Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles—a role of the endocytic–exocytic pathway

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co-culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome-mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin-1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol-binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein–lipid assemblies might be the essential structural determinant in the release process of prominin-1. Collectively, these data support the concept that prominin-1-containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.

2 resulting pellets were resuspended in Laemmli buffer and analyzed by immunoblotting.
An aliquot (1/10 th ) of the 400,000 x g supernatant was analyzed in parallel.

Sucrose gradient centrifugation
The 200,000 x g pellet was resuspended in 30 µl of PBS plus 1 mM CaCl 2 , 0.5 mM MgCl 2 and protease inhibitors (Complete, Roche Diagnostics), placed on top of a equilibrium sucrose gradient (0.1-1.2 M) and centrifuged at 65,000g for 5 hours, as described previously (Huttner et al, 1983). After centrifugation, 500 µl fractions were collected from the top to the bottom of the gradient using a pipette whereas the pellet was resuspended in 500 µl buffer A (150 mM NaCl, 2 mM EGTA, 50 mM Tris-HCl pH 7.5, 10 µg ml -1 aprotinin, 2 µg ml -1 leupeptin and 1 mM PMSF) containing 1% Triton X-100. 450 µl aliquots of each fraction were concentrated using methanol/chloroform (2:1) precipitation and analyzed by immunoblotting. The remaining 50-µl aliquot of each fraction was used to determine the sucrose concentration by measuring the refractive index.

Detergent lysis
HSPCs and the 200,000 x g pellet recovered after differential centrifugation were lysed for 30 min on ice in 60 µl or 30 µl, respectively, of ice-cold buffer A containing either 0.5% Triton X-100 or Lubrol WX (Lubrol 17A17, Serva). Detergent lysates were centrifuged at 4°C for 1 hour at 100,000 x g. The entire supernatant and pellet were analyzed by SDS-PAGE followed by immunoblotting.

Immuno-isolation of prominin-1-containing membrane vesicles
The immuno-isolation of prominin-1-CMV was performed at 4°C using immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The conditioned medium collected from either HSPCs growing on MSCs (24 ml) or, as control, MSCs alone (12 ml) was centrifuged at 1,200 x g for 20 min to remove cells.
Supernanants were concentrated to 400 µl by Centricon PL-30 (Millipore) and 400 µl of ice-cold PBS were added prior centrifugation at 10,000 x g for 30 min. The 10,000 x g supernatants were pre-incubated with 150 µl of goat-anti-mouse IgG-magnetic-beads (Miltenyi Biotec; per 12 ml of initial starting material for 1.5 hours at 4°C end-over-end. The supernatants were then applied to ice-cold PBS conditioned MS-columns (Miltenyi Biotec) placed into a magnetic field, and the flowthrough material was collected. Half of the HSPC/MSC medium fraction and the control fraction were then incubated each with 50 µl of mouse mAb CD133-magnetic-beads (Miltenyi Biotec;. The other half of HSPC/MSC medium fraction was incubated with 150 µl of goat anti-mouse IgG-magnetic-beads as an additional control. After 1 hour incubation at 4°C end-over-end, samples were subjected to a magnetic separation. The MS-columns were washed 3 times with 3 ml ice-cold PBS. Vesicles retained in the MS-columns were eluted by taking out the column from the magnet, and flushing with 100 µl PBS. Laemmli buffer (4x) was added and the samples were stored at -20°C. The recovered flow-through fractions were centrifuged at 200,000 x g for 1 hour to obtain the unbound materials. The pellets of the 200,000 x g centrifugation were resuspended in Laemmli buffer (1x) and stored at -20°C.

Radioactive labelling, lipid extraction and thin layer chromatography
6-days-old HSPCs cultured on MSCs plated on Petri dishes were collected and centrifuged at 1,200 x g for 5 min. Recovered cells were resuspended and replated onto the same MSCs in fresh HSPC medium containing 10 µCi C 14 / 100-mm Petri dish. The cells were cultured at 37°C in a humidified 5% CO 2 atmosphere for 3 additional days.
Lipids from hematopoietic cells and immunomagnetic-isolated prominin-1-CMV (see above) were extracted as follows: cells and the prominin-1-containing vesicle fractions were each put into 2 ml of MeOH/CHCl 3 (2:1), mixed, and centrifuged for 5 min at 3,200 x g. The supernatants were transferred to a fresh tube, to which 0.5 ml of 20 mM acetic acid and 0.5 ml of CHCl 3 were added, mixed and centrifuged again for 2 min at 3,200 x g. The lower phase was transferred to another tube, while 25 µl of 1 M citric acid and 1 ml of CHCl 3 were added to the upper phase, mixed and centrifuged for 2 min at 3,200 x g. The lower phase was transferred to the same tube as the previous lower phase and the CHCl 3 was evaporated under a stream of nitrogen.
Dried lipids were dissolved in MeOH/CHCl 3 (1:2) and applied to silica TLC plates (Merck) using a capillary. To separate the lipids, the plates were run in chloroform/ethanol/water/triethylamine (35/50/10/35) until 2/3 of the distance, then the plates were dried and subsequently placed and run in iso-hexane/ethylacetate (5:1) for the full distance and dried again. TLC plates were exposed to image plates (Fuji), which were analyzed by the BAS 1800 II phosphoimager after 14 days. Bands were identified by comparison with lipid standards and quantified using ImageGauge V4.23.
In the case of cell surface versus intracellular labelling, cells were first cell surface labelled with mAb CD133/1 (1 µg/ml) in the cold prior to 4% PFA fixation, as described previously (Corbeil et al, 1999), followed by the saturation of remaining mouse epitopes with unconjugated AffiniPure rabbit-α-mouse IgG (H+L) Fab fragment. Afterwards, cells were post-PFA-fixed, saponin-permeabilized, and finally labelled with mAb CD133/1 as described above for anti-CD63. As controls, no signal was observed when either anti-CD133 and anti-CD63 primary antibodies were omitted or only the CD133 immunoreactivity was detected when the second primary antibody was omitted, indicating that the first mouse primary antibody was fully saturated (data not shown).
When primary antibodies were derived from different species, fixed, saponin- In all cases, nuclei were labelled with either Hoechst 33258 (Invitrogen, Karlsruhe, Germany) or 4,6-diamidino-2-phenylindole (DAPI; 1 µg/ml; Molecular Probes). The cells were mounted in Mowiol 4.88. Images were captured using a Leica SP5 upright confocal microscope or a Zeiss LSM 510 META. Settings were such that photomultipliers were within their linear range and the same optical section thickness was 6 set for all channels. Individual sections (0.5/1.0 µm interval) or a composite of 10/16 optical sections are shown. The images shown were prepared from the confocal data files using Adobe Photoshop software. Ratio of intracellular versus surface immunofluorescence of prominin-1 was calculated from total image pixel intensity using CellProfiler 2.0 software (Broad Institute, Cambridge, MA).

Immunoelectron microscopy
Membrane vesicles -The 200,000 x g pellet obtained after differential centrifugation (see above) of 12 ml conditioned HSPC/MSC medium was resuspended with 1 ml PBS containing 1 mM CaCl 2 , 0.5 mM MgCl 2 and protease inhibitors (Complete, Roche Diagnostics) and re-centrifuged at 200,000 x g. The resulting pellet was resuspended in 40 µl of 4% PFA in phosphate buffer. The suspension was applied for 2 min onto 400mesh grids with formvar-and carbon-coating prior to washing with PBS. The samples were then blocked for 5 min with 0.1% glycin in PBS and twice for 10 min with 0.2% gelatine, 0.5% bovine serum albumin in PBS (PBG). The grids were incubated for 1 hour at RT with either mAb AC141 (0.46 mg/ml, Miltenyi Biotec) directed against human prominin-1 (Yin et al, 1997) or mAb PeliCluster CD63 (1:15, Sanquin) and after washing with PBG, samples were incubated for 1 hour with goat anti-mouse IgG coupled to 10nm gold (British Biocell). After subsequent washing steps with PBG and PBS the grids were post-fixed for 5 min in 2% glutaraldehyde (GA) in PBS. The samples were negatively contrasted with 0.15% uranyl acetate in 1.9% methylcellulose and viewed in a Morgagni electron microscope (FEI Company). Micrographs were taken with a MegaviewII camera and AnalySIS software (Soft Imaging Systems).
HSPCs -HSPC/MSC co-cultures grown for 5 days on Transwell filters were gently washed with PBS and fixed with 4% PFA in PBS for 15 min at RT. Filters were then rinsed with PBS and incubated for 10 min in 50 mM NH 4 Cl. The fixed cells were permeabilized and blocked with 0.2% saponin, 2% FCS in PBS (blocking solution) for 15 min at RT. Cells were incubated for 30 min at RT with mAb CD133/1 (1 µg/ml) followed by goat anti-mouse secondary antibody coupled to ultra-small gold (Aurion, Wageningen, The Netherlands) in blocking solution. Filters were rinsed sequentially with blocking 7 solution and PBS followed by fixation in 2% GA. The samples were rinsed in water and processed for silver enhancement using R-GENT SE-EM silver enhancement kit (Aurion) for 90 min at RT prior to post-fixation in 1% osmium tetroxide for 15 min at RT.
Dehydration was performed by a graded series of ethanol. Samples were infiltrated in EMBed resin (Science Services). 70-nm ultrathin sections were cut on a UCT ultramicrotome (Leica Microsystems) and post-stained with uranyl acetate and lead citrate.

Flow cytometry
HSPCs co-cultured on MSCs on fibronectin-coated Petri dishes for either 4, 9 or 14 days were recovered by centrifugation at 300

Internalization of prominin-1-containing membrane vesicles
The materials recovered in 200,000 x g fraction were resuspended in 300 µl of PBS, and incubated with 1,0 µM 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO; Vybrant Cell-Labeling Solution, Molecular Probes) for 15 min at 37ºC. Afterward, DiO-labelled vesicles were immuno-isolated based on prominin-1 as described above. Isolated DiOlabelled prominin-1-CMV were then incubated with co-cultured HSPCs/MSCs growing on fibronectin-coated coverslips either for 1 or 6 hours. Samples were processed for immunofluorescence for SARA. Cells were observed using a Leica SP5 upright confocal microscope.   PFA-fixed, saponin-permeabilized HSPCs growing on MSCs were doubleimmunolabelled for prominin-1 (red) with either GM130 or TGN46 (both in green) followed by appropriate secondary antibodies. Lipid droplets and nuclei were visualized by BODIPY (green) and Hoechst (blue) staining, respectively. Labelled cells were analyzed using a confocal laser-scanning microscope. Single optical x-y-plane confocal sections revealed that prominin-1 is not concentrated in either the cis-Golgi apparatus (GM130), the TGN (TGN46) or lipid droplets (BODIPY). DIC, differential interference contrast.