Subplate neurons can be derived in vitro from a male hPSC line (RUES1) via a preplate stage and their fate is also modulated post-mitotically via WNT signaling.
Description
Subplate neurons can be derived in vitro from a different hPSC line (RUES1) via a preplate stage and their fate is also modulated post-mitotically via WNT signaling. Related to Figure 2. a. Immunostaining of day 55 neurons co-expressing CTIP2 and TBR1, but not NURR1. This resembles the profile of PCW11 preplate neurons. b. Quantification of the data in a. c. Quantification of immunostaining at day 75 (blue bars) and day 120 (red bars) is shown in the right panel. Data for day 75 is from e and for day 120 is from f. d. Immunostaining of day 75 neurons showing widespread nuclear co-expression of CTIP2, TBR1, SATB2, and NURR1 in SPN like cells. This is the profile of PCW15 frontal subplate neurons. e. NOR1 expressing cells are also found in day 75 cultures and rarely overlap with NURR1 cells. They co-express SATB2 and CTIP2 (not shown). f. Immunostaining of day 120 neurons for CTIP2 and SATB2 showing mutually exclusive expression. This is consistent with a profile of mature deep layer projection neurons. g. Immunostaining of day 90 CHIR-treated cultures for CTIP2, TBR1, SATB2, and NURR1. CHIR treatment from days 75-90 causes a loss of SATB2 and NURR1 cells. Most cells express CTIP2 and TBR1, indicating a commitment to corticofugal fate. h. Immunostaining of day 120 IWR1e-rescued cultures for CTIP2, TBR1, SATB2, and NURR1. A partial rescue of SATB2 expression is observed at D120. i. Quantification of SATB2, NURR1, and TBR1 expressing cells on immunostaining in control (blue) and CHIR-treated (red) cultures at D90. j. Bar plot of fold-change in SATB2 positive cells in IWR1e rescued cultures compared to D90 and D120 CHIR-treated cultures. Data in b and c was derived from n=3; error bars represent s.d. Scale bars: a, d, f, g, and h, top panels: 100 µm, bottom panels: 20 µm, and e, 20 µm.