Journal: The Journal of Cell Biology

Article Title: N-cadherin signaling via Trio assembles adherens junctions to restrict endothelial permeability

doi: 10.1083/jcb.201802076

Figure Lengend Snippet: Cdh2 deletion in ECs and pericytes increases junctional endothelial permeability. (A and B) Permeability times surface area (PS), a measure of transendothelial 125 I-albumin permeability, in lung (A) and brain (B) of Cre-negative (Cre − ) control and Cdh2 iEC-KO littermates. The data are expressed as the rate of 125 I-albumin leakage normalized to the dry weight of the tissue; n = 4–8 mice per group. *, P < 0.05, a two-tailed, unpaired t test. (C and D) Measurement of lung (C) and brain (D) transendothelial permeability using 10-kD and 70-kD fluorescent dextran tracers. Data are presented as ratio of volume of fluorescent tracer to volume of lung or brain tissue for Cre − , Cdh2 iEC-KO, and Cdh2 iPC-KO mice. n = 10–15 fields from three to four mice per group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ANOVA with Tukey’s post hoc test. (E) 3D projected images of lung tissue from Cre − , Cdh2 iEC-KO, and Cdh2 iPC-KO mice stained for endothelial (PECAM1; green), pericyte (PDGFRβ; magenta), and nuclear (DAPI; blue) markers. Bar, 10 µm. (F) Quantification of pericyte coverage of lung microvessels expressed as pericyte area per vessel area (PDGFRβ/PECAM1) using images in E; n = 12–16 fields from three to four mice per group. ns, ANOVA with Tukey’s post hoc test. (A–D and F) Data are shown as mean ± SD. See also Fig. S1.

Article Snippet: The pellet was suspended in pericyte media (1231; ScienCell) containing pericyte growth supplement (1252; ScienCell), 10% FBS, penicillin/streptomycin, and heparin (5.5 U per 1 ml), and centrifuged at 1,000 g for 5 min at 4°C.

Techniques: Permeability, Control, Two Tailed Test, Staining

Journal: Cellular and Molecular Bioengineering

Article Title: Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation

doi: 10.1007/s12195-019-00574-3

Figure Lengend Snippet: HGF is secreted from cultured perivascular cells. (a) Quantification of HGF secretion from smooth muscle cells or pericytes by ELISA (mean ± SEM; two-way ANOVA, Bonferroni correction; p ≤ 0.0001). (b) HGF expression by perivascular cells was confirmed in vitro in the pre-vascularized engineered constructs. Immunofluorescence staining of HGF at day 5 shows that HGF (red) co-localizes with alpha-smooth muscle cell actin (aSMA, perivascular cell marker, green) but not lectin (endothelial cell marker, blue).

Article Snippet: Human smooth muscle cells (hSMCs) and human pericytes were grown in complete medium 231 (M231500, GIBCO) and pericyte media (PM1201, ScienCell Research Laboratories), respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Construct, Immunofluorescence, Staining, Marker

Journal: Fluids and Barriers of the CNS

Article Title: Investigating receptor-mediated antibody transcytosis using blood–brain barrier organoid arrays

doi: 10.1186/s12987-021-00276-x

Figure Lengend Snippet: High-throughput blood–brain barrier organoid formation with microwell hydrogel arrays. a Schematic of microwell plates in 96-well plate format and representative phase contrast image of a GRi3D organoid array within a single well. Scale bar, 1 mm. b Quantification of blood–brain barrier organoid diameter in GRi3D microwell arrays within 24- or 96-well plate compared to organoids grown on agarose substrate. Each dot represents a single organoid, while different colors represent independent experiments. c Representative images of glass-mounted blood–brain barrier organoids labelled with cell type-specific markers showing the spatial distribution of brain endothelial cells (P-gp, magenta), pericytes (NG2, green) and astrocytes (CellTracker Red, cyan) within blood–brain barrier organoids grown in Gri3D arrays. Nuclei are labelled with DAPI (grey). Upper panels show single confocal sections below the spheroid surface. Scale bar, 100 μm. The yellow line shows the position of the orthogonal cross-section shown in the lower panel. Scale bar, 50 μm. Graphs show the mean line profile intensity fluorescence of each cell-type marker within organoids. Distance in μm is measured from left to right across the ROI shown within the dotted box. d Representative fluorescent image of a live/dead staining showing the viability of blood–brain barrier organoids grown on GRi3D arrays. Calcein AM in green labels live cells whereas ethidium homodimer-1 in magenta labels dead cells. Scale bar, 1 mm. Images on the right show a higher magnification of the boxed area. Scale bar 500 μm

Article Snippet: Human brain microvascular pericytes (HBVP, ScienCell Research Laboratories) were cultured in Pericyte Growth Medium (PGM) composed of Pericytes Medium (PM, ScienCell Research Laboratories) supplemented with 2% FBS, 1% Pericytes Growth Supplement (PGS, ScienCell Research Laboratories) and 1% penicillin/streptomycin.

Techniques: High Throughput Screening Assay, Fluorescence, Marker, Staining

Journal: bioRxiv

Article Title: Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

doi: 10.1101/2023.09.29.560066

Figure Lengend Snippet: (A) Phase contrast bright 4x magnification images of iPSCs, HBVPs, mesoderm iPericytes and neural crest iPericytes. Scale = 200µm. (B) Fold change gene expression measured by qPCR of pericyte genes PDGFRB, CSPG4, ACTA2 and pluripotency genes OCT4 and NANOG by iPSCs, neural crest iPericytes, mesoderm iPericytes and HBVPs (n = 3 per cell type). Data are normalised to HBVP cells, and comparisons were made using a one-way ANOVA: PDGFRB (F (3, 8) = 103.1, p < 0.0001), CSPG4 (F (3, 8) = 4671, p < 0.0001), ACTA2 (F (3, 8) = 9.340, p < 0.0054), OCT4 (F (3, 8) = 1686, p < 0.0001) and NANOG (F (3, 8) = 606.4, p < 0.0001). Post-hoc comparisons performed using Dunnett’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001. Data are shown as mean ± SD. (C) Immunocytochemistry showing expression of pericyte proteins CD13, NG2 and PDGFRβ (green) by HBVP, mesoderm iPericytes and neural crest iPericytes. Nuclei counter-stained with DAPI (blue). Scale = 20 µm.

Article Snippet: Similar results were observed when complete pericyte media (CPM), containing specialised pericyte growth supplement (ScienCell, USA), was used compared to PM ( ).

Techniques: Gene Expression, Immunocytochemistry, Expressing, Staining

Journal: bioRxiv

Article Title: Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

doi: 10.1101/2023.09.29.560066

Figure Lengend Snippet: (A) Principal components analysis showing separate clustering of mesoderm iPericytes and iPSCs from n = 3 different cell lines. (B) Heat map showing relative expression levels in iPSCs and mesoderm iPericytes of key genes typically expressed by iPSCs, pericytes, endothelial cells (EC), microglia (MG), oligodendrocyte precursor cells (OPCs), oligodendrocytes (OL), astrocytes (AST) and neurons (NEU). Warmer colours indicate higher expression, cooler colours indicate lower expression.

Article Snippet: Similar results were observed when complete pericyte media (CPM), containing specialised pericyte growth supplement (ScienCell, USA), was used compared to PM ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

doi: 10.1101/2023.09.29.560066

Figure Lengend Snippet: (A) iPericytes were incubated in basal pericyte media (PM) and treated with PDGF-BB (PM + PDGF-BB) while being exposed to 100 µM imatinib (PM + PDGF-BB + 100µM imatinib). Proliferation was measured using an EdU uptake assay. iPericytes that are EdU-positive are indicated by magenta, while total number of iPericytes were measured by DAPI (blue). Scale bar = 50 µm. (B) Quantification of HBVPs, neural crest iPericytes and mesoderm iPericytes proliferating (as indicated by EdU-positive staining) as a percentage of total cells following 24 h exposure to PM, complete pericyte media with pericyte growth factors (CPM) or PM + PDGF-BB (n = 8 per condition). Data were analysed using a one-way ANOVA: HBVP (F (2, 21) = 35.52, p < 0.0001); neural crest iPericyte (F (2, 21) = 30.85, p < 0.0001); mesoderm iPericyte (F (2, 21) = 191.4, p < 0.0001). (C) Quantification of changes to PDGF-BB-induced proliferation with increasing concentrations of imatinib over 24 h in HBVPs, neural crest iPericytes and mesoderm iPericytes (n = 8 per condition). Data were analysed using a one-way ANOVA or Kruskal-Wallis test: HBVP (F (3, 26) = 259.2, p < 0.0001); neural crest iPericyte (H = 24.41, p < 0.0001); mesoderm iPericyte (F (3, 28) = 221.5, p < 0.0001). For (B) and (C), post-hoc comparisons were performed using Dunnett’s multiple comparisons or Dunn’s test: * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001. Data shown as mean ± SD. (D) Heat map of key genes involved in pericyte proliferation in the PDGF-BB: PDGFRβ signalling pathway in HBVP, neural crest iPericytes and mesoderm iPericytes selected from Sweeney et al. 2016 .

Article Snippet: Similar results were observed when complete pericyte media (CPM), containing specialised pericyte growth supplement (ScienCell, USA), was used compared to PM ( ).

Techniques: Incubation, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: (a) Immunostaining showed that AKAP12 was not expressed in OPCs (PDGF-R-α positive cells) in the corpus callosum region in wild-type mouse brain sections. Instead, AKAP12 was confirmed to be expressed in pericytes (PDGF-R-β positive cells) in the corpus callosum region in wild-type mouse brain sections. Importantly, AKAP12-expressing pericytes were closely located to OPCs in the corpus callosum region. Bar=100 μm. (b) Double-staining of PDGF-R-β and AKAP12 using confocal microscopy confirmed that pericytes express AKAP12. Bar=20 μm. Please see Suppl Figure S6 for additional data from immunostaining with another pericyte marker CD13.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Immunostaining, Expressing, Double Staining, Confocal Microscopy, Marker

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: (a) Cultured pericytes expressed AKAP12 and the AKAP12 expression was suppressed by AKAP12 siRNA. β-actin was used as an internal control. (b) AKAP12 knockdown did not affect pericyte survival assessed by LDH assay. LDH amounts in the culture media were not different between control-siRNA-treated pericytes and AKAP12-siRNA-treated pericytes. Values are mean ± SD. N=5.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Cell Culture, Expressing, Control, Knockdown, Lactate Dehydrogenase Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: Proteome Profiler™ Human XL Cytokine Array using pericyte-conditioned-media showed that AKAP12 knockdown changed the pattern of secreting factors from pericytes. LIF, BDNF, GRO-α, and HGF are known to promote OPC differentiation. Those factors were downregulated in the AKAP12-deficient pericyte cultures. Please see Suppl Figure S8 for other factors from pericyte cultures.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Knockdown

Journal: Stem cells (Dayton, Ohio)

Article Title: A-Kinase Anchor Protein 12 is required for oligodendrocyte differentiation in adult white matter

doi: 10.1002/stem.2771

Figure Lengend Snippet: Cultured OPCs were maintained in control media, pericyte-conditioned media (P-CM), or conditioned media from AKAP12-deficient pericytes (P−CMAKAP12-). Five days later, those OPCs were used for immunostaining or western blotting analyses. (a-b) Immunostaining with anti-MBP (oligodendrocyte marker) antibody showed that P-CM increased the number of MBP−positive cells, but P-CMAKAP12- showed less effect on OPC maturation. Bar=100 μm. Values are mean ± SD. N=5. *P<0.05 vs control, and #P<0.05 vs P-CM. (c-d) The levels of MBP expression in P-CM-treated OPCs were also lower compared to ones in P−CMAKAP12- -treated OPCs. Values are mean ± SD. N=4. *P<0.05 vs P-CM.

Article Snippet: Human brain vascular pericytes [ 30 ] were cultured in pericyte basal medium (Sciencell research laboratories) containing 2% fetal bovine serum and pericyte growth supplement (Sciencell research laboratories) onto poly-l-lysine-coated plates.

Techniques: Cell Culture, Control, Immunostaining, Western Blot, Marker, Expressing