Journal: EMBO Molecular Medicine
Article Title: Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles--a role of the endocytic-exocytic pathway
Figure Lengend Snippet: Association of prominin-1, but not CD34, with lipid raft-membrane vesicles released by HSPCs A. One-week-old co-culture HSPC/MSC conditioned medium was subjected to differential centrifugation for 5 min at 300 × g , 20 min at 1200 × g , 30 min at 10,000 × g , 60 min at 200,000 × g and 60 min at 400,000 × g . The resulting pellets were analysed by immunoblotting for either prominin-1 (top panel, arrow) or CD34 (bottom panel, arrowhead). Proteins in the 400,000 × g supernatant (Sup.) were analysed in parallel. B. The 200,000 × g pellet recovered after differential centrifugation (panel A) was subjected to equilibrium sucrose gradient (0.1–1.2 M) centrifugation. Equal volumes of the recovered fractions and the pellet were analysed by immunoblotting for prominin-1 (arrow). C,D. Negative EM analysis of the 200,000 × g pellet revealed the presence of small membrane vesicles (∼40–80 nm; arrows) and larger dense structures (∼200–600 nm; arrowhead). E. Negative staining EM of prominin-1 immunogold-labelled membrane vesicles recovered in the 200,000 × g pellet. Scale bars, 100 nm [(C–E)]. F. Haematopoietic cells (Cells) growing for 1 week in the co-culture system and 200,000 × g pellets (200,000 × g pellet) recovered upon differential centrifugation (panel A) were lysed for 30 min at 4°C in either 0.5% Triton X-100 or Lubrol WX or without detergent (PBS) and centrifuged for 60 min at 100,000 × g . The resulting supernatants (S) and pellets (P) were analysed by immunoblotting for prominin-1 (arrows). G. Lipid composition analysis of prominin-1-CMV was performed by C 14 -acetate labelling of HSPCs co-cultured with MSCs followed by TLC analysis of either haematopoietic cells (Cells) or the released prominin-1-positive vesicles recovered by a paramagnetic isolation with mAb CD133 (prominin-1-CMV). Percentages of cholesterol (Ch) and sphingomyelin (SM) of the total lipid composition are indicated. •, sample loading point.
Article Snippet: Again, additional investigation is urged to reveal whether prominin-1-CMV modify the biochemistry of the recipient MSCs (Freund et al, ) as it was elegantly proposed upon the direct contact of HSPCs with osteoblasts (Gillette et al, ).
Techniques: Co-Culture Assay, Centrifugation, Negative Staining, Cell Culture, Thin Layer Chromatography, Isolation