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: Synergistic induction of advanced neural stem cells (ANSCs) from PSCs by TTNPB and CHIR99021. (A), Schematic of the generation of advanced neural stem cells (ANSCs) from pluripotent stem cells (PSCs). (B), Morphology and alkaline phosphatase staining of PSCs (passage 20) and ANSCs (passage 50). Scale bar: 100 µm. (C), Immunofluorescence staining of pluripotency markers ( OCT4, SOX2, NANOG ) and lineage markers ( SOX17, TBXT, PAX6 ) of PSCs and ANSCs. Scale bar: 50 µm. (D), Volcano plot showing differentially expressed genes (DEGs) between ANSCs and PSCs (|log 2 FC| > 1, P < 0.05). (E), Heatmap illustrating the expression levels of pluripotency genes, neuroectodermal genes, genes associated with retinoic acid (RA) signaling, and downstream target genes of the RA signaling pathway in ANSCs and PSCs. (F), Hierarchical clustering of transcriptomic profiles from PSCs, NSCs, and ANSCs (distance metric: 1‐ Spearman correlation coefficient). (G), Schematic and morphology on day 9 of ANSCs during spontaneous differentiation in N2B27 medium. Scale bar: 100 µm. (H), RT‐qPCR analysis of SOX2, SOX1, SOX10, HOXA1, PAX6, TUBB3 , and NESTIN expression in ANSCs before (Day 0) and after spontaneous differentiation (Day 9). Data were normalized to GAPDH . Error bars represent mean ± SD. (n = 3 biological replicates). P values were determined using two‐tailed Student's t ‐tests. (I), Representative images of mouse 8‐cell embryos at 24 and 48 h post‐injection with ANSCs or PSCs. Yellow arrows indicate the injected cells carrying the tdTomato fluorescent protein. Scale bar: 100 µm. (J), Cell counts of ANSCs and PSCs contributing to mouse embryos were performed separately. (K), Representative images of ANSCs treated with TTNPB alone (CHIR99021 withdrawal). T: TTNPB; CHIR: CHIR99021. Scale bars: 100 µm. (L), Representative images of NSCs treated with LIF alone (CHIR99021 withdrawal). L: LIF (leukemia inhibitory factor). Scale bars: 100 µm. (M), Representative images of NSCs treated with CHIR99021 alone (LIF withdrawal). C: CHIR99021. Scale bars: 100 µm.

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: Staining, Immunofluorescence, Expressing, Quantitative RT-PCR, Two Tailed Test, Injection