Journal: Scientific Reports

Article Title: Multicellular Self-Assembled Spheroidal Model of the Blood Brain Barrier

doi: 10.1038/srep01500

Figure Lengend Snippet: (Trans-well): Is currently the standard setup where the BECs are grown on a filter insert with small pores to allow permeability studies between the two chambers. In co-culturing conditions, pericytes in normally grown on the opposite side of the filter potentially allow some cell-cell interaction through the pores and the astrocytes are grown in the bottom of the lower chamber to possibly influence the other cells by secreted factors into the media. (Matrigel): Cells are seeded in a gel structure to support migration an assembling. In this setup the BECs form tube-like structures on which pericytes preferentially adhere to, while the astrocytes are more loosely attached to the cell junctions. (Spheroidal): This model is completely independent of any supportive scaffold material for cell organization. The cells are allowed to freely self-assemble based on the intrinsic properties of each cell type. In our novel MCS-BBB setup, three key cell types of the neurovascular unit spontaneously form the spheroidal structure. The BECs form an outer cell monolayer representing the lumen side. The astrocytes accumulate in the core of the spheroids that could be perceived as the parenchyma space. Finally, pericytes line up in between to mediate a three cellular layer arrangement reproducing the morphological arrangement of the different cell types in the BBB.

Article Snippet: HpPs were grown in pericyte growth supplemented medium (ScienCell, USA) and hpAs were grown in astrocyte growth supplemented medium (ScienCell, USA).

Techniques: Permeability, Migration

Journal: mAbs

Article Title: A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

doi:

Figure Lengend Snippet: Anti-pDGFRβ/VeGF-A scFv-Fc-scFv inhibits endothelial sprouting and pericyte/endothelial association in a coculture sprouting assay. Bevacizumab, anti-pDGFRβ mAb e9899 or the anti-pDGFR/VeGF-A scFv-Fc-scFv were used as antagonists in a endothelial:pericyte sprouting coculture assay as described in Methods. (A) Representative figures from each of the four groups (25 nM concentration). (B) Quantitative data using metamorph software for the four groups (25 nM concentration). (C) Dose response curve for bevacizumab alone or anti-VeGF-A scFv-Fc-scFv. the anti-mAb did not have any effect on endothelia sprouting in the assay (data not shown).

Article Snippet: Human brain vascular pericytes (HBVPs) were seeded in 96 well flat-bottom plates (Falcon, Colorado Springs, CO) at a density of 500 cells/well in complete media (ScienCell Pericyte Media (PM) plus ScienCell supplements Fetal Bovine Serum, Pericyte Growth Supplement, and Penicillin-Streptomycin) at 37°C in 5% CO 2 for 24–48 hours.

Techniques: Co-culture Assay, Concentration Assay, Software

Journal: International Journal of Nanomedicine

Article Title: Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3

doi: 10.2147/IJN.S438229

Figure Lengend Snippet: Pericytes declined in age-related bone resorption and bone resorption caused by estrogen deficiency ( A ) Representative immunostaining images of femurs from OVX and female WT mice. NG2 + (red) pericytes, endomucin + (green) endothelial cell, and nuclear (blue) DAPI staining. Scale bar, 50 μm. ( B ) Quantification of NG2 + pericyte number in subepiphyseal zone of femurs from OVX and female WT mice (n = 6). ( C ) Representative TRAP staining images of femurs from OVX and female WT mice. Scale bar, 100 μm (lower panels); 500 μm (upper panels). ( D ) Quantification of osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone perimeter (Oc.N/B.Pm) in subepiphyseal zone of femurs from OVX and female WT mice (n = 6). ( E ) Analysis of the Pearson correlation between NG2 + cell number and Oc.N/B.Pm in femurs from female wild-type mice of different ages (6, 12 and 18 months). Data are presented as the mean ± S.E.M. P- values < 0.05 were considered statistically significant, as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: The pericytes were cultured as previously described using Pericyte Medium (#1201; PM, ScienCell, Carlsbad, CA, USA) supplemented with 1% Pericyte Growth Supplement (#1252; PGS, ScienCell), 1% penicillin-streptomycin (#15140122; PS, Gibco), and 2% fetal bovine serum (#10270106; FBS, Gibco).

Techniques: Immunostaining, Staining

Journal: International Journal of Nanomedicine

Article Title: Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3

doi: 10.2147/IJN.S438229

Figure Lengend Snippet: Genetic ablation of pericytes led to reduced bone mass ( A ) Schematic of NG2-tk mice modeling. Six-week-old female NG2-tk mice were intraperitoneally injected with GCV (50 mg/kg) once a day for 30 consecutive days in order to delete NG2 + pericytes. ( B ) Representative micro-CT images displaying the three-dimensional architectures of femurs from WT mice and NG2-tk mice (after GCV treatment). Scale bar, 1mm (left panels); 500 μm (right panels). ( C ) Micro-CT analysis of BV/TV, SMI, Tb.Th, and Tb.N in femurs of WT mice and NG2-tk mice (after GCV treatment, n = 6). ( D ) Representative H&E staining images of femurs from WT mice and NG2-tk mice (after GCV treatment). Scale bar, 500 μm. ( E ) Quantification of bone volume fraction (BV/TV) in femurs from WT mice and NG2-tk mice (after GCV treatment, n = 6). Data are presented as mean ± S.E.M. P -values < 0.05 were considered statistically significant, as follows: * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The pericytes were cultured as previously described using Pericyte Medium (#1201; PM, ScienCell, Carlsbad, CA, USA) supplemented with 1% Pericyte Growth Supplement (#1252; PGS, ScienCell), 1% penicillin-streptomycin (#15140122; PS, Gibco), and 2% fetal bovine serum (#10270106; FBS, Gibco).

Techniques: Injection, Micro-CT, Staining

Journal: International Journal of Nanomedicine

Article Title: Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3

doi: 10.2147/IJN.S438229

Figure Lengend Snippet: Genetic ablation of pericytes activated bone resorption ( A ) Representative immunostaining images of femurs from WT mice and NG2-tk mice (after GCV treatment). NG2 + (red) pericytes, endomucin + (green) endothelial cell, and nuclear (blue) DAPI staining. Scale bar, 50 μm. ( B ) Quantification of NG2 + pericyte number in subepiphyseal zone of femurs from WT mice and NG2-tk mice (after GCV treatment, n = 6). ( C ) Representative TRAP staining images of mouse femurs from WT mice and NG2-tk mice (after GCV treatment). Scale bar, 500 μm (upper panels); 100 μm (lower panels). ( D ) Quantification of osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone perimeter (Oc.N/B.Pm) in subepiphyseal zone of femurs from WT mice and NG2-tk mice (after GCV treatment, n = 6). Data are presented as mean ± S.E.M. P -levels < 0.05 were considered statistically significant, as follows: ** P < 0.01; **** P < 0.0001.

Article Snippet: The pericytes were cultured as previously described using Pericyte Medium (#1201; PM, ScienCell, Carlsbad, CA, USA) supplemented with 1% Pericyte Growth Supplement (#1252; PGS, ScienCell), 1% penicillin-streptomycin (#15140122; PS, Gibco), and 2% fetal bovine serum (#10270106; FBS, Gibco).

Techniques: Immunostaining, Staining

Journal: International Journal of Nanomedicine

Article Title: Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3

doi: 10.2147/IJN.S438229

Figure Lengend Snippet: Traf3 in PC-EVs mediates the inhibition of osteoclast differentiation by pericytes ( A ) The images of PC-EVs under TEM. Bar, 100 μm. ( B ) NTA results of PC-EVs. ( C ) The characterization of PC-EVs by Western blotting. ( D ) Representative TRAP staining images of monocytes after 5 days of osteoclast differentiation. RAW264.7 cells were induced with a medium containing RANKL (100 ng/mL) and non-contact co-cultured with PC-EVs (5–10 μg/mL). Scale bar, 100 nm. ( E ) Quantification of osteoclast number (Oc.N) and area of osteoclast (area of oc) of monocytes after 5 days of osteoclast differentiation (n = 6). ( F ) GO analysis map of total RNA sequencing of PC-EVs. ( G ) The coding gene of osteoclast inhibitory protein. Data are presented as mean ± S.E.M. P -levels < 0.05 were considered statistically significant, as follows: **** P < 0.0001.

Article Snippet: The pericytes were cultured as previously described using Pericyte Medium (#1201; PM, ScienCell, Carlsbad, CA, USA) supplemented with 1% Pericyte Growth Supplement (#1252; PGS, ScienCell), 1% penicillin-streptomycin (#15140122; PS, Gibco), and 2% fetal bovine serum (#10270106; FBS, Gibco).

Techniques: Inhibition, Western Blot, Staining, Cell Culture, RNA Sequencing

Journal: International Journal of Nanomedicine

Article Title: Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3

doi: 10.2147/IJN.S438229

Figure Lengend Snippet: Traf3 inhibits osteoclast differentiation through the non-canonical NF-κB pathway ( A ) Relative mRNA expression of Traf3 in vascular pericytes transfected with Traf3 siRNA (n = 4). ( B ) Representative blots of exosome-derived TRAF3 and CD63, and quantification of the ratio of TRAF3/CD63 (n = 3). PC-EV-(siNC), exosomes generated from pericytes treated with NC siRNA. PC-EV-(siTraf3), exosomes generated from pericyte treated with Traf3 siRNA. ( C ) Representative TRAP staining images of monocytes after 5 days of osteoclast differentiation. RAW264.7 cells were induced with a medium containing RANKL (100 ng/mL) and non-contact co-cultured with exosomes (5 μg/mL). Bar, 100μm ( D ) Quantification of osteoclast number (Oc.N) and area of osteoclast (area of oc) of monocytes after 5 days of osteoclast differentiation (n = 6). ( E ) Representative blots of TRAF3, TRAP, and CTSK of monocytes after 5 days of osteoclast differentiation. ( F ) Quantification of the ratio of TRAF3, TRAP, and CTSK/GAPDH in monocytes after 5 days of osteoclast differentiation (n = 3). ( G ) Representative blots of NIK, P100, P52, and RElB in monocytes after 5 days of osteoclast differentiation. ( H ) Quantification of the ratio of NIK, P100, P52, and RElB in monocytes after 5 days of osteoclast differentiation (n = 3). Data are presented as mean ± S.E.M. P -levels < 0.05 were considered statistically significant, as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The pericytes were cultured as previously described using Pericyte Medium (#1201; PM, ScienCell, Carlsbad, CA, USA) supplemented with 1% Pericyte Growth Supplement (#1252; PGS, ScienCell), 1% penicillin-streptomycin (#15140122; PS, Gibco), and 2% fetal bovine serum (#10270106; FBS, Gibco).

Techniques: Expressing, Transfection, Derivative Assay, Generated, Staining, Cell Culture

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