cell density of murine IL-3–dependent 32D cell line cultured in 2D with AML-MSCs (red line, n = 6), in the presence of IL-3 (black dotted line, n = 6) or without IL-3 (blue line, n = 8), compared with 32D cell line cocultured with h-MSCs (green line, n = 6). *P < .05, **P < .01, ***P < .001, ****P < .0001.