Hello everyone,
in our team we have established the use of NGN2 to differentiate hiPSC into neurons. We do so by transducing neurons with two lentiviruses, one with inducible NGN2 and puro resistance and another one with rtTA with neo resistance. We then do a 5 day selection with 2ug/mL of puro and 250ug/mL of G418 for two days and then 3 more days with 1ug/mL and 250ug/mL G418.
We start the differentiation by seeding the cells with mtesr with 4ug/mL of doxy and then next day we move to N2 media (dmem, NEA, N2, bdnf, hnt3, laminin, doxy). Next day we add rat astrocytes and then the next day we change to B27 media with AraC. Adapted from here: Article Rapid Neuronal Differentiation of Induced Pluripotent Stem C...
The thing is that during the culture with N2 media we have some clusters of hiPSC developing in the culture that only disappear after treatment with AraC (logic), leaving a lot of debris in the culture. Also it limits us a lot with the densities we can work, because the higher the density, the bigger the clusters and the dirtier the culture.
Has anyone the same problem? Anyone has any recommendation to get rid of these cells? We think that they are cells with basal expression of the neo resistance but with not enough expression to trigger the differentiation once we add the doxy. How would you address this?
Thank you guys.
Hi Pedro Miguel Belio Mairal,
I am also working with NGN2 neurons. I mainly know the clustering from cultures without astrocytes, so I am not sure why this is happening for you.
The only things I can recommend is to make sure your iPSC are really a single cell suspension when seeding for the differentiation. Then wash each day with PBS when doing the media change.
What are you doing with the cells? Are you replating or doing pellets or are you imaging them in some way directly in the format you start your differentiation?
Best of luck for your experiments,
Selene
Hello Selene,
Thanks for your answer. I don’t have problems of neuronal cluster, but rather from clusters of hiPSCs remaining in pluripotency and making hiPSC colonies. However, these cells are resistant to the antibiotics I used for selection. And they die when I use AraC, confirming they are mitotic cells. I think it is linked to low levels of expression of the transgene in some cells, sufficient for antibiotic resistance but not enough to trigger differentiation.
Thanks again.
Hello Pedro,
I see, okay. I also always have some iPSCs that don't (fully) differentiate. Like you mention, I get rid of them with the AraC treatment. As I am replating my neurons, I get rid of any debris that didn't come off with washes at that step.
Like you mentioned though, the lower the seeding density, the lower the number of these cells. In my experience this is the main factor to avoid undifferentiated cells.
Maybe also the DOX concentration could be something to change? Although I am not sure about this as I am using 3 ug/mL, so yours is already higher.
I hope you can manage somehow.
Selene
Thank you Selene, at least I know I’m mot the only one :)
I have had this same thing happen in the past with this differentiation protocol. The thing that worked for me was to keep doxycycline on the cells for at least 24 hours before starting selection to ensure the resistance genes are being expressed fully. However, that might be difficult for you to do if you don't want to start the differentiation process until later. The other option is to increase the concentration of your selection antibiotics (puro and G418).
Good luck!
Hello Sterling. In our case we have constitutive expression of the resistance gene. Thus, it shouldn’t really matter when I add doxycicline…