The overall goal of this procedure is to generate cortical interneuron progenitors in post-mitotic interneuron precursors for mouse embryonic stem cells using a modified embryoid body to monolayer method. This method can help answer key questions in the neuroscience field, such as how sub-types of cortical interneurons achieve their individual fates during development. The main advantage of this technique is that it enables efficient generation of Nkx2.1 expressing interneuron progenitors, as well as methods to enrich for parvalbumin or somatostatin interneuron subgroups.
To begin this procedure, plate mitotically inactive MEF feeder cells in MEF media onto tissue culture treated plates. Allow at least 12 hours for them to settle in a cell culture incubator at 37 degrees Celsius before plating the mESCs. If not used immediately, replace the MEF media every three days.
Next, add mESCs containing mouse leukemia inhibitory factor to the MEF plates. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide. Passage mESCs using trypsin-EDTA once the dish becomes 70 to 80%confluent, and maintain the mESCs on a MEF feeder layer in mESC media to keep pluripotency.
Before starting differentiation, passage the cells at least once onto gelatin-coated plates without MEFs to dilute them out. To do so, prepare gelatin-coated plates by adding 0.1%gelatin and PBS with calcium and magnesium to a tissue culture treated dish and incubate at 37 degrees Celsius for at least 1 hour. Plate 1.5 to two times 10 to the sixth cells on each ten centimeter plate and allow them to expand for two days before beginning differentiation.
On DD0, after the mESCs have grown on the gelatin coated plates for two days, aspirate the media and wash the cells once with PBS without calcium and magnesium. Then, add enough trypsin-EDTA to cover the surface of the plates. And place the cells back into the incubator at 37 degrees Celsius for four minutes.
After four minutes, quench trypsin-EDTA using two times its volume of mESC media. Transfer the cells to an appropriately sized centrifuge tube, and centrifuge the cells at 200 times g for five minutes. Next, remove the tube and aspirate the media without disturbing the pellet.
Resuspend the pellet in one millileter of KSR-N2 media. Subsequently, measure the cell concentration using a hemocytometer, or an automated cell counter. Start growing the cells as embryoid bodies by adding 75, 000 cells per millileter in KSR-N2 media in the non-adherent tissue culture dishes.
And incubate them at 37 degrees Celsius. On DD1, prepare for cell landing by coating the tissue culture treated dishes with poly-L-lysine for at least one hour or overnight at 37 degrees Celsius. On DD2, aspirate the poly-L-lysine and coat the plates with laminin overnight at 37 degrees Celsius.
On DD3, before beginning EB dissociation, aspirate laminin and allow the plates to completely dry in a tissue culture hood. Afterward, transfer the EB's with the media into a 15 milliliter tube, and centrifuge for three to four minutes at 15 times g or until the EB's have pelleted. Aspirate the media, and add three milliliters of cell detachment solution containing DNase.
Then, incubate the sample at 37 degrees Celsius for 15 minutes. Gently flick the tube every three minutes to aid in EB dissociation. Once the EB's are no longer visible, or 15 minutes have elapsed, add six milliliters of KSR-N2 containing DNase, and centrifuge for five minutes at 200 times g.
Then, plate the cells in KSR-N2 containing LDN193189, XAV939, and the ROCK inhibitor, Y27632, at 4.5 to five times ten to the fourth cells per centimeter squared. On DD5, change media with KSR-N2 containing FGF2, IGF1, and SSH. Next, prepare tissue culture plates for replating on day 8, using the instructions outlined in the procedures on DD1 and DD2.
On DD7, change the media with KSR-N2 containing FGF2, IGF1 and SHH. On DD8, replate the cells by detaching them from the plates with trypsin-EDTA for five minutes at 37 degrees Celsius. Quench trypsin-EDTA using two times its volume with KSR-N2.
And centrifuge at 200 times g for five minutes. Filter the cells through a 40 micron filter tube to remove any clumps. Then, replate the cells at 250, 000 cells per centimeter squared in N2-KSR containing FGF2, IGF1 and Y27632 with SHH.
On DD10, change the media with KSR-N2 containing SHH. Wash the cells once with PBS without calcium and magnesium, and add pre-warmed, non-trypsin containing cell dissociation reagent, containing DNase to the cells. Place them in the incubator at 37 degrees Celsius for 10 to 30 minutes, or until the cells have detached.
Then, gently tap the plates every five minutes to aid the dissociation. On DD5, change the media with KSR-N2 containing FGF2 and IGF1. Next, prepare tissue culture plates for replating on DD8.
On DD7, change the media with KSR-N2 containing FGF2 and IGF1. On DD8, replate the cells with smoothened agonist and add atypical PKC inhibitor. On DD10, change the media with KSR-N2 containing aPKCi.
On DD11, detach the cells from the plates for FACS. If the cells were not detached for FACS on DD11, change media on DD12 with KSR-N2 containing aPKCi. On DD14, change the media with KSR-N2 containing aPKCi.
And on DD16, change the media with KSR-N2 containing aPKCi. Then, on DD17, collect the Nkx-2.1:mCherry positive cells. Shown here are the representative images of immunostaining for Nkx2.1, Nkx2.1:mCherry, and Lhx6:GFP from a differentiation day 12 culture.
Note that these images are overlapping channels from the same field of view. This graph shows the quantification of the average percentage of DAPI positive nuclei that express Nkx2.1 at DD12 in culture. And this is a representative FACS plot, demonstrating the four different cell populations that can be isolated using our dual reporter mESC line.
Note that mCherry is on the x-axis, and GFP is on the y-axis. Thus, the top right box represents cells that are mCherry-GFP double positive. Here is the timecourse of Nkx2.1:mCherry and Lhx6:GFP induction from DD6 to 16, expressed as the percentage of cells in culture that are either mCherry only expressing, GFP only expressing or mCherry-GFP co-expressing.
Once mastered, this protocol can be completed within 17 days from start to finish, if performed properly. While attempting this procedure, it's important to remember to always start with pleuripotent, undifferentiated, healthy appearing stem cells. Following this procedure, other methods, like transplantation into neonatal or adult mouse neocortex can be performed in order answer additional questions regarding interneuron fate determination or the effect of interneuron transplantation on circuit function and animal behavior.
After its development, this technique paved the way for researchers in the field of developmental neuroscience to explore factors driving interneuron fate determination in mice, and by extension, patients affected by interneuron related disorders. After watching this video, you should have a good understanding of how to derive Nkx2.1 expressing progenitors, and their post-mitotic progeny from mouse embryonic stem cells. Thanks for watching, and good luck with your experiments.
