Journal: bioRxiv

Article Title: Spatial programming of fibroblasts promotes resolution of tissue inflammation through immune cell exclusion

doi: 10.1101/2024.09.20.614064

Figure Lengend Snippet: a) Dotplot of key marker genes and GO terms in deconvoluted (BayesSpace) fibroblast-rich spots from each niche. b) Heatmap showing alignment of fibroblast clusters from scRNAseq analysis of disaggregated synovial tissue (Zhang et al. 2023) to genes associated with fibroblast-rich spots in each niche and a module of the top 40 genes defining the C11 immune cell (IC)-interacting fibroblasts and C4 endothelial cell (EC)-interacting fibroblasts (Korsunsky et al. 2022). c) Representative IF image showing perivascular localisation of fibrotic marker POSTN. d) Feature plots and spatial gene expression maps demonstrating expression of the immune cell (IC)- and endothelial cell (EC)-interacting gene modules across a synovial tissue section. E) Dotplot showing expression of gene modules of IC-interacting fibroblasts and EC-interacting fibroblasts in fibroblasts from each niche. f) Correlation between Krenn histological score and expression of the IC- and EC-interacting gene program in fibroblast-rich spots. Each dot represents a sample. P value calculated using Pearsons’s correlation in ggpubr::ggscatter. g) Representative spatial gene expression maps of 5 gene modules defined by genes upregulated in human RA fibroblasts stimulated in vitro with indicated cytokines compared to non-stimulated control RA fibroblasts (Tsuchiya et al., 2021). h) Heatmap of average scaled expression of cytokine response gene modules in fibroblast-rich spots in each niche. i) Correlation between expression of cytokine response gene modules in fibroblast-rich spots and Krenn score. Each dot represents a sample. P calculated using Pearsons’s correlation in ggpubr::ggscatter.

Article Snippet: The apical and basal surfaces of transwell filters were coated with 50 µl of Collagen Type I (50 µg/mL; CC050, Sigma-Aldrich), 50 µl of Collagen Type IV (50 µg/mL; C6745-1ml, Sigma-Aldrich) and 50 µl of Collagen Type VI (50 µg/mL; 009-001-108, Rockland) and equilibrated with Endothelial Cell Growth media (C-39210, PromoCell) for 24 hours at 37 °C.

Techniques: Marker, Expressing, In Vitro, Control

Journal: bioRxiv

Article Title: Spatial programming of fibroblasts promotes resolution of tissue inflammation through immune cell exclusion

doi: 10.1101/2024.09.20.614064

Figure Lengend Snippet: a) Representative immunofluorescence images of areas around vasculature (around n=6 CD31+ vessels from a single sample) with low or high immune infiltration in synovial tissue. Scale bar: 50 µm. b) Quantification of immunofluorescence staining intensity of immune and collagen markers. N=6 for high/low-immune perivascular, measured from the same sample. ns, p>0.05; ****, p < 0.0001 (determined by Two-Way ANOVA with Tukey’s HSD Post Hoc tests). Data is mean ± standard deviation. c) Annotation of cell types based on marker gene expression in multiplex IF staining (Leica Cell DIVE) of synovial tissue. Scale bar: 100 µm. d) Heatmap showing proximity analysis of annotated cell types in multiplex IF. e) Schematic overview of transwell migration experiment. f) Quantification of transmigrated cells from flow cytometry analysis. N=4 for each condition, across different donors. **, p<0.01; *, p<0.05 (determined by Brown-Forsythe ONE-way ANOVA test with Dunnett’s T3 multiple comparisons test). Data is mean ± SEM. g) Representative multiplex immunofluorescence image of synovial tissue analysed using GeoMx® Digital Spatial Profiler (NanoString) showing masks applied to exclude CD45+ cells and select CD90+ fibroblasts to facilitate of collection of transcripts from fibroblasts in regions of interest. Scale bars: 50 µm. Detection of CD45+ cells (red) within 30µm of the endothelial and non-endothelial cells are in circled in grey (I). Detection of CD90+ fibroblasts area selected for RNA probe assessment (greyed area) (II). h) Heatmap of expression of indicated genes across regions of interests, showing difference in fibroblast gene expression from areas of high collagen versus low collagen expression. N=18 collagen high regions and n=19 collagen low regions from seven donors. i) Correlation between expression of selected genes and number of CD45+ cells x10 /µm within each region of interest of synovial tissue samples. P value calculated using Pearson’s correlation in ggpubr.

Article Snippet: The apical and basal surfaces of transwell filters were coated with 50 µl of Collagen Type I (50 µg/mL; CC050, Sigma-Aldrich), 50 µl of Collagen Type IV (50 µg/mL; C6745-1ml, Sigma-Aldrich) and 50 µl of Collagen Type VI (50 µg/mL; 009-001-108, Rockland) and equilibrated with Endothelial Cell Growth media (C-39210, PromoCell) for 24 hours at 37 °C.

Techniques: Immunofluorescence, Staining, Standard Deviation, Marker, Expressing, Multiplex Assay, Migration, Flow Cytometry

Journal: bioRxiv

Article Title: Spatial programming of fibroblasts promotes resolution of tissue inflammation through immune cell exclusion

doi: 10.1101/2024.09.20.614064

Figure Lengend Snippet: a) Distance analysis showing expression of TGF-β fibroblast response module genes from vasculature towards the lining layer. b) Dotplot showing expression of TGF-β response module and fibrotic fibroblast marker genes in synovial organoid data from Wei et al., 2020. c) Dotplot showing expression of selected collagens in organoid data from Wei et al., 2020 (Fibroblast only: organoid containing fibroblasts only; Fibroblasts+EC: organoids containing fibroblasts and ECs; Fibroblasts+EC+DAPT: organoids containing fibroblasts and ECs treated with a NOTCH inhibitor). d) Immunofluorescence images (I) Scale bars: 20µm, (II) Scale bars: 200µm and e) quantification of POSTN, Col VI or Col I staining in organoids containing fibroblasts only or fibroblasts and ECs, either “close” or “far” in respect to ECs. N=3 for each condition. **, p<0.001; NS, p>0.05, as determined by Kruskal-Wallis test with Dunn’s post-hoc test. EC, endothelial cell.

Article Snippet: The apical and basal surfaces of transwell filters were coated with 50 µl of Collagen Type I (50 µg/mL; CC050, Sigma-Aldrich), 50 µl of Collagen Type IV (50 µg/mL; C6745-1ml, Sigma-Aldrich) and 50 µl of Collagen Type VI (50 µg/mL; 009-001-108, Rockland) and equilibrated with Endothelial Cell Growth media (C-39210, PromoCell) for 24 hours at 37 °C.

Techniques: Expressing, Marker, Immunofluorescence, Staining

Journal: JCI Insight

Article Title: HTATIP2 regulates arteriogenic activity in monocytes from patients with limb ischemia

doi: 10.1172/jci.insight.131419

Figure Lengend Snippet: ( A and B ) Representative bright-field images of tubule formation following coculture of HUVECs with proarteriogenic Mo/MΦs isolated from age-matched controls ( A ) and patients with CLTI ( B , n = 5/group). ( C and D ) The length ( C ) and area ( D ) of EC tubules formed in the coculture assay were quantified using an ImageJ macro. Fold-change in tubule expression is relative to that of assays containing HUVECs only. ( E ) Laser Doppler images of paw perfusion at days 3, 7, and 14 following induction of hindlimb ischemia in nude, athymic mice. ( F ) The ischemic limbs of mice were injected with proarteriogenic Mo/MΦs from controls (left) or patients with CLTI (right) ( n = 7/group). Perfusion ratio calculated by comparison with contralateral limb. ( G – K ) Gastrocnemius muscle from the ischemic leg was analyzed for expression of CD31 (red) and laminin (green, G ) and adductor muscle for α-SMA (red, H ) to quantify capillary/fiber ratio ( I ) and α-SMA + arteriole number ( J ) and diameter ( K ) ( n = 5–7/group). * P < 0.05. Scale bar: 10μm. ( C , D , H , J , and K ) Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 (Mann-Whitney U test). ( F ) Data were analyzed by 2-way ANOVA and post hoc Bonferroni test. *** P < 0.0001. Mo/MΦ, monocyte/macrophage; CLTI, chronic limb threatening ischemia; EC, endothelial cell; α-SMA, α-smooth muscle actin.

Article Snippet: HUVECs and EA.hy926 cells were expanded in culture using endothelial basal media supplemented with 1 μg/mL hydrocortisone, 100 μg/mL penicillin, 100 μg/mL streptomycin sulphate, 250 ng/mL amphotericin B, 10 ng/mL recombinant human EGF, 3 ng/mL bFGF, 3 μg/mL heparin, and 2% fetal calf serum (Promocell).

Techniques: Isolation, Co-culture Assay, Expressing, Injection, Comparison, MANN-WHITNEY

Journal: JCI Insight

Article Title: HTATIP2 regulates arteriogenic activity in monocytes from patients with limb ischemia

doi: 10.1172/jci.insight.131419

Figure Lengend Snippet: ( A and B ) Representative bright-field images of endothelial cells cocultured with si Control siRNA or si Htatip2 siRNA iBMMs ( n = 6/group). ( C and D ) Quantification of EA.hy926 cell tubule length ( C ) and area ( D ) following coculture. Fold-change in tubule expression is relative to that of assays containing EA.hy926 cells only. ( E ) SMC proliferation was assessed when cells were exposed for 24 hours to conditioned media from si Htatip2 - or si Control -iBMMs ( n = 9/group). ( F and G ) The potential of Htatip2-silenced ( n = 6) or si Control -iBMMs ( n = 7) to promote reperfusion in the ischemic hindlimb of C57BL/6 mice was quantified by laser Doppler imaging over 21 days. P < 0.001 by repeated-measures 2-way ANOVA and * P < 0.05, ** P < 0.01, *** P < 0.001 by post hoc Bonferroni test. ( H – L ) Histological analysis of ischemic limb muscle from si Htatip2- and si Control- iBMM–treated animals for CD31 (red) and laminin (green) staining ( H ) and α-SMA staining ( J , red) to quantify capillary/fiber ratio ( I ), arteriole count ( K ), and arteriole diameter ( L ) respectively. Scale bar: 10 μm. ( C – E , I , K , and L ) Data are presented as mean ± SEM. ( C – E ) Data were analyzed by paired t test. * P < 0.05, ** P < 0.01. ( H – L ) Data were analyzed by unpaired t test. ** P < 0.01, *** P < 0.001. ( C and D ) Data are expressed as fold-change compared with tubule formation of EA.hy926 cells alone. ( G ) Data were analyzed by 2-way ANOVA and post hoc Bonferroni test. * P < 0.05, ** P < 0.01, *** P < 0.0001. Mo/MΦ, monocyte/macrophage; iBMM, immortalized bone marrow macrophage; HTATIP2, HIV-1 Tat interactive protein-2; SMC, smooth muscle cell; α-SMA, α-smooth muscle actin.

Article Snippet: HUVECs and EA.hy926 cells were expanded in culture using endothelial basal media supplemented with 1 μg/mL hydrocortisone, 100 μg/mL penicillin, 100 μg/mL streptomycin sulphate, 250 ng/mL amphotericin B, 10 ng/mL recombinant human EGF, 3 ng/mL bFGF, 3 μg/mL heparin, and 2% fetal calf serum (Promocell).

Techniques: Control, Expressing, Imaging, Staining

Journal: ACS biomaterials science & engineering

Article Title: Integration of Primary Endocrine Cells and Supportive Cells Using Functionalized Silk Promotes the Formation of Prevascularized Islet-like Clusters.

doi: 10.1021/acsbiomaterials.9b01573

Figure Lengend Snippet: Figure 2. Morphology, viability and function of xenogenic co-clusters on selected silk matrices a) Representative micrographs of xenogeneic co-clusters (rodent isolated MIP endocrine cells and human supportive cells) after culture for 7 days on WT, RGD, FN and 2R silk matrices. The endothelial cell marker CD31 (upper row) and the mesenchymal cell marker CD44 (lower row) is stained in red. Nuclei are stained in blue (DAPI) and insulin-positive cells are seen in green (GFP). Scale bars = 100µm.

Article Snippet: Mouse dermal microvascular endothelial cells (MDMEC, Cell Biologics, USA) were cultured in complete endothelial cell media MV (PromoCell) and used at passage 3-12.

Techniques: Isolation, Marker, Staining

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Substrate stiffness influences BM‐MSC EV production and bioactivity. (a) EV production as quantified by EVs per cell from BM‐MSCs seeded on Sylgard 184 PDMS substrates with different base‐to‐crosslinker ratios. EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted ( n = 3). (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in EV treatments or growth or basal endothelial media, seeded in Matrigel‐coated wells, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. All data are representative of at least three independent experiments ( n = 3). Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: To measure in vitro angiogenesis, 48‐well plates were coated with 60 μL of growth factor reduced Matrigel (Corning; 356230) and incubated at 37°C for 30 min. P4 HUVECs were then seeded at 35,000 cells/well with either endothelial growth media (PromoCell; C‐22121) with 1% penicillin–streptomycin (positive control), endothelial basal media (negative control), or endothelial basal media (PromoCell; C‐22221) with 0.1% FBS and 1% penicillin–streptomycin with 5E9 EVs/mL.

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Softer 184:527 PDMS substrates improve the angiogenic bioactivity of BM‐MSC EVs. (a) EV production quantified as EV per cell from BM‐MSCs seeded on each substrate made with different ratios of Sylgard 184 and Sylgard 527 ( n = 2). EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted. (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in the different EV treatments or growth or basal endothelial basal media, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: To measure in vitro angiogenesis, 48‐well plates were coated with 60 μL of growth factor reduced Matrigel (Corning; 356230) and incubated at 37°C for 30 min. P4 HUVECs were then seeded at 35,000 cells/well with either endothelial growth media (PromoCell; C‐22121) with 1% penicillin–streptomycin (positive control), endothelial basal media (negative control), or endothelial basal media (PromoCell; C‐22221) with 0.1% FBS and 1% penicillin–streptomycin with 5E9 EVs/mL.

Techniques: Isolation, Microscopy