To begin, seed the human embryonic stem cell suspension on a Matrigel-coated P24 well plate with 500 microliters of media. Incubate overnight to allow cell attachment. The following day, infect the attached cells at a multiplicity of infection of 10, along with eight micrograms per milliliter of polybrene.
Incubate the cells at 37 degrees Celsius for eight hours. After incubation, replace the media with fresh media supplemented with penicillin streptomycin, but without rock inhibitor. When the cells reach 90%confluency, supplement the media with 0.8 micrograms per milliliter of puromycin to start the selection process.
After around four to six days, split the stable cells at a one to four ratio and expand them for cryo-preservation and further analysis. To perform single cell selection, prepare a cell suspension equivalent to 500 cells in 10 milliliters of complete growth media. Seed 100 microliters of this suspension into each well of a 96 well plate.
Leave the cells undisturbed in an incubator for three days, then observe them for further growth. Then mark the wells containing single clones. Change the media every other day and incubate until the colonies reaches sufficient size for further expansion, cryo-preservation and analysis, which typically takes two weeks.
To begin, obtain human embryonic stem cells differentiated towards neuroectoderm fate. When the cells reach 90%confluency, treat them with 200 nanomoles of LDN193189, and 10 micromoles of SB431542 in 100%knockout serum replacement media. After the initial 24 hours, incubate the cells with two micromoles of XAV939 for an additional two days.
After three days, gradually reduce the percentage of knockout serum replacement media over a period of eight days from 75%to 50%then to 25%and finally, to 100%N2 media. On day 12, fix the cells for immuno staining using Pax6 as a neuroectoderm marker. For mesoderm fate determination studies, treat 70%confluence cells with three micromoles of CHIR 99021 in definitive endoderm media for 24 hours.
Fix the cells afterward for immunostaining using brachyury as a mesoderm specific marker. To study the endodermal stage, add definitive endoderm media without CHIR 99021 and culture the cells for an additional 24 hours. For the embryoid body formation assay, seed the cells on low attachment cell surfaces omitting Matrigel to culture them in suspension conditions.
The knockout cell lines A1, A5, B4 showed no change in pluripotency potential as determined by immunostaining for OCT4, NANOG and SOX2 markers compared to the control cells. Immunostaining for the cell proliferation marker, KI67, showed no change in the proliferation rate of knockout cell lines compared to the control. Colony and embryoid body formation were not affected in knockout cell lines compared to the control.
Knockout cell lines A1, A5, B4 retained the ability to differentiate into three germ layer cells as confirmed by immunostaining for Pax6, brachyuri, and FOXA2 markers with no change in differentiation potential compared to the control cells.
