Figure 5
From: Bone marrow stromal cell-derived hepcidin has antimicrobial and immunomodulatory activities
Hepcidin-producing BMSCs are in close proximity to hematopoietic progenitors in both mouse and human BM. Mouse (A–D) and human (E) BM sections are shown demonstrating the close proximity of hepcidin-producing BMSCs to hematopoietic precursors in both species. 16 µm thick sections were cut in a cryostat following decalcification of femurs from Nestin-GFP mice. (A) GFP in these mice labels BMSCs that support hematopoesis in the BM. (B) Hepcidin was visualized using a specific antibody (Abcam 81289) at 1:100 at 4 °C overnight followed by an anti-rabbit Alexa 594 (1:1000). (C) Hematopoietic stem cells (HSCs) were labelled using a biotinylated antibody to c-kit (BAF1356, R&D, 1:500) and developed the staining with a streptavidin-conjugated far red (Alexa-547, 1:1000). The sections were imaged with a Leica DMI6000 inverted fluorescence microscope. A Z-stack was taken at 0.5 µm intervals and the image was deconvoluted by the LAX software. Arrows point at double labelled Nestin/Hepcidin BMSCs, while arrowheads point at c-kit positive HSCs localized next to hepcidin-positive BMSCs. The star points to the lumen of a sinusoid in the BM. (D) shows the overlay of the three colors and the cell nuclei are stained with DAPI (blue). (E) Similar area from a human BM, where leptin receptor-positive BMSCs (green) also produce hepcidin (red) and are in very close proximity to CD34 -positive hematopoietic stem and progenitor cells (HSPC)s (white-far red, some pointed out by arrows). The lumen of the sinusoids is indicated with stars. This section was imaged by spectral imaging performed on a Nikon A1R + confocal microscope equipped with an A1-DUS 32 channel spectral detector. Scale bars: 50 µm.
