Figure 1.

PAX6⁺ cortical progenitors form both deep- and upper-layer cortical neurons

(A) Schematic showing dual-SMAD neural induction and FGF2 expansion to generate dorsal telencephalic progenitors from hPSCs. HES3:PAX6^mCherry progenitors were FACS isolated at day 20 (D20) and matured until D55 for analysis.

(B) H9-derived cortical progenitors express forebrain (OTX2⁺) and dorsal (PAX6⁺) markers and self-organize into rosettes with ZO-1⁺ lumens and basal PH3⁺ mitoses.

(C) Asynchronous maturation generates cultures of SOX2⁺ progenitors and TUJ1⁺ neurons.

(D) Quantification of TBR1⁺ (layer VI), CTIP2⁺ (layer V), and BRN2⁺ (layers II–IV) neurons for two hPSC lines (n = 3–4 independent experiments, data are mean ± SEM).

(E) The HES3:PAX6^mCherry reporter mirrored PAX6 protein expression in vitro.

(F) FACS isolation and quantification of PAX6⁺ cortical progenitors at D20 (n = 5).

(G–I) Long-term culture (D55) yielded cortical neurons expressing markers of layers VI (G), V (H), and II–IV (I).

(J) Glial differentiation (GFAP/SOX2) was observed after extended culture (>D80).

Scale bars, 100 μm.