Journal: Advanced Science

Article Title: TTNPB Promotes Human Pluripotent Stem Cell‐to‐Neural Stem Cell Transition via Modulation of Chromatin Accessibility and the S‐(5′‐adenosyl)‐L‐homocysteine/Choline Metabolic Network

doi: 10.1002/advs.202515648

Figure Lengend Snippet: NSCs and ANSCs transplantation ameliorates depressive‐like behaviors and remodels hippocampal transcriptomes in LPS‐ and CUMS‐induced rat models. (A), Schematic illustration of the experimental design for the establishment of LPS‐ and CUMS‐induced depressive‐like rat models and stereotactic transplantation of NSCs or ANSCs into the hippocampus. (B), Body weight (BW) was measured weekly in rats from each group (n = 6). P values were determined using Welch's t ‐tests. (C), Spleen coefficient of rats from each group at the experimental endpoint (day 28, n = 6). P values were determined using Welch's t ‐tests. (D), Sucrose preference test (SPT) assessing sucrose consumption in rats from each group before and after PBS, NSCs, or ANSCs injection (n = 6). P values were determined using Welch's t ‐tests. (E), Open field test (OFT) and elevated plus maze (EPM) behaviors of rats were recorded before and after PBS, NSCs, or ANSCs injection and analyzed using an automated animal behavior analysis system. (F), Total distance traveled during the OFT in each group. P values were determined using Welch's t ‐tests. (G), Percentage of time spent in the open arms of the EPM in each group. P values were determined using Welch's t ‐tests. (H), Representative immunofluorescence micrographs of the rat hippocampus. Red: tdTomato, green: GFAP. Scale bar, 100 µm. (I), Representative immunofluorescence micrographs of the enlarged rat hippocampus. Nuclei were stained with DAPI (blue), and transplanted cells were identified by tdTomato (red). Scale bar, 50 µm. (J), The number of tdTomato + cells with identifiable nuclear was quantified in representative microscopic fields of the NSC and ANSC group of rat hippocampal sections. (K), Detection of human‐derived transcripts in rat hippocampal RNA‐seq data using human‐specific gene sequences. Open circles indicate samples without detectable human‐specific sequences, whereas filled circles indicate samples with detectable human‐derived transcript fragments. Heatmap showing normalized read counts of detected human‐specific transcript fragments in rat hippocampal RNA‐seq data. (L), Principal component analysis (PCA) of hippocampal transcriptomes from CON, Depressed, Sham, NSC, and ANSC groups. (M), Hierarchical clustering of hippocampal transcriptomes from CON, Depressed, Sham, NSC, and ANSC groups (distance metric: 1‐Spearman correlation coefficient).

Article Snippet: The primary antibodies used included: rabbit polyclonal OCT4 (Novus Biologicals, #NBP2‐15053, 1:200), rabbit polyclonal NANOG (PeproTech, #P236, 1:200), goat polyclonal SOX2 (R&D Systems, #AF2018, 1:200), rabbit monoclonal NESTIN (Boster, #PB9874, 1:200), rabbit polyclonal PAX6 (Elabscience, #E‐AB‐61653, 1:200), rabbit polyclonal N‐Cadherin (Abcam, #ab76057, 1:200), goat polyclonal Brachyury (R&D Systems, #AF2085, 1:100), goat polyclonal SOX17 (R&D Systems, #AF1924, 1:200), mouse monoclonal NeuN (CST, #93972, 1:100), mouse monoclonal TUBB3 (Bioss, #BSM‐33177M, 1:200), rabbit polyclonal GFAP (Bioss, #bs‐0199R, 1:200), and rabbit monoclonal CDX2 (Biogenex, #Cdx2‐88).

Techniques: Transplantation Assay, Injection, Immunofluorescence, Staining, Derivative Assay, RNA Sequencing