Figure 5. Production of human cortical excitatory neurons from PSCs in vitro recapitulates in vivo development.

A. Diagram of classes of cortical projection neurons in the layers of the adult cortex, based on mouse data, with transcription factors expressed in each class of neuron as indicated. CPNs, callosal projection neurons.

B. The order of cortical neurogenesis from human ES cells recapitulates normal development. Deep and upper layer neurons are generated in a temporal order from hESCs, with layer 5 and 6 neurons (Tbr1 and CTIP2) generated before layer 2/3 neurons (Brn2). N=3 experiments scored for each marker. Error bars, s.e.m.

C-E. Differentiation of early born cortical neurons from hESCs. Corticothalamic projection neurons of layer 6 (Tbr1/CTIP2-expressing neurons, A-C) and corticospinal motor neurons of layer 5 (CTIP2-positive, Tbr1-negative neurons, white arrows in C) are both present. Scale bars, 50 μm.

F-H. Upper layer, later born cortical neurons differentiate in this system several weeks after the early born, deep layer neurons. These neurons express Cux1, Satb2 and Brn2. Scale bars, 50 μm.

I-K. Reproducible generation of deep and upper layer projection neurons by four different iPSC lines.

L, M. Birthdating by BrdU administration demonstrates that upper layer, Satb2-expressing neurons are generated from cortical stem/progenitor cells at late stages (as late as day 90) in this system. BrdU addition for 48 hours at day 50, followed by harvesting 7 days later (L), found that the majority of Tbr1-expressing, deep layer neurons have already been generated at this stage, with only a small number of Tbr1+/BrdU+ neurons found (white arrows, L), whereas no Satb2-expressing neurons have differentiated. In contrast, BrdU administration at day 90, followed by cell analysis at day 100, found that large numbers of Satb2-expressing neurons are generated from mitotic stem/progenitor cells at this stage (yellow arrows, N). Scale bar, 50 μm.

N. Relative proportions of different classes of cortical projection neurons generated from humans ES and iPS lines. Approximately equal proportions of deep and upper layer neurons are generated from all lines. See methods for technical details of cell counting.