how does your astrocytes look? I presume these are uninduced, I cannot see neurons anywhere, have they died off?

No there are many neurons in the well (not sure about the astrocytes as the culture is very dense and now seems more like in 3D), but these flat cells were clustered away from the others. Here is another pic from the same culture

I also observed this sometimes on my iPSC-derived neuron.

My guess the starting NSC material is contaminated by neural crest stem cell (NCSC). But I don't know how to specifically get rid of these cells. And as far as I know, NCSC also stain positive for Nestin, so it can be really annoying to find this surprise after 40+ days of differentiation.

Can you please share how do you induce your iPSC to NSC (embryoid body? stromal cell (PA6) feeder? or dual SMAD inhibition)? And how did you separate the NSC from the undifferentiated iPSC and other contaminating cells (rosette picking, FACS, or other)?

Usually I throw away these cells. I am also wondering how to solve this problem.

I'm looking forward for your answer. Many thanks!

Hi Yanuar,

I used the protocol from a paper where they treat the iPSC-derived EBs with some 'small molecules' and in about 2-3 weeks NSC start emerging. Meanwhile, I observed the rosette formation which is a good sign for differentiation. Without any selection/sorting, iPSC dies out in a few passages, and the remaining population is thereotically  NSPC. In my case it does not take as long as yours (>40 days), which protocol do you use?

I stained my cells for TUJ1 (neuronal marker), O4 (oligondedrocyte), and GFAP (astrocyte). These cells I could not identify did not stain for any of those markers, so I am still puzzled what they could be. 

Hi Goutam,

I managed to get oligodendrocytes after 4 weeks in the next try. I am not sure when they start emerging but based on the protocol I was using, it was suggested to culture for 2 weeks before staining. Since I did not have any oligodendrocytes after 2 weeks, I was advised to keep them for another two weeks, and in total 4 weeks was enough to have neurons, astrocytes, and oligodendrocytes.

Hello Ceren, 

We are using http://www.nature.com/nprot/journal/v7/n10/full/nprot.2012.116.html?elq=1cd01223bc6f45fba11303db4791a6b0, and have observed those flat cells consistently (around day 30) in one cell line obtained from Coriell while other (>15 lines) did not show such cells. Same cells line in another Student worked well once and we are repeating the protocol for it.

Best regards,

Ashutosh