Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Immunostaining of unedited hPSC-EPCs and hPSC-CECs with lentiviral constitutive N1ICD overexpression: Immunocytochemistry (ICC) analysis of various markers in hPSC-EPCs treated with DMSO, CHIR (hPSC-CECs), CHIR and GFP LV, or CHIR and N1ICD LV on D11 after 6 days of culture in hECSR supplemented with DMSO or CHIR and 4 days following transduction with either GFP or N1ICD LV. (A) Representative images of ICC analysis for caveolin-1, CD31, and GFP in various treatment conditions. Only LV transduced cells expressed GFP. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (B) Quantification of caveolin-1 mean gray value (MGV) in different conditions from (A), normalized to Hoechst MGV. (C) Representative images of ICC analysis for PLVAP and CD31 in indicated treatment conditions. GFP staining not shown. Hoechst nuclear counterstain overlaid in all images. Scale bar: 200 μm. (D) Quantification of PLVAP MGV in indicated conditions from (C), normalized to Hoechst MGV. (E) Representative images of ICC analysis for GLUT-1 and CD31 in indicated treatment conditions. Hoechst nuclear counterstain overlaid in all images. Scale bar: 200 μm. (F) Quantification of GLUT-1 MGV in indicated conditions from (C), normalized to Hoechst MGV. In (B) , (D) , and (F) , points represent n = 3 biological replicates from one differentiation of IMR90-4 iPSC-derived EPCs. Horizontal bars indicate mean. Hoechst-normalized relative fluorescence for each of the three markers was further normalized within each analysis such that the mean of the DMSO condition was equal to 1. Statistical analyses were performed on Hoechst-normalized data; P-values: One-way ANOVA followed by Tukey’s HSD test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Immunostaining, Over Expression, Immunocytochemistry, Transduction, Staining, Derivative Assay, Fluorescence

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Differentiation and validation of PB-TRE-N1ICD hPSC-CECs with Dox-inducible N1ICD overexpression: (A) Protocol for differentiation of hPSCs to EPCs, followed by MACS sorting, hygromycin reselection, CHIR treatment to derive CECs and doxycycline treatment to overexpress N1ICD . (B) Schematic of the doxycycline inducible N1ICD overexpressing transposon construct. The N1ICD cassette follows a TRE3G doxycycline inducible promoter. A cassette encoding the Tet-On 3G protein follows an EF-1α core promoter, resulting in constitutive expression of this gene. Tet-On 3G must associate with doxycycline to bind to the TRE3G inducible promoter. Also following the EF-1α promoter and Tet-On 3G are self-cleaving 2A peptide linker (T2A) followed by a hygromycin resistance gene (HygR). The top portion of the schematic indicates that during differentiation of hPSCs to CECs, the TRE3G promoter is not active due to the absence of doxycycline. In the bottom portion, hPSC-CECs that have been differentiated and sorted can be treated with doxycycline to activate N1ICD overexpression. (C) Representative flow cytometry plots of D5 hPSC-EPCs derived from a population of N1ICD overexpressing IMR90-4 iPSCs with heterogeneous copy numbers of the integrated construct shown in (B). Graphs show percentage of CD31+/CD34 + EPCs before (pre-MACS) and after (post-MACS) sorting. (D) Western blotting analysis of D15 hPSC-derived PB-TRE-N1ICD 10(+ CR2) clonal EPCs treated with CHIR (hPSC-CECs), with or without doxycycline. Membranes blotted for Notch1 full length (N1 FL) protein and intracellular domain (N1ICD), as well as β-actin. Predicted approximate molecular weights of each detected protein are indicated on the right-hand side. (E) N1 FL and (F) N1ICD, normalized to respective input control (β-actin) band intensities. In all Western blot analyses, points represent n = 4 biological replicates from one differentiation of IMR90-4 PB-TRE-N1ICD 10(+ CR2) iPSC-derived CECs. Bars indicate mean ± SD. β-actin-normalized band intensities were further normalized within each analysis such that the mean of the CHIR + PBS condition was equal to 1. Statistical analyses were performed on β-actin-normalized data; P-values: Student’s t -test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Biomarker Discovery, Over Expression, Construct, Expressing, Flow Cytometry, Derivative Assay, Western Blot, Control

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Immunostaining of PB-TRE-N1ICD hPSC-CECs: Immunocytochemistry (ICC) analysis of indicated markers in PB-TRE-N1ICD hPSC-CECs on D15 after 10 days of culture in hECSR supplemented with CHIR and 1:1000 diluted DPBS from D5 to D11 and 4 days of CHIR and doxycycline (1 µg/mL) or PBS treatment from D11 to D15. (A) Representative images of ICC analysis for caveolin-1 and CD31. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (B) Quantification of caveolin-1 mean gray value (MGV) in different conditions from ( A ), normalized to Hoechst MGV. (C) Representative images of ICC analysis for PLVAP and CD31. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (D) Quantification of PLVAP MGV in different conditions from (C), normalized to Hoechst MGV. (E) Representative images of ICC analysis for GLUT-1 and CD31. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (F) Quantification of GLUT-1 MGV in different conditions from ( C ), normalized to Hoechst MGV. In ( B ), ( D ), and ( F ), points represent n = 4 biological replicates from one differentiation of IMR90-4 PB-TRE-N1ICD 10(+ CR2) hPSC-CECs. Horizontal bars indicate mean ± SD. Hoechst-normalized relative fluorescence for each of the three markers was further normalized within each analysis such that the mean of the PBS condition was equal to 1. Statistical analyses were performed on Hoechst-normalized data; P-values: Student’s t test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Immunostaining, Immunocytochemistry, Fluorescence

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: FACS sorting and bulk RNA-sequencing of hPSC-CECs with doxycycline-inducible N1ICD overexpression: (A) Schematic of culture timeline and FACS sorting for PB-TRE-N1ICD 10(+ CR2) hPSC-CECs treated with or without doxycycline to overexpress N1ICD . Cells in each PBS- ( n = 4) and Dox-treated ( n = 3) sample were sorted by FACS into subpopulations based on CD144 (VE-cadherin) expression. Lower boundary for CD144 high gate was set based on majority of PBS-treated hPSC-CECs and unedited hPSC-CECs cultured in the presence of CHIR alone. Lower boundary for CD144 low gate was set based on CD144 antibody-stained undifferentiated hPSCs and isotype control antibody-stained edited hPSC-CECs. Gated plots show representative distributions of PBS- and Dox-treated edited hPSC-CECs into CD144 high and CD144 low expressing subpopulations. (B) PCA plot showing relative differences in transcriptomic profiles of bulk RNA-sequenced subpopulations from (A), including PBS-treated CD144 high (PBS_CD144hi), Dox-treated CD144 high (Dox_CD144hi), and Dox-treated CD144 low (Dox_CD144lo). Raw bulk RNA-sequencing data were pre-processed using the Galaxy pipeline (usegalaxy.org) discussed in Materials & Methods and analyzed in RStudio using the DESeq2. (C) Normalized expression (transcripts per million, TPM) of NOTCH1 and HEYL from each bulk RNA-sequenced subpopulation discussed in ( A ) and ( B ). Statistics calculated by one-way ANOVA with post-hoc Tukey’s test, significant comparisons highlighted in red. (D) Heatmap of row-normalized TPM expression for Notch signaling-related genes that are significantly differentially expressed (FDR < 0.05) between the 3 subpopulations. Selected genes obtained from KEGG and Hallmark Notch signaling gene sets via MSigDB ( https://www.gsea-msigdb.org/gsea/msigdb ). Row z-scores were calculated for each gene by subtracting mean expression across all samples from normalized expression for a specific sample and dividing the result by the standard deviation. Row z-scores range from bright red, indicating high expression, and dark blue, indicating low expression. Heatmap was produced in RStudio using the pheatmap package

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: RNA Sequencing, Over Expression, Expressing, Cell Culture, Staining, Control, Standard Deviation, Produced

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Transcriptomic comparison of PB-TRE-N1ICD hPSC-CECs ± Dox to hPSC-derived, primary or immortalized endothelial and non-endothelial cell types and to in vivo BMECs: (A) Analysis of bulk transcriptomic data of subpopulations of PB-TRE-N1ICD 10(+ CR2) hPSC-CEC subpopulations using PACNet classification scores to identify similarity to generic cell types in a training data set. Sample replicates from left to right: PBS-treated, CD144-high (PBS_CD144hi); doxycycline-treated, CD144-high (Dox_CD144hi); doxycycline-treated, CD144-low (Dox_CD144lo); random cell type (rand) auto-generated by PACNet software. Classification score colors ranging from black to yellow indicate no transcriptomic resemblance to 100% transcriptomic match, respectively, to training data cell types indicated on y-axis. (B) PACNet analysis of primary human brain microvascular endothelial cells (HBMEC), immortalized hCMEC.D3 brain endothelial cells (hCMEC_D3), and primary human aortic endothelial cells (Haortic) relative to training set cell types. All analyses in (A) and (B) were performed using the PACNet webtool ( https://cahanlab.org/resources/agnosticCellNet_web/ ). (C) Heatmap of transcript abundance (log 2 [TPM + 1]) of indicated endothelial, mesenchymal, fibroblast, and epithelial genes across samples with different degrees of N1ICD overexpression. Heatmap columns indicate sample/replicate and rows indicate genes. Cell color indicates transcript abundance, ranging from blue (lowest expression) to red (highest). Heatmaps were generated using the pheatmap package in R. (D) PCA plot combining several in vivo and in vitro transcriptomic datasets, including: pseudo-bulked in vivo adult brain capillary endothelial cell scRNA-seq data (Yang et al., 2022) ; pseudo-bulked in vivo embryonic brain capillary endothelial cells scRNA-seq data (Crouch et al., 2022) ; in vitro primary/immortalized brain endothelial and primary peripheral endothelial cells (similar to those presented in [B]) bulk RNA-seq data (Qian et al., 2017) ; in vitro Passage 1 hPSC-derived endothelial progenitor cells (EPCs) treated with CHIR and hPSC-derived smooth muscle-like cells (SMLCs) bulk RNA-seq data (Gastfriend et al., 2021) ; in vitro primary cells representing different developmental lineages bulk RNA-seq data (Pandey, 2022, GSE190615 ); in vitro PB-TRE-N1ICD hPSC-CECs with or without doxycycline bulk RNA-seq data (this study). PCA plot was generated using the DESeq2 package in R

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Comparison, Derivative Assay, In Vivo, Generated, Software, Over Expression, Expressing, In Vitro, RNA Sequencing

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Normalized expression of selected BBB and cell identity-related genes from bulk RNA-seq of edited PB-TRE-N1ICD 10(+ CR2) hPSC-CECs ± Dox subpopulations: (A-M) Comparison of normalized gene expression (TPM) between bulk RNA-sequenced PBS- and Dox-treated subpopulations for indicated genes. In all analyses, points represent n = 3 or n = 4 biological replicates depending on treatment condition from one differentiation of IMR90-4 PB-TRE-N1ICD 10(+ CR2) hPSC-CECs. Bars indicate mean ± SD. Relative gene expression values are shown in TPM, and statistical analyses were performed on TPM data; P-values < 0.05 by one-way ANOVA with post-hoc Tukey’s test are highlighted in red

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Expressing, RNA Sequencing, Comparison, Gene Expression

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: N1ICD overexpression reduces fluorescent albumin accumulation by inhibiting caveolae-mediated endocytosis: (A) Representative live cell epifluorescence microscopy images of 10(+ CR2) PB-TRE-N1ICD hPSC-CECs cultured in hECSR + CHIR, with PBS or Dox, and PBS-cultured cells with methyl-β-cyclodextrin (MβCD, inhibitor of caveolae-mediated endocytosis) pretreatment that were incubated at 37 °C with bovine serum albumin, or BSA, conjugated to AlexaFluor 647 (albumin-AF647). CD144 (VE-cadherin) and AlexaFluor 647 are shown in the 488 and 647 nm channels, respectively. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (B) Representative flow cytometry plots for isotype control, PBS- and Dox-treated cells from the fluorescent albumin accumulation assay showing gating of the CD144 high (CD144hi) and CD144 low (CD144lo) subpopulations. (C) Quantification of albumin-AF647 accumulation in gated subpopulations incubated at 37 °C. Statistical comparisons shown between accumulation for each individual subpopulation. (D) Quantification of albumin-AF647 accumulation in all CD144-positive cells from each condition, with or without MβCD pretreatment. Conditions incubated at 37 °C as well as 4 °C control were included. Selected statistical comparisons are shown among PBS- or Dox-treated conditions in the presence and absence of MβCD-pretreatment. Statistical analyses for ( C ) and ( D ) were performed on raw MFI data; P-values < 0.05 by one-way ANOVA with post-hoc Tukey’s test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Over Expression, Epifluorescence Microscopy, Cell Culture, Incubation, Flow Cytometry, Control

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: N1ICD overexpression reduces abundance of total and caveolae-associated vesicles: Transmission electron microscopy (TEM) analysis of PBS- or Dox-treated PB-TRE-N1ICD 10(+ CR2) hPSC-CECs. (A) Representative ultrastructural TEM images. Red arrowheads indicate locations of vesicles. Scale bar: 400 nm. (B) Quantification of vesicle density relative to total cell area for each image. (C) Quantification of vesicle count per diameter range across all 12 images for PBS- or Dox-treated conditions. (D) Representative immunolabeled TEM image of Nanogold particles bound to caveolin-1 in PBS-treated cells. Scale bar: 100 nm. (E) Quantification of area fraction occupied by Nanogold particles relative to total cell area. (F) Quantification of caveolin-1/Nanogold-associated vesicles relative to total cell area. For ( C ), ( E ), and ( F ), points represent 3 images measured per biological replicate for n = 4 replicates in each treatment condition. Horizontal bars indicate mean ± SD. Statistical analyses were performed on cell area-normalized counts for ( C ) and ( F ) and on area fraction for ( E ). All P-values < 0.05 by Student’s t -test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Over Expression, Transmission Assay, Electron Microscopy, Immunolabeling