Figure 4. Regulation of HSC maintenance by nestin⁺ MSCs.

a, Expression and regulation of core HSC maintenance genes by CD45⁻ Nes-GFP⁺ cells. Q-PCR for Cxcl12, stem cell factor/kit ligand (Kitl), angiopoietin-1 (Angpt1), interleukin-7 (Il7), vascular cell adhesion molecule-1 (Vcam1) and osteopontin (Spp1) in CD45⁻ Nes-GFP⁺ and CD45⁻ Nes-GFP⁻ cells sorted from the bone marrow of mice injected with G-CSF or vehicle (n = 6). b–k, Bone marrow (BM) and spleen nucleated (b, f), Lin⁻ CD48⁻ (c, g), CD48⁻ LSK (d, h) and CD150⁺CD48⁻ LSK (e, i) cells 3–16 days after tamoxifen and diphtheria toxin administration in Nes-cre^ERT2/iDTR double- and control iDTR single-transgenic mice (n = 6–12). j, Long-term culture-initiating cell assay using limiting dilutions of bone marrow cells from Nes-cre^ERT2/iDTR (red) or control iDTR mice (black) 1 month after tamoxifen and diphtheria toxin treatment; the percentage of culture dishes in each experimental group that failed to generate colony-forming units is plotted against the number of test bone marrow cells; bone marrow HSC frequencies are indicated; Pearson chi-squared test (n = 4–6). k–m, Nestin⁺ cells are required for the homing of haematopoietic stem and progenitor cells. k, Bone marrow homing of haematopoietic progenitors (CFU-C) in tamoxifen- and diphtheria-toxin-treated Nes-cre^ERT2/iDTR and control iDTR mice (n = 4–8). l, m, HSCs rapidly home near GFP⁺ cells in the bone marrow of Nes-Gfp transgenic mice. l, Average shorter distances between bone marrow HSCs, Nes-GFP⁺ cells and the bone surface 2 h (n = 16), 48 h (n = 30) and 96 h (n = 14) after HSC transplantation into lethally irradiated mice. m, Representative DyD-stained (red) HSC, Nes-GFP⁺ (green) cell and bone matrix (blue). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed t-test. All error bars indicate s.e.m.