Hi, Gwen,

I guess it is important to first figure out whether they actually died by undergoing apoptosis or they simply detached due to the problem of the coating. Gelatin coating from different vendors are NOT equally effective. Our experience is that the ones from StemCell are much more effective than others.

Also, I personally suggest identifying the components you are suspecting to cause cell death by removing one of them each time and see if that helps. This will take some time but it will help you have consistent results in the long term!

I am not expert on neuronal differentiation, but I noticed that small details like glucose level in your basal medium, L-glutamine concentrations may all affect the outcome, so you may consider going through all those things to find the culprit!

Best luck!

you can also add ITS, vitmin C and adjusting the concentraiton of BSA or FBS. Here I want to ask you question, you culture mouse stem cell on the gelatine-coating, ES-2i medium , when will you differentiate mouse stem cells? how many density of your mouse stem cell will be cultured on lif medium? I am not sure about this. because I also do not know whether my stem cells begin to differentiate on lif medium.

I just culture my mouse stem cells and differentiate my mouse stem cells into insulin-producing cells.

Thanks for your answers!

I´m not sure if the coating is a problem because the ESCs are growing perfectly fine on the gelatine? But I will also try other gelatine/PDL coating prior to differentiation to see if there is a difference.

As far as I understood it is natural that there is quite some cell death , however some cells should stay attached (which isn´t the case with our mESCs).

I´ll try to modify the medium according to your suggestions and let you know if something is working!

Yucheng Zhang: I´m culturing 1-2x10² cells/cm² in ES-2i on gelatine, next day I change to differentiation medium. So far I´ve also tried to have them without LIF / inhibitor for one day prior to differentiation but it doesn´t seem to change something.

I am also seeing similar cell death problem in my neural differentiation using the Gaspard protocol. I followed exactly as the nature protocol says. Cells typically live 4 days from the initial neural induction and then begin to die. Some colonies were able to live up to 6-7 days but with that small amount of cells, I could not do any experiments. I am trying to figure out what might cause these massive cell death. Any suggestions?

The protocol says buffer should be used within 7 days. I am thinking making new buffer more frequently.

Dear Jinjin,

I was (and still am) seeing the same.

On day 4 of neural induction I have the most cell death but usually when there are still cells left on day 6 I get some neuronal cells in the end. However, I also had some cells of other lineages (mesodermal) that started forming cluster and were contracting.

Since I couldn´t get rid of it I now try to establish NPC lines and further differentiate these cells.

For NPC line generation I use the "STEMdiff™ Neural Rosette Selection Reagent" to detach the cells on day 12. Afterwards I keep the cells in DDM medium + EGF and FGF. After several splits I get a nice NPC culture. Maybe this is also working for you?

I usually just prepare 50ml of the medium and most of the time it is used up within one week but even when I was keeping it longer I couldn´t see a difference.

Thank you very much, Gwen.

I am working on several mESC mutants and trying to neural differentiate them, so I couldn't use the established NPC lines. Maybe I will try some other protocols that use modified N2 later, but I know they might make different type of NPCs other than the cortical ones.

Good luck with your research!

Jinjin Zhu did you have any success in differentiating the mESCs? I am currently trying this and have the same issue as you :-/

Melanie Maerken I ended up using 2007 Bibel protocol (2007;2(5):1034-43. doi: 10.1038/nprot.2007.147.). With cellular aggregates, the cell survive better. I think my cells are not very healthy so they are very sensitive to medium change, which caused a lot cell death with the Gaspard protocol.

Hello, I have participated in the study by Nicolas Gaspard and use this method for more than 10 years. I agree with you, there could be dead cells. As written in our protocol, it is important to change ESC with fresh ESC media one hour before splitting and to air-dry gelatin-coated plates for at least 10 minutes. In my hand, it works better with 60 or 100 mm plates than in 6WP, 12 or 24 WP. Also, remember that we were not using 2i but FBS/Lif so I guess that ESC are less stuck in pluripotency and mighty be easier to be neuralized. And how course, the lot of FBS can make a lot of difference. I confirm, the N2 should be used within one week. I have used many ESC lines and it works fine with some lines (in particular with the E14 line, A2loxCRE, Oct4-GFP, Sox1-GFP, Tau-GFP...) but not with other lines (I work on parental imprinting and need hybrid ESC lines).

To improve cell survival, this is what I did:

1. Use a different coating (mouse laminin or matrigel)

2. Add B27 (with no vitamin A) since the start of differentiation

3. Omit Cyclopamine (for those not interested in having 'pure' cortex)

Good luck!

Tristan Bouschet Thank you so much for updating! I have a very naive question, what is the difference between DDM from the Gaspard protocol and N2B27. I see the original Ying 2003 protocol is using N2B27. Since you mentioned that we can add B27 from the start, does that mean neurobasal is not good for early neural differentiation from ESCs?

Dear Jinjin,

You are very welcome! You are right, the protocol resembles a lot that of Ying and co-workers. B27 was omitted to have a medium as simple as possible 'by default'. B27 (we use B27 without vitamin A, as it is the precursor of retinoic acid that promotes non cortical fate) is good for early neural differentiation. Thus, you can add B27 from the start (so use DDM+B27= Yiing's medium). The differentiated cells will be neural, but I did not chararterize them as deeply as we did in Gaspard et al, 2008, so I cannot confirm they are truly cortical cells. Neurobasal is a neuronal media, so I will wait that neurons are well present in the dish (around day 12-14) before adding it. Good luck!

Thank you very much! Tristan Bouschet