Cell culture

You can see the protocol for culturing the cells in the uploaded file. This information was provided by the coauthors of the article Femke MS de Vrij, Bas Lendemeijer, and Steven A Kushner. For more information about isolation and differentiation of the cells see the articles which we mention in the section "Cell culture" of Materials and methods ((Gunhanlar et al., 2018) (Dupin and Coelho-Aguiar, 2013; Lee et al., 2007)). The CD271+ cells are sorted after EB stage and sorting is based on this paper: http://dx.plos.org/10.1371/journal.pone.0017540 . I believe this provides enough information to be able to culture the cells. 

NPC protocol

NPCs grow on laminin coating: 1ml DMEM + 20 µl laminin (1mg/ml, dilute from supplier stock) in incubator for 30 minutes minimum (for example, 3 ml DMEM + 60 µl laminin for 10 cm dish). Before plating NPCs remove coating, no need to rinse.

Plating NPCs:

• Spin down NPCs in 5 ml NPC medium without FGF, 3 minutes 1000G.
• Resuspend pellet in appropriate amount of NPC medium with 1:500 FGF (for example, 7 ml for 10 cm dish + 14 µl FGF)
• Plate cells on coated dish/well and distribute cells by shaking sideways

Growing NPCs:

• Refresh every other day (Monday, Wednesday and Friday) with NPC medium +1:500 FGF
• When full either passage cells or freeze down (approximately 1 per week split 1:4)

Passaging/Freezing:

• Remove medium, rinse once with PBS and remove PBS
• Add 1 ml accutase for a 10 cm dish
• In incubator for +/- 1 minute
• Add 5 ml NPC medium without FGF to collect cells
• Spin down for 3 minutes at 1000 G
• Resuspend pellet in NPC medium for culturing or in FBS + 10% DMSO to freeze cells

NPC medium:

Advanced DMEM F-12            

N2 supplement 1%                    

B27 supplement 2%         

Penicilin-streptomycin 1%                                              

Laminin 0.1%

Reagents:

Advanced DMEM F-12               12634028      Life Technologies Europe Bv

N2 supplement                          17502048      Life Technologies Europe Bv

B27 supplement                         12587010      Life Technologies Europe Bv

Penicilin-streptomycin               15140122      Life Technologies Europe Bv

Accutase (100 ml)                      A1110501      Life Technologies Europe Bv

FGF                                            GF003AF        Millipore

Laminin                                      L2020            Sigma