Journal: bioRxiv

Article Title: Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

doi: 10.64898/2026.04.14.718471

Figure Lengend Snippet: (A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression of HOXC9 and KDR on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).

Article Snippet: Following primary antibodies were used; TBXT (Abcam, ab209665, 1:1000), ISL1 (DSHB, 39.4D5, 1:200), HAND1 (R&D Systems, AF3168, 1:100), PITX1 (Novus Bio, NBP1-88644, 1:500), HOXC9 (Abcam, ab50839, 1:200), KDR (R&D Systems, AF357, 1:1000), TP63 (Abcam, ab124762, 1:200).

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Comparison

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: PRND is selectively expressed in tip endothelial cells ( Tip EC) and promotes tip cell–like behavior. (A) UMAP projection of tumor‐associated endothelial cells (ECs), colored by inferred EC subtypes: arterial, venous, stalk‐like, tip‐like, and transition. (B) Overlay of PRND expression on the UMAP shows selective enrichment in Tip EC. (C) Violin plot showing PRND expression across EC subtypes, with highest level detected in Tip EC. (D) Volcano plot comparing gene expression between tip‐like and stalk‐like ECs. PRND is among the most significantly upregulated genes in tip‐like ECs. Genes with adjusted p < 0.05 and Log2 fold change > 0.25 were considered differenially expressed. (E) Quantification of double (CD34 + Dll4 + ) and triple (CD34 + Dll4 + CXCR4 + ) gated Tip EC after 24 h treatment with recombinant Doppel (rDpl, 1 µg mL −1 ) in HDMECs. Dots represent independent experiments, n = 3 per condition. Two‐tailed Student's t ‐test; p * < 0.05. (F) Representative images of VEGF‐induced endothelial sprouting from HUVEC spheroids treated with increasing concentrations of rDpl. White asterisks indicate HUVEC sprouts. Scale bar, 100 µm. Right panel: Quantification of sprouts per spheroid. n = 5 experimental replicates; 40–50 spheroids were counted per experiment. See Also Figure . Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple‐comparison test; p ** < 0.01, nonsignificant comparisions not shown. (G) Heatmap of the top 20 differentially expressed genes (DEGs) between PRND + and PRND − ECs. A total of 1500 randomly selected cells per group were analyzed. Rows represent genes; columns represent individual cells. Expression values were log‐normalized and Z‐scored. Color scale: red = high expression; blue = low expression. (H) WikiPathways enrichment analysis of DEGs in PRND + versus PRND − ECs, highlighting VEGF/VEGFR2 signaling as the top enriched pathway. Dot size indicates gene count; color represents adjusted p ‐value. All data are presented as mean ± standard deviation (SD).

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Expressing, Gene Expression, Recombinant, Two Tailed Test, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: Doppel promotes tip cell morphology and cytoskeletal remodeling. (A) Volcano plot showing differentially expressed genes (DEGs) between PRND + versus PRND − within the Tip EC population. Genes with adjusted p < 0.05 and Log2 fold change > 0.25 were considered significant. (B) Gene Ontology enrichment analysis of DEGs in PRND + versus PRND − Tip ECs. Dot size indicates gene count; color represents adjusted p‐ value. Pathways related to actin cytoskeleton and cell motility are highlighted. (C) Quantification of EC length in HDMECs transfected with either mock‐ or Doppel‐constructs. Doppel‐transfected cells exhibit elongated morphology and filopodia‐like protrusions., n = 6 experimental replicates. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple‐comparison test; p * < 0.05, nonsignificant comparisions not shown. Corresponding images are shown in Figure . White arrows indicate filopodia. Scale bar, 100 µm. (D) Representative confocal images of HDMECs transfected with Doppel cDNA or mock vector. Doppel‐PLA, red dots; phalloidin, green; DAPI, blue. Scale bar, 10 µm. (E) Representative confocal image showing subcellular distribution of VEGFR2 in Doppel‐ and mock‐transfected HDMECs. In Doppel‐expressing cells, VEGFR2 localizes toward the leading edge. VEGFR2‐PLA, red dots; Phalloidin, green; DAPI, blue. Scale bar, 10 µm. Right panel: Number of VEGFR2‐PLA dots either in cytoplasm or leading‐edge. n = 5 experimental replicates; 50–100 cells were counted per experiment. Statistical analysis was performed using two‐tailed Student's t ‐test; p **** < 0.0001, p ** < 0.01. (F) RTK phospho‐array analysis of Hu Dpl cells transfected with scramble or Doppel siRNA. Right panel: Quantification by pixel density. n = 3 experimental replicates. (G) Western blot analysis for phosphorylated and total Src in ECs isolated from SCC7 and CT26 tumors following transfection with either Doppel or scramble siRNA. Doppel protein levels confirm knockdown efficiency. (H) Western blot analysis for active and total CDC42 and RhoA in Hu Dpl cells following transfection with either Doppel or scramble siRNA under VEGF stimulation. All data are presented as mean ± standard deviation (SD).

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Transfection, Construct, Comparison, Plasmid Preparation, Expressing, Two Tailed Test, Western Blot, Isolation, Knockdown, Standard Deviation

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: Doppel facilitates Tip EC mobility and stability through VEGFR2‐mediated signaling. (A) Schematic of a bioengineering platform with alternating VEGF‐rich (V + ) and VEGF‐depleted (V − ) stripes for directional endothelial cell migration. (B) Brightfield and confocal microscopy images showing VEGFR2 localization relative to VEGF‐A gradients in HUVEC and Hu Dpl cells. n = 9∼10 experimental replicates. VEGFR2, red; Phalloidin, green; DAPI, blue. Scale bar, 10 µm. Right panel: Ratio of VEGFR2‐proximal PLA dots in V + and V − areas for both HUVEC and Hu Dpl cells. (C) Schematic of a bioengineered angiogenesis platform incorporating ECs to visualize Tip EC migration and representative fluorescent image showing the sprouting of Hu Dpl cells along a V + stripe. CD31, red; VEGFR2, green; DAPI, blue. Scale bar, 100 µm (D) Confocal image of Tip EC cells expressing activated integrin in Hu Dpl sprouts. Activated αVβ3 integrein, red; VEGFR2, green; DAPI, blue. Scale bar, 100 µm (E) Quantification of sprouting length in Hu Dpl versus HUVEC spheroids per V + stripe. n = 10 sprouts. (F) Quantification of Tip EC number in Hu Dpl versus HUVEC spheroids per V + stripe. n = 20 individual fields. (G) Representative confocal image of Dll4 expression in Tip EC from Hu Dpl versus HUVEC. Dll4, purple (left)/ grayscale (right); VEGFR2, green; DAPI, blue. Scale bar, 25 µm. Right panel: Quantification of Dll4‐positive signals per nucleus in Hu Dpl versus HUVEC sprouts in the V + stripes. n = 20 individual fields. (H) Schematic model illustrating Doppel's role in Tip EC differentiation, filopodia extension and directed migration through VEGFR2 signaling. Doppel enhances VEGFR2 localization to the leading edge and activates Dll4 and downstream Src signaling pathways that stabilize tip cell identity and motility. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using two‐tailed Student's t ‐test unless otherwise stated; p **** < 0.0001, p *** < 0.001, p ** < 0.01.

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Migration, Confocal Microscopy, Expressing, Protein-Protein interactions, Standard Deviation, Two Tailed Test

Journal: Advanced Science

Article Title: Selective Targeting of Tip Endothelial Cells as a Therapeutic Strategy for Tumor Angiogenesis

doi: 10.1002/advs.202512975

Figure Lengend Snippet: Selective depletion of Tip ECs via Doppel blocking antibodies. (A) Tumor growth curves following treatment with anti‐Dll4 (navy), anti‐VEGFR2 (yellow), anti‐Doppel (red) antibodies, as well as control (gray). Mice were treated at 10 mg kg −1 once every 4 days, n = 7. Two‐way ANOVA followed by Tukey's multiple‐comparison test on day 12. (B,C) Flow cytometric quantification of endothelial subtypes in tumors: CD31 + CXCR4 + Tip ECs (B) and CD31 + SELP + stalk ECs (C). (D,E) Proportion of VEGFR2 + cells in Tip EC (D) and stalk EC (E) subsets. (F) Schematic representation of Tip EC states based on relative VEGFR2 expression. Top: Highly differentiated Tip ECs exhibiting a high tip/stalk VEGFR2 + ratio. Bottom: Competing or unstable Tip ECs characterized by a low tip/stalk VEGFR2 + ratio, indicating phenotypic plasticity and competition with neighbouring Stalk EC. (G) Quantification of VEGFR2 + Tip EC/ Stalk EC ratio in tumors from each group. (H) Representative confocal images of tumor vasculature stained for endothelial and Tip EC markers. White asterisks indicate CD31 + CXCR4 + Tip ECs. CXCR4, green; CD31, red; DAPI, blue. Scale bar, 20 µm. (I) Quantification of Tip ECs per vessel. Dots indicate individual vessels. (J) Representative immunofluorescence images of liver tissue sections of mice treated with anti‐VEGFR2 antibody, anti‐Dll4 antibody, and anti‐Doppel antibody, as well as control. red; CD31, green; DAPI, blue. (K) Tumor growth of 4T1 orthotopic tumors treated with control IgG (gray), anti‐Doppel antibody (red), and anti‐VEGFR2 (yellow). Mice were treated at 10 mg kg −1 once every 4 days, n =7. Two‐way ANOVA followed by Tukey's multiple‐comparison test. (L) Flow‐cytometric quantification of tip endothelial cells ( Tip EC: CD31 + CXCR4 + ). All data are presented as mean ± standard deviation (SD). Arrows indicate the treatment initiation and tumor size at that time. Statistical analysis was performed using one/two‐way ANOVA followed by Tukey's multiple‐comparison test unless otherwise stated; p **** < 0.0001, p *** < 0.001, p ** < 0.01, p * < 0.05, nonsignificant comparisons not shown.

Article Snippet: For VEGFR2 staining, goat anti‐mouse VEGFR2 polyclonal antibody (1:200, AF644, R&D Systems) followed by Alexa 488‐linked donkey anti‐goat antibodies were used.

Techniques: Blocking Assay, Control, Comparison, Expressing, Staining, Immunofluorescence, Standard Deviation