Timing of Wnt Inhibition Modulates Directed Differentiation of Medial Ganglionic Eminence Progenitors from Human Pluripotent Stem Cells

Figure 3

Effect of timing of Wnt inhibition on generation of MGE progenitors in the RD protocol. (a) Schematic timeline of the RD protocol showing stages of MGE progenitor generation, involved techniques, medium composition, and time of treatment with SHH and IWP-2 and medium change. (b) Confocal images of NPC double stained with SOX2/NESTIN and FOXG1/MAP2 antibodies. NPC collected at D25 and plated on 8-well chambers were fixed and stained two days later. Scale bar: 100 μM. (c) Quantitative RT-PCR analysis of various markers (expression relative to nontreated cells, ) in NPC on D25 of directed differentiation. Data are presented as . and difference between treatment with SHH alone and SHH supplemented with Wnt inhibition during NSC stage (stage 1) and/or NPC stage (stage 2) and between Wnt inhibition in stage 1 and stage 2. (d) Immunocytochemistry analysis of NPC for ventral, NKX2.1, and dorsal, PAX6, forebrain markers in response to treatment with SHH alone and SHH supplemented with Wnt inhibition during NSC stage (stage 1) and/or NPC stage (stage 2). Scale bar: 100 μM. (e) Quantification of data in (d). Graph showing the NKX2.1/PAX6 ratio in untreated cells (NT) and cells treated with SHH alone and SHH supplemented with Wnt inhibition during the NSC stage (stage 1) and/or NPC stage (stage 2). Data are presented as . difference between treatment with SHH alone and with SHH + IWP-2.

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