Agrin controls ST-HSC functions through the α-DG receptor. (A) Representative confocal images of LSK cells obtained from the BM of control mice and stained for α-DG (green), Sca-1 (light blue), c-kit (red), and nuclear DNA (blue). Scale bars, 5 and 10 μm. The graph shows α-DG MFI analysis on a representative LSK sample (ctrl neg, n = 20; α-DG, n = 45).(B) In vivo competitive reconstituting ability of sorted CD34⁺CD135⁻ LSK cells (CD45.2) pretreated with anti–α-DG blocking antibody IH6C or with isotype control or untreated. A mixture of 60% (CD45.2) in competition with 40% (CD45.1/45.2) untreated sorted CD34⁺CD135⁻ LSK cells was transferred into lethally irradiated recipients (CD45.1) together with 1 × 10⁵ Lin⁺ feeder BM cells from control adult mice mice (CD45.1). BM cells were analyzed by flow cytometry 8 days after the transfer; data are expressed as the relative percentage of CD45.²⁺ cells on total CD45.²⁺ plus CD45.1/45.²⁺ gated cells; n = 14, *P < .05, **P < .01. (C) Sorted control CD34⁺CD135⁻ LSK cells (45.1) were cultured on MSC monolayers from either control (ctrl) or Musk-L;Agrn^−/− mice, or, where indicated, control CD34⁺CD135⁻ LSK cells were pretreated with isotype or blocking antibody IH6C4 (+IH6C4) and cultured on control MSC monolayers. Proliferation of CD45.1⁺ cells was measured 5 days later. Data are shown as mean, n = 3; **P < .01, ***P < .0001. (D) Total BM cells from control mice were preincubated with α-DG blocking antibody IIH6C4, or its isotype control, for 40 minutes at room temperature. Cells were than stimulated with 10 μg/mL recombinant agrin (R&D) and the ERK phosphorylation was detected on gated CD34⁺CD135⁻ LSK cells. Changes in ERK phosphorylation were expressed as fold increase of mean fluorescence intensity (MFI) of stimulated over unstimulated cells (time 0); n = 3. Statistical analysis was performed comparing isotype versus IIH6C4 treated cells (**P < .01, ***P < .0001). In all panels, error bars represent SEM.