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First published online 2 July 2003
doi: 10.1242/jcs.00639

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Regulation of neural markers nestin and GFAP expression by cultivated bone marrow stromal cells

S. Wislet-Gendebien1,*, P. Leprince1, G. Moonen1 and B. Rogister1,2

1 Center for Cellular and Molecular Neurobiology, University of Liège, 17 Place Delcour, B-4020 Liège, Belgium
2 Department of Neurology, University of Liège, C.H.U. (B35) Sart Tilman, B-4000 Liège, Belgium



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  		Fig. 1. Immunological and functional characterization of undifferentiated rSCs. FACS demonstrates that the cells are negative for CD11b (A) and CD45 (B), two lymphohematopoietic surface markers. Immunofluorescent labelling shows that rSCs are positive for Thy1.1 (C) and p75 NGF receptor (D). Adipocyte induction was revealed by accumulation of lipid vacuoles that were stained with Oil Red O (E). Chondrocytes were obtained by culturing rSCs as a pellet for 20 days in chondrocytic induction medium. Sections of the paraffin-embedded cells were stained with toluidine blue. The formation of cartilaginous structures is characterized by differentiated chondrocytes in lacunae surrounded by extracellular matrix (F). When rSCs were placed in osteocyte induction medium, cells formed nodules (G) with multi-layered regions, and a significant increase in alkaline phosphatase was observed (H). Enzyme activity of the cells was measured in triplicate cultures with the alkaline phosphatase colorimetric test. In this test, the variation of the absorbency was measured as a function of time at 405 nm. Scale bars: (C-E) 40 µm; (F,G) 150 µm.

 


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  		Fig. 2. Regulation of nestin expression by rSCs by the serum. rSCs were cultivated in different culture medium: DEM/F12+N2+B27 (A), DEM/F12 (B), DEM/F12+N2 (C) or DEM/F12+B27 (D) and DEM/10% FBS (E). Cultures were labeled with anti-nestin antibody (green) and cell nuclei were counterstained with ethidium homodimer (red). Scale bar: (A-E) 40 µm. Nestin expression is present in cells grown in all serum-free media, and this result was confirmed by western blotting (F). Radial glial cells (RGCs) were used as nestin-positive control and ß-actin was used as protein-loading control.

 


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  		Fig. 3. Regulation of nestin expression by rSCs, by the number of passages. rSCs express nestin weakly between passage 0 and passage 6 (A, rSCs passage 4). The expression increases between passage 6 and passage 10 (B, rSCs passage 10). Beyond passage 11, ~75% of rSCs express nestin in serum-free medium (C, rSCs passage 15). Cultures were labeled with nestin antibody (green labelling) and cell nuclei were counterstained with ethidium homodimer (red labelling). (D) The evolution of nestin expression as function of the number of passages of rSCs. This graphic was established from results of immunocytochemical analysis on passage 4, 6, 7, 8, 9, 11, 14 and 15. In this experiment, results are expressed as percentages of nestin-positive cells in each passage in serum-free condition (n=4, representing a minimum of 1500 counted cells in each passage). These results were confirmed by western blotting on passage 4 (P4), 10 (P10) and 15 (P15) (E). Radial glial cells (RGCs) were used as nestin-positive control and actin was used as protein-loading control. No alteration of the capacity of differentiation: (F) P10 adipocyte, (G) P18 adipocyte, (H) osteocytes can be observed even in the long-term cultures. Scale bar: (A-C,F,G) 40 µm.

 


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  		Fig. 4. Simultaneous nestin expression and aggregates formation. After 72 hours incubation in serum-free medium, the cells were trypsinised and resuspended in DEM/F12 with N2 and B27 for 24 hours. During that time, passage 15 cells aggregated to form clusters or aggregates resembling neurospheres (A), whereas passage 4 rSCs remained in suspension (B). Nestin-negative passage 15 cells immediately adhere on the culture dish (C). The spheres obtained in condition A were then placed on polyornithine for 5 days in DEM/F12, N2 and B27 (E) where they exhibited a different morphology than that observed in DEM/20% FBS medium (D). Nestin immunocytochemistry (green) reveals that only 15% of the cells still express nestin under these conditions (F). Scale bar: (A-F) 40 µm.

 


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  		Fig. 5. Effect of co-culture on neural differentiation. Heterogenous spheres of nestin-positive rSCs stained with DiD VybrantTM cell-labelling solution (red) (Molecular Probes) and GFP-positive NSCs (M.1,M.2) are placed on polyornithine-coated dishes for 5 days. Some rSCs have differentiated into GFAP-positive cells (indicated by arrowheads). GFAP (red) is expressed by a large fraction of cells (A), of which some also contained green fluorescent protein (B), and thus originate from mNSC. Triple labelling (including nuclei stained by EtD1 in blue) allow the identification of non-green rSCs that express GFAP (C). Co-culture of passage 4 nestin-negative rSCs with GFP-positive mNSC demonstrated that only a small percentage of GFAP-positive cells were derived from MSCs (D-F). Astroglial differentiation of passage 15 rSCs were confirmed with Glast marker (red) (G-I). Pure GFP-positive mNSC were used as a control to demonstrate that all GFAP (red)-positive cells from mNSC remain GFP-positive (green) (J-L). A double-labelling with M2 (blue) and GFAP (red) antibodies allows the confirmation of the mesenchymal origin of some GFAP-positive cells (O). rSC-derived GFAP-positive cells are not recognized by the M2 antibody. The GFP-positive astrocytes (green astrocytes) that differentiate from mNSC are recognized by the M2 antibody (N). rSCs, mNSC and co-cultured rSCs and mNSC were stained with propidium iodide and subjected to FACS analysis (P). Arrowheads in C,E,H,O indicate the mesenchymal-derived cells that express neural markers. Scale bars: (A,B,D,F,G,I,M.1) 150 µm; (C,E,H,J,K-M.2) 40 µm; (N,O) 60 µm.

 

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