Journal: Acta Neuropathologica

Article Title: HIF-1α is involved in blood–brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis

doi: 10.1007/s00401-020-02174-2

Figure Lengend Snippet: Junctional localization of S. pneumoniae at the BBB in vitro and in vivo. a Primary mouse brain ECs (MBMEC) were infected with S. pneumoniae and subjected to immunofluorescence staining for claudin-5 to mark endothelial junctions (red) and co-stained with an anti-pneumococcal antibody (green) to label S. pneumoniae . Confocal fluorescence microscopy of EC monolayers indicated a predominant localization of bacteria at the cell–cell borders co-localizing with claudin-5, an endothelial tight junctions marker as shown by b quantification that indicated a significantly higher number of bacteria close to the junctions. N = 3 independent preparations of MBMEC from 2–3 mice each time. Two independent wells were counted for each set comprising approximately 25 cells/field at 60X magnification (scale bar: 20 μm). Bacteria within a distance of 1 bacterium equivalent size from the junction were considered close to the junction. Data presented as mean ± SEM, ** p < 0.01, 2-tailed paired t test. c–i Transmission electron microscopy (TEM) analysis was performed on brain sections from hematogenously infected mice post-perfusion with PBS/PFA and fixation in PFA/glutaraldehyde. S. pneumoniae either directly or in protective membrane bound vesicles were localized at the endothelial tight junctions both in meninges and cortex in several mice analyzed (representative images from N = 6 mice subjected to TEM). Artificial coloration was performed for better visualization and to highlight the localization of S. pneumoniae . SPN- S. pneumoniae , EC-endothelial cell, PC-pericyte, LU-lumen, BL-basal lamina, AEF-astrocytic endfeet, ERY-erythrocyte. j–k Primary human brain ECs (HBMEC) were also infected with S. pneumoniae and stained with anti-pneumococcal antibody and claudin-5 and subjected to super resolution microscopy using Nikon structured illumination microscopy (N-SIM). In 2 different preparations of HBMEC, the localization of bacteria was primarily at the junctions with super resolution images demonstrating engagement of S. pneumoniae with the endothelial junctions (Figures to the right both in j, k ). Scale bar: 5 μm. l Primary mouse brain ECs (MBMEC) were infected with GFP-labeled S. pneumoniae strain (MOI 10) 2 days post-isolation and subjected to live-cell imaging. MBMECs as well as bacteria were imaged in brightfield and GFP fluorescence channel every 10 s starting 1 h post-infection for a total of 2 h. Five time-lapse images (10-s interval) capture transmigration of few bacteria across cell–cell borders (whites lines), which demonstrate paracellular route for transmigration of S. pneumoniae across brain endothelial cells. Representative images from 1 preparation from N = 3 MBMECs preparations (1 animal/set) infected with GFP-labeled D39 strain. Scale bar: 10 μm.

Article Snippet: Primary human pericytes (passage #5–8, ScienCell, USA) and primary mouse astrocytes (isolated from pups as described previously [ , ], passage #1–2) were cultured in their respective media (pericyte medium, ScienCell) or astrocyte medium comprising DMEM (high glucose, GlutaMAXTM, pyruvate, Gibco®), 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: In Vitro, In Vivo, Infection, Immunofluorescence, Staining, Fluorescence, Microscopy, Bacteria, Marker, Transmission Assay, Electron Microscopy, Membrane, Super-Resolution Microscopy, Labeling, Isolation, Live Cell Imaging, Transmigration Assay

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: (A) Schematic of the brain pericyte differentiation protocol developed by Stebbins et al . (B) Images of differentiating cells from iPSC to day 42 (D42) of pericyte differentiation. NCSC priming (D0-D15) results in a heterogeneous population of cells including larger cells at the colony border (white arrows). NCSCs are isolated and grown in pericyte differentiation medium, at which point a more homogenous population of cells can be seen (D21). Differentiating cells acquire an elongated morphology over the period of pericyte differentiation (D15-D42). This morphology is comparable to the morphology seen in human primary foetal pericytes. Scale bar = 200μm.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Isolation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: (A) RT-qPCR shows high levels of expression of pluripotency gene expression (OCT3/4, SOX2, and NANOG) in iPSCs, with lower levels of expression seen in iPSC-derived pericytes. These pluripotency genes are also expressed at low levels in human primary foetal (HPF) pericytes. (B) Immunofluorescence images demonstrating pluripotency marker protein expression (OCT3/4, SOX2, and NANOG) in iPSCs, but not iPSC-derived pericytes. (C) RT-qPCR shows gene expression of pericyte markers (PDGFRβ, NG2, CD13, and αSMA) in iPSC-derived and HPF pericytes. (D) Immunofluorescence images demonstrating pericyte marker protein expression (PDGFRβ, CD13, and αSMA) in day 42 iPSC-derived pericytes. High levels of αSMA protein expression are observed in iPSCs and day 21 iPSC-derived pericytes (E) RT-qPCR shows gene expression of brain-specific pericyte markers (FOXF2, FOXC1, and vitronectin) in day 42 iPSC-derived pericytes and HPF pericytes. Gene expression of FOXF2 is absent in iPSCs and NCSCs. The dotted line on all RT-qPCR graphs indicates a ΔCt of 30, demonstrating the minimum ΔCt threshold of expression in these experiments. “Neg. Con.” refers to the negative control that didn’t receive primary antibody. Scale bar = 100μm in all images. Error bars represent standard deviation between the three experimental repeats.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Immunofluorescence, Marker, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating nuclear translocation of NFκB in response to increasing concentrations of IL-1β in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) brain pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which demonstrate NFκB translocation at an EC 50 of 6.76pM in day 21 iPSC-derived pericytes (B, dotted line), and 4.64pM in day 42 iPSC-derived pericytes (dotted line, D). Images of HPF pericytes are quantified using MetaXpress, showing an EC 50 of 2.26pM (dotted line, F). Data presented is one representative experiment of two experimental repeats with the iPSC-derived pericytes, and one representative experiment of four experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear NFκB. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Immunofluorescence, Translocation Assay, Derivative Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating the subcellular localisation of STAT1 in response to increasing concentrations of IL-1β in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) brain pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which demonstrate STAT1 translocation at a potent EC 50 of 0.74pM in day 21 iPSC-derived pericytes (B, dotted line), but not in day 42 iPSC-derived pericytes. Images of HPF pericytes are quantified using MetaXpress, showing STAT1 translocation at an EC 50 of 4.19pM (dotted line, F) in a minor subset of HPF pericytes. Data presented is one representative experiment of two experimental repeats with the iPSC-derived pericytes, and one representative experiment of three experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear STAT1. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Immunofluorescence, Derivative Assay, Concentration Assay, Translocation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating nuclear translocation of NFκB in response to increasing concentrations of TNF in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) brain pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which demonstrates NFκB translocation at an EC 50 of 14.2pM in day 21 iPSC-derived pericytes (B dotted line). The concentration-response curve did not plateau in day 42 iPSC-derived pericytes due to a lack of cell viability at the highest treatment concentration (D). Images of HPF pericytes are quantified using MetaXpress, showing NFκB translocation at an EC 50 of 1.84pM (dotted line, F). Data presented is one representative experiment of two experimental repeats with the day 21 iPSC-derived pericytes, one experimental repeat with the day 42 iPSC-derived pericytes, and one representative experiment of three experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear NFκB. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Immunofluorescence, Translocation Assay, Derivative Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating the subcellular localisation of STAT1 in response to increasing concentrations of TNF in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which shows no STAT1 translocation in response to TNF treatment. The day 42 iPSC-derived pericytes lacked cell viability at the highest treatment concentration (C, D). Images of HPF pericytes are quantified using MetaXpress, showing very potent STAT1 translocation in a minor subset of cells at an EC 50 of 0.289pM (dotted line, F). Data presented is one representative experiment of two experimental repeats with the day 21 iPSC-derived pericytes, one experimental repeat with the day 42 iPSC-derived pericytes, and one representative experiment of three experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear STAT1. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Immunofluorescence, Derivative Assay, Concentration Assay, Translocation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: (A) Immunofluorescence images comparing the abundance of phagocytosed fluorescent beads in human primary foetal (HPF) pericytes (left) and iPSC-derived pericytes (right). (B) Flow cytometry histo-plots show cultured primary cells to contain phagocytic (red) and non-phagocytic (black) cells. The auto-fluorescent threshold is denoted by the vertical red line. The gating strategy for flow cytometric analysis can be found in Figure S1. (C,D) Quantification of histo-plots shows a significant reduction in percentage of phagocytic HPF pericytes with IL-1β treatment, but no change in either day 21 or 42 iPSC-derived pericytes. No change in mean fluorescent intensity (MFI) was observed with either IL-1β or TNF treatment, though day 21 and day 42 iPSC-derived pericytes exhibited more phagocytic activity than HPF pericytes (D). Quantitative data presented is averaged from three to five experimental repeats. Significance is determined using a 2-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Two different primary pericyte cell suppliers were used in this study: Creative Bioarray Primary Human Brain Cortex Pericyte Cells (catalogue#: CSC-C4387X, Creative Bioarray), and ScienCell Human Brain Vascular Pericytes (catalogue#: 1200, Sciencell).

Techniques: Immunofluorescence, Derivative Assay, Flow Cytometry, Cell Culture, Activity Assay

Journal: Advanced Science

Article Title: A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood–Brain Barrier Transport Properties

doi: 10.1002/advs.202004856

Figure Lengend Snippet: Linear regression analysis and goodness‐of‐fit ( R 2 ) values of generated sizes of BBB spheroids after six days postseeding, including human primary astrocytes (hA), human primary pericytes (hP), and immortalized hCMEC/D3 (BEC) in a ratio of 1:1:3 in respect to initial seeding densities. Statistical significance of respective slopes was determined by analysis of covariance (ANCOVA). Data are expressed as mean value ± SD for n = 6. Underlined values are considered as the most optimal seeding density

Article Snippet: Human primary pericytes (hP; SC‐1200, Provitro AG, Germany) were cultivated in pericyte medium PM (ScienCell, USA) supplemented with 2% FBS (ScienCell, USA), 1% of penicillin/streptomycin (ScienCell, USA), and 1% pericyte growth supplement (ScienCell, USA).

Techniques: Generated

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: (A) Schematic of the brain pericyte differentiation protocol developed by Stebbins et al . (B) Images of differentiating cells from iPSC to day 42 (D42) of pericyte differentiation. NCSC priming (D0-D15) results in a heterogeneous population of cells including larger cells at the colony border (white arrows). NCSCs are isolated and grown in pericyte differentiation medium, at which point a more homogenous population of cells can be seen (D21). Differentiating cells acquire an elongated morphology over the period of pericyte differentiation (D15-D42). This morphology is comparable to the morphology seen in human primary foetal pericytes. Scale bar = 200μm.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Isolation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: (A) RT-qPCR shows high levels of expression of pluripotency gene expression (OCT3/4, SOX2, and NANOG) in iPSCs, with lower levels of expression seen in iPSC-derived pericytes. These pluripotency genes are also expressed at low levels in human primary foetal (HPF) pericytes. (B) Immunofluorescence images demonstrating pluripotency marker protein expression (OCT3/4, SOX2, and NANOG) in iPSCs, but not iPSC-derived pericytes. (C) RT-qPCR shows gene expression of pericyte markers (PDGFRβ, NG2, CD13, and αSMA) in iPSC-derived and HPF pericytes. (D) Immunofluorescence images demonstrating pericyte marker protein expression (PDGFRβ, CD13, and αSMA) in day 42 iPSC-derived pericytes. High levels of αSMA protein expression are observed in iPSCs and day 21 iPSC-derived pericytes (E) RT-qPCR shows gene expression of brain-specific pericyte markers (FOXF2, FOXC1, and vitronectin) in day 42 iPSC-derived pericytes and HPF pericytes. Gene expression of FOXF2 is absent in iPSCs and NCSCs. The dotted line on all RT-qPCR graphs indicates a ΔCt of 30, demonstrating the minimum ΔCt threshold of expression in these experiments. “Neg. Con.” refers to the negative control that didn’t receive primary antibody. Scale bar = 100μm in all images. Error bars represent standard deviation between the three experimental repeats.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Immunofluorescence, Marker, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating nuclear translocation of NFκB in response to increasing concentrations of IL-1β in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) brain pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which demonstrate NFκB translocation at an EC 50 of 6.76pM in day 21 iPSC-derived pericytes (B, dotted line), and 4.64pM in day 42 iPSC-derived pericytes (dotted line, D). Images of HPF pericytes are quantified using MetaXpress, showing an EC 50 of 2.26pM (dotted line, F). Data presented is one representative experiment of two experimental repeats with the iPSC-derived pericytes, and one representative experiment of four experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear NFκB. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Immunofluorescence, Translocation Assay, Derivative Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating the subcellular localisation of STAT1 in response to increasing concentrations of IL-1β in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) brain pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which demonstrate STAT1 translocation at a potent EC 50 of 0.74pM in day 21 iPSC-derived pericytes (B, dotted line), but not in day 42 iPSC-derived pericytes. Images of HPF pericytes are quantified using MetaXpress, showing STAT1 translocation at an EC 50 of 4.19pM (dotted line, F) in a minor subset of HPF pericytes. Data presented is one representative experiment of two experimental repeats with the iPSC-derived pericytes, and one representative experiment of three experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear STAT1. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Immunofluorescence, Derivative Assay, Concentration Assay, Translocation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating nuclear translocation of NFκB in response to increasing concentrations of TNF in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) brain pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which demonstrates NFκB translocation at an EC 50 of 14.2pM in day 21 iPSC-derived pericytes (B dotted line). The concentration-response curve did not plateau in day 42 iPSC-derived pericytes due to a lack of cell viability at the highest treatment concentration (D). Images of HPF pericytes are quantified using MetaXpress, showing NFκB translocation at an EC 50 of 1.84pM (dotted line, F). Data presented is one representative experiment of two experimental repeats with the day 21 iPSC-derived pericytes, one experimental repeat with the day 42 iPSC-derived pericytes, and one representative experiment of three experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear NFκB. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Immunofluorescence, Translocation Assay, Derivative Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: Immunofluorescence images demonstrating the subcellular localisation of STAT1 in response to increasing concentrations of TNF in day 21 iPSC-derived pericytes (A), day 42 iPSC-derived pericytes (C), and human primary foetal (HPF) pericytes (E). Images of iPSC-derived pericytes are quantified using an ImageJ macro to generate concentration-response curves (B, D) which shows no STAT1 translocation in response to TNF treatment. The day 42 iPSC-derived pericytes lacked cell viability at the highest treatment concentration (C, D). Images of HPF pericytes are quantified using MetaXpress, showing very potent STAT1 translocation in a minor subset of cells at an EC 50 of 0.289pM (dotted line, F). Data presented is one representative experiment of two experimental repeats with the day 21 iPSC-derived pericytes, one experimental repeat with the day 42 iPSC-derived pericytes, and one representative experiment of three experimental repeats with the HPF pericytes (see Table S9). Scale bar = 200µm with exception of 40µm for all further magnified images. White arrows indicate nuclear STAT1. Error bars represent standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparisons test. * = P<0.05, ** = P<0.01, *** = P<0.001.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Immunofluorescence, Derivative Assay, Concentration Assay, Translocation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

doi: 10.1101/2024.09.16.613375

Figure Lengend Snippet: (A) Immunofluorescence images comparing the abundance of phagocytosed fluorescent beads in human primary foetal (HPF) pericytes (left) and iPSC-derived pericytes (right). (B) Flow cytometry histo-plots show cultured primary cells to contain phagocytic (red) and non-phagocytic (black) cells. The auto-fluorescent threshold is denoted by the vertical red line. The gating strategy for flow cytometric analysis can be found in Figure S1. (C,D) Quantification of histo-plots shows a significant reduction in percentage of phagocytic HPF pericytes with IL-1β treatment, but no change in either day 21 or 42 iPSC-derived pericytes. No change in mean fluorescent intensity (MFI) was observed with either IL-1β or TNF treatment, though day 21 and day 42 iPSC-derived pericytes exhibited more phagocytic activity than HPF pericytes (D). Quantitative data presented is averaged from three to five experimental repeats. Significance is determined using a 2-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The Creative Bioarray Primary Human Brain Cortex Pericyte Cells were used as a positive control for characterisation of the iPSC-derived brain pericytes, including RT-qPCR and immunocytochemistry related to characterisation.

Techniques: Immunofluorescence, Derivative Assay, Flow Cytometry, Cell Culture, Activity Assay

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: Model creation and colorectal cancer-secreted cytokine profiling ( A) Schematic of hydrogel model creation and cluster analysis of CRC cell line cytokine profiles. Media containing secreted cytokines and exosomes of Caco2, SW480, and HCT116 CRC cell lines was isolated via a centrifugation protocol. Normal human fibroblasts or microvascular pericytes were suspended in collagen I-hyaluronic acid hydrogel matrices and cultured in CRC conditioned media to study cellular response to primary tumor soluble signals. (B–C) Clustering analysis of conditioned media based on a 200 cytokine array showed distinct signatures for conditioned media produced by each cell line. Cytokines upregulated in metastatic cell-conditioned media and their expression levels are shown in (C). CM – Caco2 Media, SM – SW480 Media, HM – HCT116 Media, FM – Fibroblast Control Media, PM – Pericyte Control Media.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Isolation, Centrifugation, Cell Culture, Produced, Expressing, Control

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: CRC Secretome Differentially Activates Pericytes and Fibroblasts in 3D Hydrogel Constructs. CRC-derived factors differentially activate fibroblasts and pericytes in the hydrogel microenvironment (A) Representative images of phalloidin (green)/α-SMA (red)/DAPI (blue) stain on fibroblasts (top) and pericytes (bottom) treated for 3 days with colorectal cancer conditioned media. (B and C) Eccentricity quantification of fibroblasts and pericytes in each condition, where a score of 0 indicates circularity, and a score nearing one indicates a spindle-like shape. (D and E) Fluorescence quantification of α-SMA expression per cell. Scale bar 50 μm. Significance: ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Construct, Derivative Assay, Staining, Fluorescence, Expressing

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: Pericytes Exhibit a Relaxed Phenotype in Response to CRC-Secreted Factors CRC-derived factors promote pericyte relaxation and changes in cell-matrix interactions. (an i-iv) Representative images of pericytes stained for phosphorylated myosin light chain (green, iii.), paxillin (orange, iv.), and cell nuclei (blue, ii.) after three days of culture in conditioned media. (B) Fluorescence quantification of p -MLC. Phosphorylation of myosin light chain decreases significantly in pericytes exposed to HCT116 CM as compared to control conditions. (C) Fluorescence quantification of paxillin. No significant change in paxillin expression was observed for any condition, despite visual differences in staining patterns. Scale bar 20 μm. Significance: ∗∗∗p < 0.001. Data are represented as mean ± SEM

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Derivative Assay, Staining, Fluorescence, Phospho-proteomics, Control, Expressing

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: CRC Secretome Drives Matrix Metalloproteinase Signaling and Collagen Deposition CCSF exposure changes collagen degradation and deposition behaviors for both pericytes and fibroblasts. (A–C) SW480 conditioned media significantly increases MMPs two and nine expressions by fibroblasts. All CRC media upregulate MMP13 expression, with HCT116 media inducing the largest change. (D–F) Pericyte MMP2 expression is significantly downregulated with exposure to CCSFs, and additionally correlates with CRC cell line aggression. Pericyte MMP13 is underexpressed with CCSF exposure, and MMP9 shows no clear trend relative to media type. (G and H) HCT116 conditioned media significantly increases the quantity of collagen deposition by fibroblasts; pericytes follow the same trend but have no significant changes. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM n ≥ 3.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Expressing

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: Pericytes and Fibroblasts Reorganize Collagen Fiber Architecture and Density in the Presence of CCSFs Picrosirius red stain of collagen fibers in fibroblast and pericyte-laden hydrogels.(A) Polarized light images of picrosirius red stains were analyzed using CT-Fire software. (B) Collagen fiber density was slightly increased for all CCSF conditions in fibroblast cultures, and significantly lowered for SW480 and HCT116 conditions in pericyte cultures. (C) Collagen fiber width skewed lower in fibroblasts CCSF cultures compared to normal control and higher in pericyte SW480 and HCT116 cultures. Fiber length was not affected by CCSF exposure in either fibroblast or pericyte cultures. Collagen alignment was lower, i.e., was less organized, in fibroblast and pericyte hydrogels cultured with HCT116 media. Scale bar 50 μm. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Bar plots are shown as mean ± SEM Box and whisker plots are presented with Tukey formatting and horizontal bar at the median.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Staining, Software, Control, Cell Culture, Whisker Assay

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet:

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Recombinant, Collagen Assay, Staining, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Software

Journal: Science Advances

Article Title: PAI-1 is a vascular cell–specific HIF-2–dependent angiogenic factor that promotes retinal neovascularization in diabetic patients

doi: 10.1126/sciadv.abm1896

Figure Lengend Snippet: ( A ) Accumulation of HIF-2α in the GCL in OIR mice from P12 to P17. ( B ) Increased Hif2α mRNA expression in the GCL by RNAscope. ( C to E ) Coexpression of HIF-1α with isolectin B4 (lectin), CD31, or chondroitin sulfate proteoglycan 4 (NG2) was not detected in OIR mice retinas at P13 by immunofluorescence (IF). ( F and G ) Coexpression of HIF-2α with endothelial cell marker CD31 (e) or the pericyte marker NG2 (p) was observed in OIR mice retinas at P16. n = 4 to 6 animals; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bars, 100 μm.

Article Snippet: iHUVECs, primary HRECs, and primary human pericytes were obtained from Lonza and cultured according to the manufacturer’s protocols.

Techniques: Expressing, RNAscope, Immunofluorescence, Marker

Journal: Science Advances

Article Title: PAI-1 is a vascular cell–specific HIF-2–dependent angiogenic factor that promotes retinal neovascularization in diabetic patients

doi: 10.1126/sciadv.abm1896

Figure Lengend Snippet: ( A and B ) Retinal NV in the OIR model occurs at or above the GCL, containing resident glial cells (Müller cells, astrocytes, and microglia) and retinal ganglion cells (RGCs). Coexpression of PAI-1 with (A) GFAP (to label astrocytes and Müller cells) and (B) RBPMS (to label RGCs) was not detected. ( C to E ) Coexpression of PAI-1 with (C) IB4 (to label vascular and microglia cells), (D) CD34 (to label endothelial cells), and (E) NG2 (to label pericytes) was observed in retinal NV tissue in OIR mice by IF. ( F ) Coexpression of PAI-1 with HIF-2α in retinal NV tissue. n = 6 animals. Scale bars, 50 μm (A and C to F) and 100 μm (B).

Article Snippet: iHUVECs, primary HRECs, and primary human pericytes were obtained from Lonza and cultured according to the manufacturer’s protocols.

Techniques:

Journal: Science Advances

Article Title: PAI-1 is a vascular cell–specific HIF-2–dependent angiogenic factor that promotes retinal neovascularization in diabetic patients

doi: 10.1126/sciadv.abm1896

Figure Lengend Snippet: ( A and B ) Expression of PAI1 mRNA in iHUVEC exposed to hypoxia in vitro (A) and in OIR mice retinas in vivo (B) after treatment with polymer nanoparticle–mediated RNAi (NP-siRNA) knockdown of PAI-1. ( C ) Retinal NV (outlined) at P17 in OIR mice following the intravitreal injection with NP-pai-1 or NP-scr (as a control). ( D and E ) Quantitation of avascular retina and retinal NV at P17 after the intravitreal injection with NP-pai-1 or NP-scr. n = 6 to 8 animals. Data are shown as means ± SD. Statistical analyses were performed by one-way ANOVA with Bonferroni’s multiple-comparison test (A), two-way ANOVA with Bonferroni’s multiple-comparison test (B), or two-tailed unpaired Student’s t test (D and E). * P < 0.05; ** P < 0.01. Scale bars, 500 μm. ( F and G ) A schematic representation of therapies for retinal NV in ischemic retinal disease. In ischemic retinopathies, decreased perfusion of the inner retina (F) results in accumulation of HIF-1α and HIF-2α in hypoxic retinal Müller cells (G), resulting in the secretion of angiogenic mediators. These paracrine secretions are effectively targeted by PRP. ( H ) While anti-VEGF therapy can inhibit VEGF released by endothelial cells and pericytes, other angiogenic autocrine/paracrine mediators released by retinal vascular cells are not effectively treated with PRP or anti-VEGF therapy. Therapies targeting the HIF-2–dependent expression of PAI-1 by retinal vascular cells may be an effective adjunct therapy for the treatment of ischemia-driven retinal NV.

Article Snippet: iHUVECs, primary HRECs, and primary human pericytes were obtained from Lonza and cultured according to the manufacturer’s protocols.

Techniques: Expressing, In Vitro, In Vivo, Polymer, Knockdown, Injection, Control, Quantitation Assay, Comparison, Two Tailed Test