Fig 2: Cytoarchitecture of the human fetal spinal cord. (A) Transmission electron microscopy (TEM) images of the central canal of the human fetal spinal cord (scale bar (from left to right) = 20 µm, 10 µm, 2 µm, and 500 nm). The central canal is formed by ependymal cells organized as a pseudostratified epithelium with a large number of cilia in the apical zone (white arrow), nuclei with condensed chromatin (*), and large apical junction complexes (white arrowheads). (B) Representative coronal immunostaining images (complete coronal sections in upper panels and magnified view of the corresponding CC area in the lower panels) of human fetal spinal cords for SOX2 (yellow), Ki67 (yellow), Nestin (green), Gfap (red), NeuN (red), and Olig2 (yellow) (white bar scale= 500 µm; red bar scale = 50 µm).

Fig 3: Fluorescent immunohistochemistry of biopaper and PET membranes. PECAM-1 (red) is present at BMEC junctions in both models (a, d). GFAP (green) is distributed throughout the astrocyte, showing similar morphology across membrane materials (b, e). Merged images are shown in (c) and (f).

Fig 4: PGA-SS-FAS priming process enhances neuronal and oligodendrocyte differentiation and induces neurite outgrowth in a model of neurite retraction. (A) Representative immunofluorescence images of hfNPCs using ß-III-tubulin for immature neurons (green), GFAP for astrocytes (red), Olig2 for oligodendrocytes (green), NeuN for mature neurons (red), and DAPI (blue) (scale bar = 20 µm) (positive nuclear staining is indicated with an arrow) in the presence of the PGA-SS-FAS compound for inducing the priming process (PGA-SS-FAS) or its vehicle (control). (B) Quantification of the percentage of positive cells for each of the indicated cell markers with (black bars) or without (white bars) 24 h PGA-SS-FAS priming. Data presented as the mean ± SEM determined by Student’s unpaired t-test (n = 3). ** p < 0.01, *** p < 0.001 vs. hfNPCs. (C) Representative immunofluorescence images of ß-III-tubulin (green) and DAPI (blue) staining during the in vitro neurite retraction assay induced by lysophosphatidic acid (LPA) treatment during 24 h in hfNPC adherent cultures. Yellow arrows indicate neurite-like processes emanating from neuronal progenitors in the control condition (in the absence of LPA) and in co-treated cultures (LPA + PGA-SS-FAS); red arrows indicate rounded cells 24 h after LPA treatment and induced neurite-like retraction (scale bar = 50 µm). (D) Neurite length quantification presented as the mean ± SEM determined by one-way ANOVA with the Tukey multiple comparison test (n = 3). ** p < 0.01, *** p < 0.001 vs. control; +++ p < 0.001 vs. control.

Fig 5: 2-Dimensional Images of the Whole-Mount SAN Preparations With Triple Immunolabeling Illustrating PGCs and SAN Pacemaker Cells Imaged by Optical Slicing(A) Tiled panoramic 2-dimensional image (4 mm by 1.2 mm), illustrating the cytoarchitecture of the HCN4-immunoreactive meshwork from the SVC to the IVC; the approximate border of crista terminalis (CT) is indicated by the yellow broken line. Glial fibrillary acidic protein–positive (GFAP+) (green) and S100 calcium-binding protein B–positive (S100B+) (cyan) cells are scattered between HCN4-immunoreactive cells (red color) across the SAN. (B to E) Peripheral glial cells (PGCs) immunoreactive to GFAP and to S100B among HCN4+ cells, imaged with high optical magnification. GFAP+ was detected in higher levels than S100B+ within the branch PGCs. (E) Web of PGCs near the lumen of the blood vessels. Abbreviations as in Figures 1 and 2.