creative primary human brain cortex pericyte cells  (Bioarray Inc)

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:

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: Functional 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