A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

The overall goal of this screen is to identify small molecule protein kinase inhibitors that modulate embryonic stem cell pluripotency. This method can help answer key questions in the embryonic stem cell signaling field by identifying novel protein kinase signaling pathways that regulate embryonic stem cell pluripotency. So the main advantage of this technique is that the screen is relatively easy to perform using standard laboratory equipment and readily available reagents.

We first had the idea for this method when we realized that most research in pluripotency signaling pathways had completely ignored vast waves of the kinome. Prepare gelatin-coated 10 centimeter dishes by adding five milliliters of 0.1%gelatin to 10 centimeter plates. After incubating at room temperature for five minutes, aspirate the gelatin and let the plate dry for two minutes.

Then, plate any standard mouse ES cell line on the coated dishes in standard mouse ES cell media containing 100 nanograms per milliliter of GST-tagged LIF, 10%fetal calf serum, and 5%knockout serum replacement. Culture the cells at 37 degrees celsius 5%CO2 and replace the media every day. Passage the mouse ES cells at around 80%confluency every second day by aspirating the media and washing with five milliliters of phosphate-buffered saline per plate.

Then, add one milliliter of trypsin EDTA per plate of mouse ES cells and incubate at 37 degrees celsius for 10 minutes. Following incubation, re-suspend the trypsinized cells in four milliliters of media and centrifuge the cells at 1200 RPM for five minutes. Thoroughly re-suspend the resulting cell pellet in five milliliters of media, pipetting up and down to generate a single cell suspension.

Count the cells by combining a 10 microliter cell suspension and 10 microliters of trypan blue. Place the mixture into a cell counting chamber or use a hemocytometer and light microscope. Next, seed 3000 mouse ES cells into 0.1%gelatin-coated 96 well plates in a final volume of 100 microliters of media using a multichannel pipette.

Then, apply kinase inhibitors at a one to 100 dilution using a multichannel pipette. Gently pipette the media to mix the inhibitor and cell suspension. Then, allow the cells to settle in a tissue culture hood for one hour to ensure equal distribution across the plating surface.

Culture the cells for 48 hours without changing the media. To begin the screening analysis, wash 96 well mouse ES cell plates in 200 microliters of PBS using a multichannel aspirator and pipettes. After washing, mix cell extracts in 150 microliters of lysis buffer.

Clarify the extracts by centrifugation at 1500 times G for 30 minutes in V-bottomed 96 well plates. Then, immobilize 100 microliters of supernatants onto a nitrocellulose membrane using a 96 well vacuum dot plot manifold. Dry the membrane and stain with 40 milliliters of Ponceau S to ensure consistent transfer.

After washing the membrane with TBST, block it in TBST plus 3%milk. Then, incubate the membrane in Nanog and Dnmt3b antibodies at a dilution of one to 1000 in the blocking buffer overnight. The next day, wash the membrane three times for 10 minutes in TBST.

Then, incubate it in 30 milliliters of secondary antibodies at a dilution of one to 10, 000 in blocking buffer. Develop the membrane using a digital immunoblotting imaging system. It is critical to validate kinase inhibitors identified in the screen using conventional immunoblotting before proceeding.

This helps remove false positives before further downstream analysis. Analyze the screen data and validate inhibitors as bonafide pluripotency regulators as described in the text protocol. Shown here is a representative Nanog dot plot from the performed screen.

Positive control inhibitors which drive primed or naive pluripotency are highlighted. Also shown is a representative Dnmt3b dot plot from the performed screen. Positive control inhibitors which drive primed or naive pluripotency are again highlighted.

In this plot, the inhibitors were ranked for the Nanog to Dnmt3b ratio. A two-fold Nanog to Dnmt3b ratio threshold is set to identify inhibitors that stabilize naive or primed pluripotency. Summed Nanog and Dnmt3b signals are also provided.

In addition, kinase inhibitors selected for further validation are noted. Shown here is a conventional immunoblot validation of selected kinase inhibitors, Nanog and Dnmt3b immunoblots, as well as the Nanog to Dnmt3b ratio and the total signal are provided. After watching this video, you should have a good understanding of how to identify small molecule kinase inhibitors that modulate embryonic stem cell pluripotency.

Once mastered, this technique can be done in 72 hours if performed properly. Though this method can provide insight into the kinase signaling pathways that control pluripotency, it can also be applied to embryonic stem cell differentiation and other regulatory systems such as epigenetic modifiers. Following the procedure, other libraries of small molecule inhibitors, such as those targeting epigenetic modifiers, can be used to identify new players in pluripotency regulation.

After its development, this technique paved the way for our research group to elucidate novel pluripotency signaling pathways. We use this technique to identify a novel role for the amplified pathway in regulating pluripotency.