Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 1. The SM22α-Cre specificity in the mouse brain vasculature. mT/mG reporter mice were bred with SM22α-Cre deleter mice (mT/mG:SM22α-Cre), and brain tissues were harvested at postnatal day 6 (P6). A, Sections of cerebellum and cerebrum were detected for mG expression by fluorescence microscopy. SM22α-Cre-driven mG was specifically detected in microvessels of both cerebrum and cerebellum (arrowheads) but not in control mT/mG mice (arrows). High power images of arrowhead-indicated regions are shown on the right. n=3 mice per group. B, Cerebral sections were immunostained with anti- PDGFR-β followed by an allophycocyanin (APC)-conjugated secondary antibody with an IgG isotype as a control. mG expression was colocalized with the pericyte (PC) marker PDGFR-β in the brain microvasculature of mT/mG:SM22α-Cre (arrowhead), but not in the IgG staining or in the control mT/mG mice (arrow). n=3 mice per group. C, mG+ and mG− cell populations were isolated from P6 mT/mG:SM22α- Cre brain tissues, and gene expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with specific PC and endothelial cell (EC) markers as indicated. mG+ cells expressed PC marker genes PDGFRB (PDGFR-β, SCPG4 [NG-2], and ANPEP [CD13], but not EC marker genes PECAM1 [CD31], VEGFR2 [VEGFR2] and CDH5 [VE-cadherin]) with normalization by GAPDH. Data are mean±SEM; n=3; ***P<0.001 by unpaired 2-tailed Student t test. D and E, Mouse brain microvascular pericytes (mBMVPCs) and ECs (mBMVECs) were isolated from wild-type (WT) mice at P6, and immunostained with PC marker PDGFR-β and EC marker VE-cadherin. Phase images (D) and immunofluorescence images (E) are presented. F–I, Ccm3 deletion was specifically in mouse brain PCs but not in mouse brain ECs. mBMVPCs and mBMVECs were isolated from P6 WT and Ccm3smKO brain tissues. F, Cells were immunostained with PC marker PDGFR-β and EC marker VE-cadherin. G, Ccm3 gene expression was determined by qRT-PCR. n=3; ***P<0.001 by unpaired 2-tailed Student t test. H, CCM3 protein was determined by Western blotting. Representative blot form 3 experiments. I, CCM3 protein was determined by immunostaining using an anti-CCM3 antibody with costaining of antipaxillin antibody. n=3. Scale bar: 50 μm (A, B, and D); 25 μm (E and F); 10 μm (I).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Expressing, Fluorescence, Microscopy, Control, Marker, Staining, Isolation, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Western Blot, Immunostaining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 3 Continued. Representative images are shown in E. Quantification of % GFAP coverage on CD31+-vessel was quantified by Image J (F). G–I, Mouse brain microvascular pericytes (mBMVPCs) were isolated from neonatal WT and CCM3 smKO brains. 4×105 WT and CCM3- knockout (KO) mBMVPCs were seeded on fibronectin-coated culture slides for indicated times (0–16 h), and unattached cells were washed away. Cells were fixed with 4% paraformaldehyde (PFA) and stained with phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Representative images for each time point are shown (G). Cell area (H) and cell length (I) were measured by Image J software. Ten fields were counted and n=3 repeated experiments. J and K, RNA-seq analyses. The endogenous CCM3 was knocked out by CRISPR/Cas9. mRNA from confluent WT and CCM3-KO human brain microvascular pericytes (hBMVPCs) were subjected to RNA-seq analyses. J, Gene expression value was estimated by Cufflinks (v1.2.0) and genes with >2-fold change between WT and KO were defined as differential expression. K, Gene Ontology analysis using GOstats was performed and the significant pathways (muscle cell migration and extracellular matrix [ECM] organization) are presented. n=2. Data are means±SEM. Scale bars: 25 μm (A, C, G, and I); 500 nm (E).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Isolation, Knock-Out, Staining, Software, RNA Sequencing, CRISPR, Gene Expression, Quantitative Proteomics, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 5. Cerebral cavernous malformation (CCM)3-knockout (KO) pericytes (PCs) attenuates PC migration and endothelial cell (EC)-PC interactions. CCM3-KO human brain microvascular pericytes (hBMVPCs) were re-expressed with vector (VC), CCM3-wild type (WT), or CCM3-4KE by lentivirus infection. A, WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were harvested and subjected to Western blotting to test for adhesion complexes and RhoA-pMLC signaling. Relative protein levels were quantified and fold changes are presented by keeping WT as 1.0. B and C, 4×105 hBMVPCs were seeded on fibronectin-coated culture slides for 16 h. Cells were fixed with 4% paraformaldehyde (PFA) followed costaining with phosphor-paxillin (green) and phalloidin (red) with DAPI counterstaining (blue) (B). Number of FA per cell was measured by Image J software. Ten fields were counted and n=3 repeated experiments. D and E, Rescue PC migration by CCM3-WT but not by paxillin-defective CCM3-4KE mutant. WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were subjected to wound injury followed by incubation for 24 h. D, Representative images of cell migration are shown. Dashed lines indicate the remaining gaps. E, Quantitation of EC migration. The percentage of unhealed wound was quantified, n=3. F and G, EC-PC interactions in 3-dimensional spheroid sprouting assay. Human brain microvascular ECs (hBMVECs) were infected with EGFP (enhanced green fluorescent protein)-expressing retroviruses, whereas WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were infected with mCherry-expressing lentiviruses. ECs and PCs (2:1 ratio) were seeded to beads and coated with microbeads, embedded in fibrin gels and grown in EGM-2 endothelial growth medium for 4 d. A representative image of 10 beads for each sample is shown in F and percentage of PC coverage of sprouts is quantified in G. n=10, *P<0.05; **P<0.01 (1- way ANOVA). Additional 2 independent experiments were performed. Error bars indicate SEM. Scale bar: 10 μm (B); 100 μm (D and F).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Knock-Out, Migration, Plasmid Preparation, Infection, Western Blot, Software, Mutagenesis, Incubation, Quantitation Assay, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 6. Cerebral cavernous malformation (CCM)3 loss in pericyte (PC) induces extracellular matrix (ECM) deposition in CCM. A and B, Increased ECM deposition in CCM3-deficient human brain microvascular pericytes (hBMVPCs). Wild-type (WT) and CCM3-KO hBMVPCs were cultured confluently on fibronectin-coated culture slides for 16 h. Cells were subjected to costaining with fibronectin (green) and Col IV (red) with DAPI counterstaining (blue). Mean fluorescence intensity (MFI)/per cell were measured by Image J software. Ten fields were counted and n=3 repeated experiments. C and D, P6 WT and Ccm3smKO mouse brain sections for staining of CD31 with fibronectin or NG-2. IgG isotype was used as a control. Representative images are shown (C). Normalized fibronectin MFI were measure by Image J (by taking WT as 1.0). Scale bar: 50 μm (A and C). E, A model for CCM3-depleted PC in promoting CCM lesion progression. The brain microvessels have an extraordinarily high PC to endothelial cell (EC) ratio, and PCs have multiple slender processes extending longitudinally to cover capillary EC for vascular integrity. Integrin-mediated matrix interactions and PC migration are critical in this process. Focal adhesion- mediated cell adhesion is important for cell migration, but the extent of adhesion can govern migration speed. We observed that CCM3- deficient PCs exhibit excess adhesion due to enhanced ECM deposition, ITG-β1 (integrin β1) activation and paxillin-mediated focal adhesion. We propose that CCM3 loss in PCs enhances PC adhesion while reducing PC protrusion and migration along EC, leading to the disruption of PC-EC interactions and resulting in CCM lesion formation.

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Cell Culture, Fluorescence, Software, Staining, Control, Migration, Activation Assay, Disruption

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: Micromachines

Article Title: Recent Progress in Microfluidic Models of the Blood-Brain Barrier

doi: 10.3390/mi10060375

Figure Lengend Snippet: Cellular constituents of the blood-brain barrier (BBB). The BBB is formed by brain microvascular endothelial cells (BMECs), which are connected by tight junctions. The endothelium, together with the basal lamina, pericytes, and astrocytic end-feet forms the neurovascular unit. Some substances diffuse freely into and out of the brain parenchyma, others such as nutrients need specific transporters, while molecules such as insulin, leptin and transferrin are transported by receptor- mediated transcytosis. (Reprinted from Reference in accordance with the Creative Commons Attribution).

Article Snippet: Ref. [ ] , High-throught, the model harbors 96 or 40 chips in a 384-well plate. In each chip, a perfused vessel of BMECs was grown against an extracellular matrix gel, astrocytes and pericytes were added on the other side of the gel to complete the BBB model. , Human TY10 cell line (isolated from normal brain tissue from a patient with meningioma) , Human hBPCT cell line pericytes from brain tissue of a patient t. Human hAst cell line astrocytes from human primary astrocytes distributed by Lonza. , No artificial membranes, using extracellular matrix gel , Collagen-I , No electrode , claudin-5, VE-cadherin, PECAM-1 (immunofluorescence staining) , Not measured , FITC-dextran (20 kDa) , Developed a high-throughput plate-based model, and used to assess passage of large biopharmaceuticals across the BBB..

Techniques:

Journal: Micromachines

Article Title: Recent Progress in Microfluidic Models of the Blood-Brain Barrier

doi: 10.3390/mi10060375

Figure Lengend Snippet: The main characters of some microfluidic blood-brain barrier (BBB) models.

Article Snippet: Ref. [ ] , High-throught, the model harbors 96 or 40 chips in a 384-well plate. In each chip, a perfused vessel of BMECs was grown against an extracellular matrix gel, astrocytes and pericytes were added on the other side of the gel to complete the BBB model. , Human TY10 cell line (isolated from normal brain tissue from a patient with meningioma) , Human hBPCT cell line pericytes from brain tissue of a patient t. Human hAst cell line astrocytes from human primary astrocytes distributed by Lonza. , No artificial membranes, using extracellular matrix gel , Collagen-I , No electrode , claudin-5, VE-cadherin, PECAM-1 (immunofluorescence staining) , Not measured , FITC-dextran (20 kDa) , Developed a high-throughput plate-based model, and used to assess passage of large biopharmaceuticals across the BBB..

Techniques: Permeability, Immunofluorescence, Staining, Derivative Assay, Diffusion-based Assay, Marker, In Vivo, Western Blot, Microscopy, Isolation, Impedance Spectroscopy

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: Journal of Neuroinflammation

Article Title: Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease

doi: 10.1186/s12974-014-0219-y

Figure Lengend Snippet: Time course analysis of human cytomegalovirus-GFP (HCMV-GFP) infection of IBRB (inner blood-retinal barrier) cells. (A) Top panel: phase contrast images of human mock and infected retinal microvascular endothelial cells, retinal pericytes and Müller cells. Bottom panel: phase contrast images of infected retinal microvascular endothelial cells, retinal pericytes and Müller cells with a fluorescent overlay showing HCMV-GFP-positive cells. Magnification = 200x. (B) A graph showing the number of infected HCMV-GFP-positive retinal microvascular endothelial cells (open bars), retinal pericytes (gray bars) and Müller cells (black bars) per 4 × 10 6 total cells over the time course of 12, 24, 48 and 96 hours post infection.

Article Snippet: Primary human retinal capillary endothelial cells, retinal pericytes, human brain microvascular endothelial cells, human brain pericytes and human astrocytes were obtained from Cell Systems Corporation (Kirkland, WA, USA) and were cultivated in Pericyte Media (PM) from ScienCell (Carlsbad, CA, USA).

Techniques: Infection

Journal: Journal of Neuroinflammation

Article Title: Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease

doi: 10.1186/s12974-014-0219-y

Figure Lengend Snippet: Time course analysis of human cytomegalovius-GFP (HCMV-GFP) infection of BBB (blood-brain barrier) cells. For comparison purposes, (A) the top panel includes phase contrast images of human mock and infected brain microvascular endothelial cells, brain vascular pericytes and astrocytes. The bottom panel shows phase contrast images of infected brain microvascular endothelial cells, brain pericytes and astrocytes with a fluorescent overlay showing HCMV-GFP-positive cells. Total magnification is 200x. (B) A graph indicating the number of infected HCMV-GFP-positive brain microvascular endothelial cells (open bars), brain pericytes (gray bars) and astrocytes (black bars) per 1.25 × 10 6 total cells over the time course 12, 24, 48 and 96 hours post infection.

Article Snippet: Primary human retinal capillary endothelial cells, retinal pericytes, human brain microvascular endothelial cells, human brain pericytes and human astrocytes were obtained from Cell Systems Corporation (Kirkland, WA, USA) and were cultivated in Pericyte Media (PM) from ScienCell (Carlsbad, CA, USA).

Techniques: Infection

Journal: Journal of Neuroinflammation

Article Title: Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease

doi: 10.1186/s12974-014-0219-y

Figure Lengend Snippet: A retinal Tricell culture model. (A) Phase contrast image of a Tricell culture mixture of human retinal microvascular endothelial cells, pericytes and Müller cells representing the inner blood-retinal barrier (IBRB). (B) A live/dead stain of the retinal Tricell culture. (C) Triple labeled immunohistochemistry (IHC) of the IBRB cell culture (arrows indicate an endothelial (EC), pericyte (PC) and Müller cell (MC)). Magnifications = 200x.

Article Snippet: Primary human retinal capillary endothelial cells, retinal pericytes, human brain microvascular endothelial cells, human brain pericytes and human astrocytes were obtained from Cell Systems Corporation (Kirkland, WA, USA) and were cultivated in Pericyte Media (PM) from ScienCell (Carlsbad, CA, USA).

Techniques: Staining, Labeling, Immunohistochemistry, Cell Culture