Impact of simulated microgravity on oligodendrocyte development: implications for central nervous system repair.

We have recently established a culture system to study the impact of simulated microgravity on oligodendrocyte progenitor cells (OPCs) development. We subjected mouse and human OPCs to a short exposure of simulated microgravity produced by a 3D-Clinostat robot. Our results demonstrate that rodent and human OPCs display enhanced and sustained proliferation when exposed to simulated microgravity as assessed by several parameters, including a decrease in the cell cycle time. Additionally, OPC migration was examined in vitro using time-lapse imaging of cultured OPCs. Our results indicated that OPCs migrate to a greater extent after stimulated microgravity than in normal conditions, and this enhanced motility was associated with OPC morphological changes. The lack of normal gravity resulted in a significant increase in the migration speed of mouse and human OPCs and we found that the average leading process in migrating bipolar OPCs was significantly longer in microgravity treated cells than in controls, demonstrating that during OPC migration the lack of gravity promotes leading process extension, an essential step in the process of OPC migration. Finally, we tested the effect of simulated microgravity on OPC differentiation. Our data showed that the expression of mature oligodendrocyte markers was significantly delayed in microgravity treated OPCs. Under conditions where OPCs were allowed to progress in the lineage, simulated microgravity decreased the proportion of cells that expressed mature markers, such as CC1 and MBP, with a concomitant increased number of cells that retained immature oligodendrocyte markers such as Sox2 and NG2. Development of methodologies aimed at enhancing the number of OPCs and their ability to progress on the oligodendrocyte lineage is of great value for treatment of demyelinating disorders. To our knowledge, this is the first report on the gravitational modulation of oligodendrocyte intrinsic plasticity to increase their progenies

Simulated microgravity increases the number of DNA-synthesizing hOPCs.

<p>Cultures of hOPCs were treated during 24 and 48-four hour pulses of 10 µM bromo-deoxyuridine (BrdU) were done during the 0G treatment as well as in parallel untreated cultures. After each BrdU pulse, cells were fixed and immunostained with anti-BrdU. (<b>A</b>) Microphotographs showing BrdU positive cells at 24 and 48 h. green: BrdU immunostaining. Scale bar = 50 µm. (<b>B</b>) The percentage of BrdU positive cells in each experimental condition was compared with respective controls. Values are expressed as mean ± SEM of two independent experiments. ***p<0.001 versus control.</p

Simulated microgravity promotes the extension of the leading process in OPCs.

<p>(<b>A</b>) Mixed glial cultures of PLP-GFP labeled OPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal microscope. Pictures show examples of living GFP-labeled OPCs that were imaged for a period of 26 h. Scale bar = 50 µm. In the insert, a single migrating OPC is shown, small yellow arrowheads point at the leading process and the cell soma is shown by the large arrowhead. Scale bar = 50 µm (<b>B</b>) The average leading process length and soma area of migrating OPCs was calculated from at least 50 cells in each experimental condition. Values are expressed as mean ± SEM of at least four independent experiments. *p<0.05, **p<0.01 versus control cells.</p

Simulated microgravity maintains the expression of early developmental markers in hOPCs.

<p>Cultures of hOPCs were treated during 24(<b>A</b>) Microphotographs showing Olig2 and Sox2 positive cells in control and 0G treated cultures. Scale bar = 50 µm. (<b>B</b>) The percentage of positive cells in each experimental condition was analyzed by confocal microscopy. Results are the means ± SEM for three independent experiments. *p<0.05, **p<0.01, versus respective control.</p

Simulated microgravity increases OPC migration.

<p>Mixed glial cultures of PLP-GFP labeled OPCs were imaged with a GFP filter at 6 min intervals for a period of 16 h. (<b>A</b>) Time-lapse series of OPCs from cultures treated during 24 h in 0G. Yellow arrowheads identify a migrating OPC. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. Cell migration speed and distances were analyzed off-line by tracing individual cells at different times, after which migratory values were statistically analyzed across the experimental conditions. (<b>B</b>) OPC migration speed was calculated from at least 60 cells in each experimental condition. (<b>C and D</b>) Total migration distance was followed for 12 h in 50 cells from each experimental condition and the percentage of migrating cells was calculated from the entire cell population. Values are expressed as mean ± SEM of at least four independent experiments. **p<0.01, ***p<0.001 versus control cells.</p

Microgravity increases OPC numbers and oligodendrocytes remained immature.

<p>Mixed glial cultures of PLP-GFP labeled OPCs were treated during 24, 48 and 72 h in 0G. (<b>A</b>) Yellow arrowheads define PLP-GFP/Sox2 double positive cells at 24, 48 and 72 h. Green: PLP-GFP, Red: Sox2 immunostaining. Scale bar = 50 µm. (<b>B and C</b>) After treatment, cells were fixed and immunostained for several oligodendrocyte stage markers and the percentage of positive cells in each experimental condition was analyzed by confocal microscopy. (<b>D</b>) Mixed glial cultures of PLP-GFP labeled OPCs were treated during 10, 15 and 30 days in 0G. After treatment, cells were fixed and immunostained for Olig1 and Olig2 and the percentage of positive cells in each experimental condition was analyzed by confocal microscopy. Results are the means ± SEM for three independent experiments. *p<0,05, **p<0,01 and ***p<0,001 vs. respective controls.</p

Proliferation of PLP-GFP-expressing OPCs is increased by simulated microgravity.

<p>Mixed glial cultures of PLP-GFP labeled OPCs were treated during 24, 48 and 72 h in 0G. Twenty-four hour pulses of 10 µM bromo-deoxyuridine (BrdU) were begun at 0 h, 24 h and 48 h. After each BrdU pulse, cells were fixed and immunostained with anti-BrdU and anti-NG2 antibodies. (<b>A</b>) Microphotographs showing NG2+/BrdU+ cells at 24, 48 and 72 h. Green: NG2 immunostaining, and Red: BrdU immunostaining. Scale bar = 50 µm. (<b>B and C</b>) The percentage of NG2+/BrdU+ and PLP-GFP+/BrdU+ cells in each experimental condition was compared with respective controls. Values are expressed as mean ± SEM of two independent experiments. *p<0.05, **p<0.01 versus respective control.</p

Cell cycle shortening in microgravity.

<p>Mixed glial cultures of PLP-GFP labeled OPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal microscope. GFP-labeled OPC clones were imaged with a specific GFP filter at 6 min intervals for a period of 26 h. (<b>A</b>) Time-lapse series of OPC∼GFP clones from cultures treated during 24 h in 0G. Yellow arrowheads designate cytokinesis events. Tracking of cells between birth cytokinesis and division cytokinesis was noted with a yellow asterisk near the cell, which was generated from frame to frame. Each frame represents a single section of a time lapse video sequence. Time is denoted in hours in the bottom right corner. Scale bar = 50 µm. (<b>B and C</b>) Estimated cell cycle times and percentage of mitotic OPCs for each experimental condition. Values are expressed as mean ± SEM of four independent experiments. *p<0.05, **p<0.01, versus respective control.</p

Like in mouse OPCs simulated microgravity shortens the cell cycle of human OPCs (hOPCs).

<p>hOPCs were incubated in a chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal inverted microscope. Cells were imaged at 6 min intervals for a period of 26 h. Estimated cell cycle times and percentage of mitotic hOPCs for control and 0G treated cultures are shown. Values are expressed as mean ± SEM of four independent experiments. *p<0.05, **p<0.01, versus respective control.</p