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First published online 13 June 2006
doi: 10.1242/jcs.03004

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Synergistic effects of CoCl₂ and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells

¹ UMR-CNRS 6185, Neurodegenerescence: models and therapeutic strategies, University of Caen, CYCERON, Bd Henri Becquerel, BP 5229, 14074 Caen CEDEX, France
² CERMN EA3915, Pharmacology and Physiology Department, University of Caen Basse-Normandie, 5 rue Vaubénard, 14032 Caen CEDEX, France

View larger version (63K): [in a new window]   Fig. 1. CoCl2 treatment provoked MSC differentiation into neuron-like cells. (A) MSC morphological changes observed after 72 hours of CoCl2 treatment by phase-contrast microscopy (objective 20x) and by fluorescence microscopy (objective 40x) after Rhodamine-labelled phalloidin staining (insets). (B) Real-time RT-PCR analyses of neuronal genes and osteogenic genes after 6 and 24 hours of CoCl2 treatment. Values are represented as fold change compared with control ± s.e.m., n=3, *P<0.05, univariate Student's t-test. (C) CoCl2-treated cells were stained for neuronal markers (nestin, Tuj1, NF200 and MAP2a,b) and nuclei were counterstained with Propidium Iodide (PI, in red). Photographs were obtained with confocal microscopy. Bars, 20 µm. (D) Quantification of nestin-, Tuj1- and MAP2ab-positive cells compared with total cells during CoCl2 treatment. *P<0.05, Student's t-test, CoCl2-treated cells were compared with control cells.

View larger version (40K): [in a new window]   Fig. 2. CoCl2 induced HIF-1 and its target gene expression in MSCs. (A) HIF-1 immunostaining observed in MSCs with confocal microscopy after 3 and 72 hours of 100 µM CoCl2 treatment. In the third image from the left, nuclei were counterstained with Propidium Iodide (PI, in red). Bars, 20 µm. (B) HIF-1 expression analysed by western blotting of cytoplasmic and nuclear extracts from MSCs after 3 hours of CoCl2 treatment. (C) Expression of HIF-1 target genes analysed by real-time RT-PCR after 6 and 24 hours of CoCl2 treatment, n=3, *P<0.05, univariate Student's t-test. (D) Western blot of EPO and VEGF expression after 72 hours of CoCl2 treatment. Actin expression was used as protein loading control. (E) MSC morphology observed after 72 hours of 100 µM CoCl2 treatment with or without 1.25 nM echinomycin (objective 20x). (F) Luciferase activity measurement after 36 hours of 100 µM CoCl2 ± 1.25 nM echinomycin treatment. Firefly luciferase activities were normalized with the internal control Renilla luciferase activity. The fold induction represents the luciferase activity observed in transfection with the HRE-pGL3SV40 plasmid relative to the empty plasmid (pGL3SV40). Values are represented as fold induction compared with control ± s.e.m., n=3, *P<0.05, PLSD Fisher.

View larger version (21K): [in a new window]   Fig. 3. CoCl2 decreased MSC proliferation. (A,B) BrdU incorporation of MSCs cultured with or without 100 µM CoCl2. BrdU was incorporated only for the last 2 hours of treatment. (A) BrdU immunostaining using DAB-nickel after 24 hours of treatment. (B) Percentage of BrdU-positive MSCs cultured with or without 100 µM CoCl2 after 6, 24 and 48 hours of treatment. Data shown are mean ± s.e.m. from three independent MSC preparations and from three different wells in each experiment. *P<0.05, Student's t-test, CoCl2-treated cells were compared with control cells. (C) Expression of cell-cycle-related genes was analysed by real-time RT-PCR after 6 and 24 hours of CoCl2 treatment, n=3, *P<0.05, univariate Student's t-test.

View larger version (77K): [in a new window]   Fig. 4. Y-27632 potentiated CoCl2 effect on MSC differentiation into neuron-like cells. (A,B) MSC morphological changes observed after 72 hours of Y-27632 (30 µM) and/or CoCl2 treatment by phase-contrast microscopy (A) and fluorescence microscopy (B) after Rhodamine-labelled phalloidin staining. (C) Effect of Y-27632 with or without CoCl2 on NSE, MAP2c and cyclin D1 expression, analysed by western blot of MSCs after 72 hours of treatment. Mouse primary-neuron-enriched cultures (11 days in vitro) were used as a positive control and actin was used as protein-loading control. (D,E) TH expression analysed by (D) immunostaining and (E) western blotting after 72 hours of Y-27632 treatment with or without CoCl2.

View larger version (16K): [in a new window]   Fig. 5. Effect of 72 hours of CoCl2 and/or Y-27632 treatment on Ca2+ influx in response to dopamine in MSCs. (A) Analysis of the percentage of cells responding to dopamine after 72 hours of CoCl2 and/or Y-27632 treatment. N=3, n=50-75 cells, *P<0.05, PLSD Fisher. (B,C) Effect of CoCl2 and/or Y-27632 treatment on the intensity of the Ca2+ influx in MSCs responding to dopamine. N=3, n=50-75 cells, *P<0.05, #P<0.0001, PLSD Fisher.

View larger version (12K): [in a new window]   Fig. 6. Electrophysiological properties of control MSCs and CoCl2/Y-27632 co-treated MSCs. Treatment with CoCl2 and Y-27632 induced a change in the currents recorded from MSCs. (A) Control cells showed typical inward-rectifying I-V relationship without any sign of voltage-dependent current at depolarizing potentials (n=3). Treated cells, by contrast, clearly exhibited outward-rectifying current similar to that found in neurons (n=10). (B) The outward-rectifying current encountered in CoCl2/Y-27632 co-treated cells was completely blocked by 10 mM TEA (n=3). Insert shows current traces obtained with a voltage step to +60 mV in the presence and absence of 10 mM TEA. Calibration bars, 250 pA 100 milliseconds.

View larger version (38K): [in a new window]   Fig. 7. Effect of Y-27632 on HIF-1 expression in MSCs with or without CoCl2 treatment. (A) HIF-1 immunostaining observed with confocal microscopy on MSCs treated with CoCl2 and/or Y-27632 for 3 hours. Bar, 20 µm. (B) HIF-1 western blot performed from nuclear extracts after 3 hours of CoCl2 and/or Y-27632 treatment. (C) Luciferase activity measurement after 3, 8, 24 and 36 hours of CoCl2 and/or Y-27632 treatment. Firefly luciferase activities were normalized with the internal control Renilla luciferase activity. The fold induction represents the luciferase activity observed in transfection with the HRE-pGL3SV40 plasmid relative to the empty plasmid (pGL3SV40). The values are represented as fold induction compared with control ± s.e.m., n=3, *P<0.05, #P<0.0001, PLSD Fisher.

View larger version (25K): [in a new window]   Fig. 8. Schematic representation of the effect of CoCl2 and Y-27632 treatments on MSC differentiation in neuron-like cells. Images of MSC morphology were obtained after Rhodamine-labelled phalloidin staining and observed by fluorescence microscopy. One arrow indicates an increase and two arrows, a larger increase.

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