The overall goal of this procedure is to screen small molecule libraries for agents that promote astroglial differentiation in glioblastoma stem cells. This method can help answer key questions in the cancer stem cell field, such as what are the signaling pathways that are required for maintaining glioblastoma stem cells in the undifferentiated state. Proper execution of this technique allows for the straightforward identification of small molecules that promote glioblastoma stem cell differentiation.
The implication of this technique extend toward therapy for Ehlers-Danlos glioblastoma because reducing the percentage of glioblastoma stem cells may decrease the likelihood of recurrence. This method can also be applied to setting differentiation into other cell lineages using other reporter constructs. Our report is the first to establish a high throughput approach to screen for agents that force glioma stem cells to differentiate.
Working with lentivirus particles in cancer cells derived from patients can be extremely hazardous. Therefore standard procedure for working with bloodborne pathogens should always be followed. To begin, seed one million glioblastoma stem cells or GSCs in two milliliters of complete neural stem cell growth medium in a six-well plate.
Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five. To the cell and virus mixture, add two microliters of polybrene such that the final concentration is eight micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5%CO2 overnight. After incubation, replace the growth medium to remove unbound virus.
And centrifuge cells at 360 times G for five minutes. Then carefully aspirate medium and plate cells back in a six-well plate. And continue to incubate the cells for 24 hours.
Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times G for five minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.
Place the plate back in the incubator to allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pippetting up and down gently.
To the dissociated cells add 800 microliters of HBSS to ensure minimal cell attachment or aggregation. Next, transfer 200 microliters of each cell suspension into wells of a 96-multiwell plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry.
Record at least 10, 000 viable cells in each acquisition. In all flow cytometric analysis use parental non-transduced cells or vector-transduced nonfluorescent cells to establish baseline fluorescence. To select and expand individual subclones seed cells at a density of 0.7 cells per well in 100 microliters of neural stem cell medium in a 96-well plate.
Maintain these clones in culture for 11 days. Look out for neurospheres with a diameter greater than 100 microns. We have previously established this cutoff size to retrospectively determine if the neuorsphere was originated by a self-renewing glioblastoma stem cell.
This cutoff size may need optimization when working with different glioblastoma cell models. Observe the cells under a fluorescence microscope equipped with a FITC filter and mark wells that contain a single neurosphere in which approximately one 1%to 5%of the cells are GFP-positive. After marking, expand individual clones until a sufficient number of cells is available for flow cytometry analysis.
Assuming the reporter clones contain 1%to 5%of GFP-positive cells, harvest 0.5 milliliters from each clone along with an equal quantity of non-transduced control cells to determine the exact percentage of GFP-positive cells. After pelleting cells at 360 times G for five minutes at room temperature, aspirate the supernatant. Add 200 microliters of dissociation reagent to each pellet and incubate the tubes in a water bath set to 37 degrees Celsius.
After incubation, triturate the cells about 20 to 30 times to achieve a single cell suspension. Then add 800 microliters of HBSS to ensure minimal cell attachment or aggregation. Now transfer 200 microliters of each cell suspension into wells of a 96-multiwell plate.
Finally, perform flow cytometry, recording at least 10, 000 viable cells in each acquisition. To assess the clonogenic capacity prepare single cell suspensions of differentiation reporter subclones by triturating the cells about 20 to 30 times. Then plate these cells in 100 microliters of complete neural stem cell growth medium at a density of about five to 500 cells per well in 96-multiwell plates.
Maintain these cells in culture for nine to 11 days. Look for neurospheres under a light microscope. A well containing at least one single large neurosphere is considered positive.
Considering parental non-transduced cells and GFP-positive lentivirus-transduced cells as controls input the total wells analyzed and the number of positive cells on the ELDA online interface. To begin screening, plate 5000 cells in 99 microliters of complete neural stem cell growth medium in a 96-well plate. Then with a 12-channel pipettor treat cells with two micromolar final concentration of diluted library compounds or with the DMSO control.
Incubate the cells for 72 hours. Now add 150 microliters of cell dissociation reagent using a 12-channel pipettor in each well and incubate at 37 degrees Celsius for 20 minutes. Then gently triturate the cells in each well about 20 to 30 times using a 12-channel pipettor until a single cell suspension is achieved.
Now perform flow cytometry and determine the percentage of cells expressing the GFAP:GFP reporter. First determine the cell size and viability from the forward and side scatters and gate the viable cell population. Then determine the GFP-positive cells within the viable cell population.
An increase of percentage of GFP-positive cells by three standard deviations over controls is considered to be a positive hit which may be adjusted to accommodate user preference of stringency. Representative images of HSR-GBM1 GSC subclones expressing low and high levels of GFAP:GFP are shown. The subclones positive for GFAP:GFP expression were selected by flow cytometry for the patient-derived neurosphere line.
Here, low GFAP:GFP indicates that less than 5%of the cells in the clone are GFP-positive, while high GFAP:GFP indicates that greater than 75%of the cells in the clone are GFP-positive. These results show that in vitro self-renewal capacity of GFAP-low subclones is higher than their GFAP-high counterparts as determined by ELDA. Therefore GH subclones are more differentiated than GL subclones.
Finally, drug libraries were screened to identify agents capable to induce astroglial differentiation in GSCs. 12 drugs were found to significantly increase the percentage of cells expressing the GFAP:GFP reporter. Once mastered, this technique can be done in a few weeks if it is performed properly.
Following this procedure other methods like alamarBlue assay, clonogenic assay in semisolid media, flank or intracranial xenograph injection can be performed in order to answer additional questions like the effect of selective drugs on cellular proliferation and clonogenicity in vitro and tumorigenicity in vivo. After watching this video, you should have a good understanding of how to identify compounds capable of inducing astroglial differentiation in glioblastoma stem cells.
