Fig 1: Expression of isthmic organizer markers in transplanted human cells. (A) Coronal sections of chicken induced optic tectum (iTeO) grafted with hESC-3d-NECs and harvested at 19 dpi after Nissl staining. Boxed areas on the left encompassing the grafted area and the induced stratified layers are shown at higher magnification on the right. (B) Immunofluorescence image depicting the area of GFP expression, containing transplanted hPSC-NECs at 19 dpi. Coronal sections were immunostained with a GFP antibody and analyzed using confocal microscopy. The dotted box delineates the laminated area without GFP positive cells. Nuclei were counterstained with DAPI (blue). (C) Immunofluorescence staining of coronal sections for the isthmic organizer (IsO)-related markers FGF18, FGF8, OTX2, WNT3A, β-catenin, LMX1A, and FOAXA2 in transplanted human GFP-positive cells. Immunofluorescence for the human-specific antigen STEM121 was used to identify transplanted hPSC-NECs. Nuclei were counterstained with DAPI (blue).
Fig 2: Grafted human NECs acquire characteristics of the isthmic organizer. (A) Log2-fold expression changes of isthmic organizer (IsO) markers genes in NEC (generated by the 3d protocol) before and at different timepoints after transplantation relative to hESC obtained from transcriptome analysis. Values were derived from data obtained in RNA-seq experiments. Shrunken Log2 fold changes relative to hESC and Benjamini–Hochberg adjusted P-values for indicated genes were extracted from differential gene expression analysis performed using DESeq2. Asterisks indicate an adjusted P-value <0.05. (B) Immunofluorescence analysis of sections for different IsO-related markers (FGF8, OTX2, LMX1A, FOXA2) at 6 dpi (days post-implantation). Staining for a human-specific antigen STEM121 was used to identify transplanted hPSC-NECs. Nuclei were counterstained with DAPI (blue).
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