Journal: bioRxiv

Article Title: Lineage tracing reveals atoh7 positive and negative retinal ganglion cell populations in the zebrafish retina

doi: 10.64898/2026.03.19.712911

Figure Lengend Snippet: (A) Transverse sections (10μm) of wild type (wt) and atoh7-/- 5 dpf atoh7: iCre; ubi: Switch larval retina with Pax6 antibody-stained amacrine cells in the INL and RGCs/amacrines in the GCL. White arrows indicate Pax6+ amacrine INL cells which colocalize with atoh7 mCherry, while yellow arrows show Pax6+ amacrine INL that are atoh7- . (B) Bar graph demonstrating both atoh7+ and atoh7 -Pax6+ amacrine cell populations in wild type (n=5) and atoh7 -/- (n=5) backgrounds, showing a significant increase in the proportion of atoh7+ cells in atoh7 -/- retinas compared to wild type (p=0.0018, two tailed unpaired t test). (C) Representative transverse section (10μm) images of 5 dpf atoh7: iCre; ubi: Switch wild type and atoh7 mutant retinas stained with Prox1 to label bipolar cells. (D) Bar graph depicting percentage of atoh7+ and atoh7 -Prox1+ bipolar cell populations within the wild type (n=5) and atoh7 mutant (n=5). Unpaired t-test analysis shows no significant difference in the atoh7+ Prox1+ bipolar cell populations between genotypes (p=0.431, two tailed unpaired t test). Scale bars, 50μm (A) and 20μm (A), respectively. *, p < 0.05, ns, not significant, p ≥ 0.05. Error bars indicate standard deviation.

Article Snippet: Primary antibodies included: goat α mCherry (AB0081-500, OriGene) 1:500, rabbit α Rbpms2 (AB181098-1001, Abcam) 1:1000, rabbit α Pax6 (12323-1-AP, Proteintech) 1:500, rabbit α Prox1 (AB5475, Millipore) 1:500, mIgG1 α Zpr1 (ANZPR-1, Zirc) 1:100, and wheat germ agglutinin (WGA) conjugated to Alexa 488 (W11261, ThermoFisher) 1:500.

Techniques: Staining, Two Tailed Test, Mutagenesis, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Generation and characterization of human induced pluripotent stem cells from neuropathologically confirmed multiple system atrophy patient-derived fibroblasts

doi: 10.3389/fimmu.2026.1641981

Figure Lengend Snippet: Evaluation of the differentiation potency of the six MSA patient-derived iPSC lines, using two different functional assays that differ in the developmental time: (A) early-stage and (B) late-stage in the induction of all the three embryonic germ layers. (A) Schematic representation of the early-stage method, in which iPSCs were singularized, cultured as a monolayer, and treated with defined differentiation commercial mediums. This protocol is based on the STEMdiff™ Trilineage Differentiation Kit. (B) Schematic representation of the late-stage method, in which embryoid bodies were generated, grown during 2–3 days, transferred in adherent conditions, and treated with defined home-made differentiation mediums. The differentiation potential was established (A) within 1 week or (B) after 4 weeks. (C-E) Representative three-channel immunofluorescence imaging and the corresponding merged obtained using the (A) method and the MSA01 cell line, (C) PAX6 combined with NES as ectoderm markers; (D) SOX17 combined with FOXA2 as endoderm markers; and (E) Brachyury (TBXT) combined with NCAM1 as mesoderm markers. (F) All early ectodermal (PAX6 and NES), (G) endodermal (SOX17 and FOXA2), and (H) mesodermal (TBXT and NCAM1) lineage markers were homogeneously and highly expressed in the six MSA patient-derived iPSC line. (I-K) Equivalently, all late markers were detected. (I) The ectoderm derivative showed cells extending projections and ubiquitously expressing the β-tubulin III (TUBB3) protein. Some of these cells were assembled as neural rosettes. (J) The induction of the endoderm was confirmed by the expression of α-fetoprotein (AFP), revealing a single layered cup-shaped epithelium. (K) For the mesodermal differentiation, cells expressed α-smooth muscle actin (ACTA2), exhibiting the distinctive contractile stress fibrils. Nuclei were counterstained with DAPI in (C-K) . Scale bars (C-K) 20 µm.

Article Snippet: Rabbit monoclonal anti-paired box 6 (PAX6) , Cell Signaling , Cat#60433; RRID: AB_2797599.

Techniques: Derivative Assay, Functional Assay, Cell Culture, Generated, Immunofluorescence, Imaging, Expressing

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

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: ANSCs exhibit distinct chromatin accessibility landscapes. (A), ATAC‐seq analysis of PSCs, NSCs, and ANSCs. ATAC‐seq signals at Refseq genes as normalized CPM (counts per million). Heatmaps showing the landscapes of peaks around the transcription start sites (TSSs). (B), Signal intensity distribution of differential chromatin accessibility between ANSCs and PSCs (sigUp: significantly upregulated; sigDown: significantly downregulated; nonSig: not significant). (C), Signal intensity distribution of differential chromatin accessibility between ANSCs and NSCs (categories as in B). (D), Integrative Genomics Viewer (IGV) snapshots showing ATAC‐seq signals at pluripotency‐ and neuroectodermal‐ associated gene loci. (E), Genomic distribution of differentially enriched ATAC‐seq peaks (ANSCs vs. PSCs). (F), Venn diagram illustrating the overlap of upregulated genes in ANSCs vs. PSCs and H9‐ANSCs vs. H9‐PSCs from ATAC‐seq data (upper panel). Venn diagram illustrating the overlap of upregulated genes in NSCs vs. PSCs and H9‐NSCs vs. H9‐PSCs from ATAC‐seq data (lower panel). (G), Scatter plot of chromatin accessibility activity scores for ANSCs vs. PSCs. (H), Gene Ontology (GO) enrichment analysis of genes showing increased chromatin accessibility in ANSCs compared to PSCs. BP: biological process; CC: cellular component; MF: molecular function. (I), Chromatin footprints of pluripotent and neuroectodermal transcription factors in ANSCs vs. PSCs. Red: ANSCs; Blue: PSCs. (J), Venn diagram showing the overlap between upregulated genes from ATAC‐seq and RNA‐seq (ANSCs vs. PSCs). (K), Heatmap showing neuroectodermal gene expression in ANSCs and PSCs. (L), IGV snapshots showing ATAC‐seq and RNA‐seq signals at the PAX6 locus.

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: Activity Assay, RNA Sequencing, Gene Expression