Retinoic acid impairs the hematopoietic stem cell niche function of BMSCs. (A) Schematic of experimental design for in vivo HSC niche function after ATRA treatment. Wild-type CD45.2⁺ mice, pretreated with vehicle or ATRA, consecutively 6 times, were lethally irradiated and transplanted with 1 × 10⁶ CD45.1⁺ BMNCs for recovery. After 8 weeks of recovery, 1 × 10⁶ recovered CD45.1⁺ BMNCs were mixed with 2 × 10⁵ fresh CD45.2⁺ BMNCs for competitive reconstitution analysis. (B-C) The percentage (i) and absolute numbers (ii) of CD45.1⁺ donor-derived hematopoietic stem and progenitor cells (HSPCs) (B) and cell cycle of engrafted CD45.1⁺HSCs (C) in the BM after an 8-week recovery in recipients pretreated with vehicle or ATRA, as indicated; n = 6 mice per group. (D) Engraftment analysis of total engrafted CD45.1⁺ donor cells (Total), B cells (B220⁺), T cells (CD3⁺), and myeloid cells (Gr1⁺Mac1⁺) at indicated weeks after transplantation; n = 3 donor mice per group and 6 recipient mice per group. (E-F) The percentage (E) and absolute numbers (F) of CD45.1⁺ donor-derived HSPCs in the BM of recipients at 4 weeks after secondary transplantation; n = 5 mice per group. (G-H) Quantitative polymerase chain reaction (qPCR) analysis of niche factors in sorted CD45^–Ter119^– stromal cells (G) and PDGFRα⁺ BMSCs (H) from control and ATRA-treated mice, respectively; n = 4 mice per group. (I) Schematic of experimental design for Scf-GFP mice treated with ATRA or control vehicle (i); absolute numbers (ii; Ctrl mice n = 8, ATRA-treated mice n = 6), and GFP mean fluorescence intensity (iii, n = 3 mice) of CD45^–Ter119^–CD31^–Scf-GFP⁺ cells in Scf-GFP mice treated with vehicle or ATRA, as indicated. (J) Schematic of experimental design for HSC in vitro expansion when cocultured with BMSCs pretreated with vehicle or ATRA, as indicated. (K) Absolute numbers of in vitro–expanded LT-HSCs, as indicated; n = 3 biologically independent replicates. (L) Engraftment analysis of total engrafted CD45.1⁺ donor cells (Total), B cells (B220⁺), T cells (CD3⁺), and myeloid cells (Gr1⁺Mac1⁺) at indicated weeks after transplantation; n = 3 donor mice and n = 5-6 recipient mice per group. (M) Schematic of experimental design for HSC cotransplantation with BMSCs sorted from control or ATRA-pretreated mice, as indicated; as seen in panels N to P. (N) Representative images of LepR-Cre; tdTomato⁺ intrafemur-injected BMSCs in the BM at 2 weeks after injection; Tomato (red), CD150 (green), and lineage/CD48 (blue) (scale bar, 15 µm). (O) The percentage (i) and absolute numbers (ii) of engrafted CD45.1⁺ donor-derived HSPCs in recipients at 8 weeks after transplantation (n = 4–5 mice). (P) The recovery of hematopoietic mononuclear cells in the peripheral blood of recipients at 4 weeks after transplantation. Error bars indicated mean ± SD. Two-tailed Student t tests were used to assess statistical significance (B, E, F, G, H, I, and O). *P < .05; **P < .01; ***P < .001. Repeated-measures 2-way ANOVA (C, D, L, and P) followed by Dunnett’s test for multiple comparisons, ^‡P < .05; ^‡‡P < .01; ^‡‡‡P < .001; ^‡‡‡‡P < .0001. LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP, multipotent progenitor.