Journal: eLife

Article Title: Statistical analysis supports pervasive RNA subcellular localization and alternative 3' UTR regulation

doi: 10.7554/eLife.87517

Figure Lengend Snippet: ( a ) Experimental setup for human pericyte cell culture with reverse-transcriptase quantitative PCR (RT-qPCR) and extracellular TIMP3 protein ELISA readouts at four-timepoints. ( b ) TIMP3 protein secretion per cell per hour does not significantly change throughout culture time, even though the total protein measured by BCA does change. ( c ) qPCR experiment design with proximal and distal qPCR primers to distinguish long and short 3’ UTR isoforms. The proximal qPCR primer can detect both long and short isoforms while the distal primer can only amplify the long 3’ UTR. ( d ) The ratio of distal to proximal primer-template abundances significantly decreases throughout culture time, implying increased usage of the short TIMP3 3’ UTR compared to the long isoform. ( e ) TIMP3 3’ UTR abundance, normalized by 18 s housekeeper abundance, fluctuates from halving to doubling between culture timepoints for both distal and proximal primers.

Article Snippet: Human brain vascular pericytes (PCs, Sciencell) were cultured up to passage 5 in low-glucose DMEM (Gibco) supplemented with 10% FBS.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Statistical analysis supports pervasive RNA subcellular localization and alternative 3' UTR regulation

doi: 10.7554/eLife.87517

Figure Lengend Snippet:

Article Snippet: Human brain vascular pericytes (PCs, Sciencell) were cultured up to passage 5 in low-glucose DMEM (Gibco) supplemented with 10% FBS.

Techniques: Software, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share glucose with astrocytes in an occludin-modulated manner. (a) Confocal microscopy of a representative live pericyte-astrocyte co-culture 30 min after plating. Pericytes were pre-loaded with 2-NBDG (green; e.g. blue thick arrows) and astrocytes with violet-BMQC (cell mask, red) but not with 2-NBDG. Colocalization of both signals (yellow-orange; e.g. white thin arrows) indicated that astrocytes had received 2-NBDG. Intensity coefficient (Ic) represents the whole 2-NBDG fluorescence intensity normalized to the surface it occupies, regardless of cell type. A larger Ic means more 2-NBDG was introduced into the system (taken up by pericytes). Transfer coefficient (Tc) represents the fraction of astrocytic surface occupied by 2-NBDG normalized against the Intensity coefficient. A larger Tc implicates more 2-NBDG was distributed across all possible astrocytes, and represents greater transferred volumes. Data correspond to the quantitation of the images shown. They are representative of three separate experiments. (b) Distribution of 2-NBDG in the same co-culture shown in (a), 24 h after plating. Pericytes are devoid of any stain (e.g. white thin arrows). 2-NBDG signal (green) colocalizing with astrocytes is seen as cyan/white (e.g. yellow thick arrows). (c) Similar co-culture as in (a) recorded 30 min post-plating; however, pericytes were treated with negative-control siRNA (SCR) before being loaded with 2-NBDG. (d) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (c), 24 h post-plating. (e) Similar co-culture as in (a) and (c), recorded 30 min post-plating; however, pericytes were treated with anti-occludin siRNA (OCC−) before being loaded with 2-NBDG. (f) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (e), 24 h post-plating. All images are representative of three separate experiments. (g) Average intensity (Ic) and H) Transfer (Tc) coefficients depicting transcellular glucose transport between pericytes and astrocytes as shown in (a) to (c). n = 3, p vs. WT.

Article Snippet: Cell culture Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37°C in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Confocal Microscopy, Co-Culture Assay, Fluorescence, Quantitation Assay, Staining, Negative Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share mitochondria with astrocytes in an occludin-mediated manner. (a) Live co-culture, 24 h post-plating, of astrocytes labeled with violet-BMQC (blue) and mitochondria-stained (TMRE in green) pericytes treated with anti-occludin siRNA (OCC−), negative-control siRNA (SCR), or non-treated (wild-type, WT). Thin white arrows exemplify TMRE-stained pericytes, while thick yellow arrows show pericyte mitochondria in the body of astrocytes (cyan signal). (b) Quantitation of TMRE intensities in astrocytes and pericytes in the same co-cultures shown in (a). Average ± SEM, n = 25 collected from three experiments, p vs. SCR. Only significant values are shown (c) Astrocytes treated with vehicle (Veh) or with endosulfan sulfate (ES, 4 h) to block their energetic metabolism. Middle image, surviving astrocytes exhibiting widened bodies and gross morphological alterations are exemplified by thin white arrows. Yellow arrowheads point to astrocytes that still retain their normal morphology. Right image: astrocytes treated with ES; however, isolated murine live brain capillaries (D shows a single brain capillary) were added to their growth medium 2 h post-treatment, and incubated for two additional hours. Note markedly improved astrocyte morphology. (e) Not-labeled human astrocytes cultured with murine live brain capillaries pre-labeled with TMRE (red) and 2-NBDG (green) for 2 h. TMRE and 2-NBDG transferred from microvessels to astrocytes (arrows) indicate transfer of mitochondria and glucose, respectively. (f) Similar TMRE and 2-NBDG transfer (arrows) in microvessel-rescued/ES-treated astrocytes after incubation with TMRE and 2-NBDG-labeled murine live brain capillaries.

Article Snippet: Cell culture Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37°C in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Co-Culture Assay, Labeling, Staining, Negative Control, Quantitation Assay, Blocking Assay, Isolation, Incubation, Cell Culture

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: Journal of thrombosis and haemostasis : JTH

Article Title: A Unique Protein Kinase C-Dependent Pathway for Tissue Factor Downregulation in Pericytes

doi: 10.1111/jth.14399

Figure Lengend Snippet: PMA actively downregulates TF in primary human pericytes. Pericyte cultures were treated with PMA for the indicated times. The mean +/− standard deviation (SD) of 3 independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001. A. Expression of TF protein was assessed by western blot. B. For each time point the relative amount of TF protein expressed by PMA-treated cells is shown as a percentage of TF expressed by vehicle-treated cells. C. Activity of total TF as measured in lysed pericytes was determined by measuring cleavage of a FXa-specific chromogenic substrate as described in “Methods”. TF activity is presented as the rate of FXa generation. D. Expression of TF mRNA was quantitated by qRT-PCR. For each time point the amount of TF mRNA expressed by PMA-treated cells is presented as the fold change relative to TF mRNA expressed by vehicle-treated control cells.

Article Snippet: Placental-derived primary human pericytes (#PER-F) and pericyte growth media (#PER-1) were purchased from Zen-bio, Inc. Pericytes were cultured according to manufacturer’s protocol.

Techniques: Standard Deviation, Expressing, Western Blot, Activity Assay, Quantitative RT-PCR, Control

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A Unique Protein Kinase C-Dependent Pathway for Tissue Factor Downregulation in Pericytes

doi: 10.1111/jth.14399

Figure Lengend Snippet: Internalized TF is degraded primarily in lysosomes. Pericytes were treated with MG132 or chloroquine prior to PMA for the indicated times. The mean + SD of 3 independent experiments is shown. **p<0.01, ***p<0.001. A. Relative amounts of internalized TF and expression of total TF were assessed by biotin labeling of cell surface proteins and western blotting as in “Methods”. B. The amounts of internalized TF are expressed as percentages relative to the total amount of TF expressed on the cell surface. C. Relative amounts of total TF expressed by PMA-treated pericytes are shown as percentages of TF expressed by control cells. D. Pericytes were stained for TF (green), the lysosomal marker LAMP-1 (red), and nuclei (blue, DAPI). E. Co-localization of TF and LAMP-1 was determined by calculating Pearson’s Correlation Coefficient with Image J, Fiji Plugin.

Article Snippet: Placental-derived primary human pericytes (#PER-F) and pericyte growth media (#PER-1) were purchased from Zen-bio, Inc. Pericytes were cultured according to manufacturer’s protocol.

Techniques: Expressing, Labeling, Western Blot, Control, Staining, Marker

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A Unique Protein Kinase C-Dependent Pathway for Tissue Factor Downregulation in Pericytes

doi: 10.1111/jth.14399

Figure Lengend Snippet: PMA shortens the half-life of TF protein while leaving degradation of TF mRNA unaffected. The mean +/− standard deviation (SD) of 3 independent experiments is shown. **p<0.01, ***p<0.001. A. Pericytes were pre-treated with Cyclohexamide (CHX) prior to addition of vehicle or PMA for the indicated times. Degradation of TF protein was assessed by western blot. B. Relative amounts of TF protein expressed by CHX-treated cells are given as a percentage of TF expressed by non-CHX-treated control cells. C. Pericytes were pre-treated with Actinomycin D (ActD) prior to receiving vehicle or PMA for the indicated times. Degradation of TF mRNA was analyzed by qRT-PCR. The amount of TF mRNA expressed by ActD-treated pericytes relative to non-ActD-treated control cells is shown.

Article Snippet: Placental-derived primary human pericytes (#PER-F) and pericyte growth media (#PER-1) were purchased from Zen-bio, Inc. Pericytes were cultured according to manufacturer’s protocol.

Techniques: Standard Deviation, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A Unique Protein Kinase C-Dependent Pathway for Tissue Factor Downregulation in Pericytes

doi: 10.1111/jth.14399

Figure Lengend Snippet: Inhibition of Protein Kinase C attenuates PMA-mediated downregulation of TF. Pericytes were pre-treated with Go 6983 or GFX prior to PMA for the indicated times. The mean + SD of 3 independent experiments is shown. *p<0.5, **p<0.1, ***p<0.001. A. TF protein expression was analyzed by western blot. B. For each time point the amount of TF protein is shown as a percentage of the TF expressed by control cells. C. Expression of TF mRNA was quantified by qRT-PCR. The amount of expressed TF mRNA is presented as the fold change relative to TF mRNA expressed by control cells. D. Activity of total TF in lysed pericytes is shown as the rate of FXa generation.

Article Snippet: Placental-derived primary human pericytes (#PER-F) and pericyte growth media (#PER-1) were purchased from Zen-bio, Inc. Pericytes were cultured according to manufacturer’s protocol.

Techniques: Inhibition, Expressing, Western Blot, Control, Quantitative RT-PCR, Activity Assay

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A Unique Protein Kinase C-Dependent Pathway for Tissue Factor Downregulation in Pericytes

doi: 10.1111/jth.14399

Figure Lengend Snippet: PMA increases internalization of TF from the pericyte surface. Where specified, pericytes were pre-treated with either Go 6983 or GFX prior to receiving PMA for the times indicated. The mean +SD of 3 independent experiments is shown. **p<0.01, ***p<0.001. A. Pericyte cultures were fixed and stained for TF (green). Prominent punctate staining for TF was seen 4 hours after PMA while TF was virtually absent 8 hours after PMA. B. The total TF content of pericytes was determined by western blotting of whole cell lysates. Internalized surface TF was determined by labeling cell surface proteins with biotin, isolating biotin-labeled proteins from cell lysates, and assessing their TF content by western blotting. C. Relative amounts of internalized TF are shown as a percentage of the total surface TF. D. Relative amounts of total TF are presented as a percentage of the total surface TF expressed by vehicle-treated control cells. E. Activity of surface TF was determined on whole cells by measuring the rate of FXa generation.

Article Snippet: Placental-derived primary human pericytes (#PER-F) and pericyte growth media (#PER-1) were purchased from Zen-bio, Inc. Pericytes were cultured according to manufacturer’s protocol.

Techniques: Staining, Western Blot, Labeling, Control, Activity Assay

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

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: 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