Generating homogenous cortical preplate and deep-layer neurons using a combination of 2D and 3D differentiation cultures.

Embryonic stem cells (ESCs) can be used to derive different neural subtypes. Current differentiation protocols generate heterogeneous neural subtypes rather than a specific neuronal population. Here, we present a protocol to derive separate two-deep layer cortical neurons from mouse ESCs (mESCs). mESCs were differentiated into mature Tbr1 or Ctip2-positive neurons using a monolayer-based culture for neural induction and neurosphere-based culture for neural proliferation and expansion. The differentiation protocol relies on SMAD inhibition for neural induction and the use of FGF2 and EGF for proliferation and it is relatively short as mature neurons are generated between differentiation days 12-16. Compared with the monolayer-based differentiation method, mESCs can be directed to generate specific deep-layer cortical neurons rather than heterogeneous cortical neurons that are generated using the monolayer differentiation culture. The early analysis of progenitors using flow cytometry, immunocytochemistry, and qRT-PCR showed high neuralization efficiency. The immunocytochemistry and flow cytometry analyses on differentiation days 12 and 16 showed cultures enriched in Tbr1- and Ctip2-positive neurons, respectively. Conversely, the monolayer differentiation culture derived a mixture of Tbr1 and Ctip2 mature neurons. Our findings suggested that implementing a neurosphere-based culture enabled directing neural progenitors to adopt a specific cortical identity. The generated progenitors and neurons can be used for neural-development investigation, drug testing, disease modelling, and examining novel cellular replacement therapy strategies.