primary human dermal lymphatic endothelial cells lecs  (PromoCell)

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: Mixed junctional state in capillary LV. (A and B) Whole mounts prepared from the upper 200 µm of the human dermis showing PDPN + LVs and the distribution of the junctional protein VE-cadherin (VE-cad). Representative images from n = 8 donors are shown. (A) Blind-ended lymphatic capillaries with oak leaf–shaped LECs (depicted by an asterisk) or more elongated LECs (depicted by an arrow) were observed in human dermis. (B) Images of LVs with oak leaf–shaped LECs joined by button junctions (depicted by an asterisk) or LVs with more elongated, zipper-like LECs (depicted by an arrow), as well as BVs joined with zipper junctions. Scale bars: 20 µm. (C) Whole mounts of human dermis (upper 200 µm) showing the presence of intracellular CCL21 in afferent LVs stained with anti-VE-cadherin or anti-PDPN antibodies. Representative images from n = 3 donors are shown. Scale bars: 20 µm. (D) GO term analysis of genes enriched in the cap2 LEC subset compared with all other LEC subsets. Selected terms for enriched GO biological and cellular processes are shown along with the −log 10 of the adjusted P value (list of marker genes for the enrichment analysis in ). The vertical line represents the adjusted P value set at an FDR of 0.05. (E) Volcano plot of DEGs between cap1 and cap2 LEC subsets. The horizontal line shows the log 2 fold change (FC) threshold set at 0.75. Vertical lines show the significance threshold for adjusted P values set at an FDR of 0.05. (F) Individual UMAP plots showing the expression of specific cap2 LEC genes, i.e., WWTR1 , ITGA9 , PTK2 , YAP1 , ITGAV , FBN1 . (G and H) Whole mounts prepared from the upper 200 µm of the human dermis showing PDPN + LVs expressing (G) the focal adhesion kinase (FAK), and (H) FAK and the chemokine CCL21. Representative images from n = 3 donors are shown (G and H). Scale bars: 20 µm. For the FAK images, a thin stack at the level of the LV was made to visualize FAK expression specifically in LECs. (I) Human dermal LECs were subjected to mechanical stretch (10% strain every 30 s for 18 h) with a bioreactor. Representative immunofluorescence images of control (static) human dermal LECs and human dermal LECs subjected to stretching are shown. VE-cadherin was used to visualize the cell boundary and subsequent elongation. Scale bar: 100 µm. (J) Aspect ratio of control LECs or stretched LECs was quantified and is represented as a box plot. Statistics were computed with the nonparametric Mann–Whitney test. Pooled data were derived from n = 3 independent replicates with 695 cells analyzed in total. ***P < 0.001. (K) RNA of control or stretched human dermal LECs was extracted, and RT-qPCR was performed. The fold change expression levels of CCL21 and LYVE1 between stretched and control samples are depicted. A fold change below one shows reduced gene expression under stretched conditions. Two independent experiments from n = 3 human dermal LEC donors, with four pooled technical replicates for each. Statistics were computed with paired Student’s t test. **P <0.01. (L) Violin plots showing differential expression of CCL21 and LYVE1 in the cap1 and cap2 clusters. Statistics: the P adjusted value is shown. ***P <0.001. FDR, false discovery rate.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: Staining, Marker, Expressing, Immunofluorescence, Control, MANN-WHITNEY, Derivative Assay, Quantitative RT-PCR, Gene Expression, Quantitative Proteomics

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: Valve population is composed of two subclusters with different gene expression. (A) Subclustering analysis of the valve cluster revealed two valve subclusters, corresponding to the LECs on the upstream sides of the valve leaflets, and LECs on the downstream sides of the valve leaflets. (B) Volcano plot of DEGs between the upstream valve LEC and downstream valve LEC clusters. The horizontal line shows the log 2 fold change (FC) threshold set at 0.75. Vertical lines show the significance threshold for adjusted P values set at an FDR of 0.05. (C) Individual UMAP plots showing the expression of DEGs in upstream valve LECs ( GJA1 , ITGA9 , NEO1 , CD24 ) and in downstream valve LECs ( GJA4 , CLDN11 , FOXC2 , ANGPT2 ). (D and E) Confocal analysis of valves present in the upper 200 µm of human dermis confirmed (D) the differential expression of FOXC2 and CD24 by LECs on different sides of the valve leaflet, and (E) the colocalization of CD24 and NEO1 by LECs on the same valve leaflet side. Representative images from n = 2–3 independent experiments (donors) are shown in D and E. Scale bars: 20 µm. (F) Schematic illustration of the structure of the valves and the positioning of the LECs within the valves, based on . (G) Schematic illustration of bulk RNA-seq of human dermal LECs subjected to laminar shear stress (4 dyn/cm 2 ), oscillatory-like shear stress (laminar shear stress at 4 dyn/cm 2 that reverses direction by 180° every 4 s), or static conditions for 48 h. The top 50 DEGs in the laminar and oscillatory conditions are shown in and . (G) Pearson correlation plot of the bulk RNA-seq described in E. (H) PCA plot of the samples described in G analyzed at three different conditions (static, oscillatory, and laminar). (I) Similarity of LEC clusters upstream and downstream of valves with the bulk RNA-seq of human dermal LECs subjected to static conditions; laminar or oscillatory flow was determined by the correlation of the top 100 DEGs of valve LEC clusters with the bulk RNA-seq dataset. FDR, false discovery rate.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: Gene Expression, Expressing, Quantitative Proteomics, RNA Sequencing, Shear

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: Bulk RNA-seq of human dermal LECs subjected to laminar or oscillatory shear stress. (A) Selected pathways from Enrichr analysis (GO terms) of DEGs upregulated in the LECs on the upstream sides of valve leaflets or upregulated in LECs on the downstream sides of valve leaflets (list of DEGs used for the enrichment analysis in ). The vertical line represents the adjusted P value set at an FDR of 0.05. (B) Flow cytometry analysis of CD31 and PDPN expression of the three human dermal LEC donors used for bulk RNA-seq. Cells were gated on single cells, followed by viable cells, and the expression histograms shown in this panel. (C) Volcano plots of significant DEGs when comparing laminar or oscillatory shear stress to static control. (D) Heatmap of expression levels of selected genes in the bulk RNA-seq study. FDR, false discovery rate.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: RNA Sequencing, Shear, Flow Cytometry, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

doi: 10.1084/jem.20242353

Figure Lengend Snippet: CD24 expression in murine and human dermal LECs in vitro and in vivo . (A) RNA expression in FPKM in three donors of human dermal LECs subjected to laminar (lam) shear stress, oscillatory (osc) shear stress, or static conditions (data from the bulk RNA-seq). (B) CD24 is not expressed at the protein level in in vitro –cultured human dermal LECs as assessed by flow cytometry. Representative flow cytometry plot of donor 431Z006.2. Donors 433Z032.3 and 439Z007.2 neither expressed CD24. (C) CD24 expression in mouse dermal imLECs. Representative flow cytometry plot of three experiments. (D and E) Whole mounts of Prox1- eGFP mouse ear showing expression of (D) CD24 in lymphatic valves visualized with Prox1 (high in valves) and (E) in combination with the cell adhesion molecule CD31. Representative images from five independent experiments. Scale bars: (D) 100 µm, (E) 25 µm. (F and G) Quantification of ITGA9 + valves was performed in 6-image Tilescans acquired in an ear skin area containing predominantly collectors (coll: CD31 + aSMA + ) or pre-collectors (precoll: CD31 + aSMA − ). (F) Representative image of a Tilescan (left) and of ITAGA9 + valves in CD31 + aSMA − pre-collecting (middle) and CD31 + aSMA + collecting vessels (right). Scale bars from left to right: 150, 100, 50 µm. (G) Quantifications of valves in the pre-collector or collector area of ear skin whole mounts from adult WT ( Cd24 +/+ ) and Cd24 −/− mice ( n = 8 WT and n = 7 Cd24 −/− ). (H and I) Lymphatic drainage assay: adult WT and Cd24 −/− mice were injected with a near-infrared dye conjugate intradermally in the ear skin. Clearance of the tracer was monitored over 24 h by IVIS imaging. (H and I) (H) Average dye clearance plots and (I) calculated half-lives in WT and Cd24 − /− mice (data from one experiment with four to five mice per group are shown). ns, not significant; FPKM, fragments per kilobase of transcript per million mapped reads.

Article Snippet: Juvenile single donor male human dermal LECs were purchased from PromoCell (C-12216, lot numbers: 431Z006.2, 439Z007.2, and 433Z032.3).

Techniques: Expressing, In Vitro, In Vivo, RNA Expression, Shear, RNA Sequencing, Cell Culture, Flow Cytometry, Injection, Imaging

Journal: The Journal of Clinical Investigation

Article Title: Organ-specific features of human kidney lymphatics are disrupted in chronic transplant rejection

doi: 10.1172/JCI168962

Figure Lengend Snippet: ( A ) Violin plots showing upregulation of IFN-inducible genes IFITM2 and IFITM3 in LECs from rejecting allografts. ( B ) UMAP of the scRNA-Seq data showing enrichment of IFN-γ within the T/NK cell cluster. ( C ) UMAP showing enrichment of an IFN-γ signature, including IFNGR1 , IFNGR2 , IFITM2 , and IFITM3 . ( D ) CellPhoneDB interaction map depicting predicted lymphatic-CD4 + T cell crosstalk in rejection. Inhibitory interactions (blue) include PVR and LGALS9; stimulatory interactions (red) are also shown. Node size reflects expression frequency; line intensity indicates interaction strength. Ligands of interest, PVR and LGALS9 , are highlighted. ( E ) Heatmap of immune checkpoint interactions between LECs and effector CD4 + T cells across disease states. Color indicates normalized CellPhoneDB interaction score. All scores were normalized for each ligand-receptor pair. ( F ) Immunofluorescence validation of PVR expression on PDPN + lymphatics (arrowhead) in rejecting kidneys ( n = 2); CD4 + T cell shown in contact (asterisk). Scale bar: 30 μm. ( G ) IFN-γ stimulation of cultured human LECs increases LGALS9 levels at 24 and 48 hours (qPCR; *** P = 0.0002, ** P = 0.0093, respectively) relative to HPRT. ( H ) LGALS9 protein secretion increased at 48 and 72 hours (ELISA; *** P = 0.0002, **** P < 0.0001, respectively) after IFN-γ stimulation of cultured human LECs. qPCR and ELISA experiments were repeated 3 times, and all assays were performed in duplicate, with each dot on the graph representing the mean data obtained for each repeat.

Article Snippet: Adult human dermal LECs (PromoCell, C-12217) were cultured in MV2 medium and treated with recombinant human IFN-γ (50 ng/mL) or unstimulated control medium for 24, 48, or 72 hours.

Techniques: Expressing, Immunofluorescence, Biomarker Discovery, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Oncogenic virus hijacks SOX18 pioneer function to enhance viral persistence

doi: 10.1101/2025.06.28.662102

Figure Lengend Snippet: A-C. HeLa cells expressing SOX18wt, mutants C240X (dominant-negative transactivation deficient) or HMGdel (DNA-binding deficient), or mCherry as a control, and thereafter infected with rKSHV.219 for 72h (KSHV-HeLa). A. IF images of the SOX18wt and mutants expressing cells labeled with anti-SOX18 antibody and a schematic of the constructs. B. Immunoblotting with anti-SOX18 antibody using β-actin as a loading control for normalization. C. RT-qPCR for the indicated viral genes in KSHV-HeLa. D. LECs infected with rKSHV.219 (KLECs) for 72 hours and treated with Sm4 or DMSO control for 24h and relative mRNA measured for indicated viral transcripts. Statistical significance was determined by one-way ANOVA with Dunnett correction for multiple comparisons; ns = non-significant. E-F. Uninfected LECs and KLECs 72h p.i. treated with DMSO or Sm4 for another 72h and E) labeled with anti-ARID1A and -BRG1 antibodies, nuclei were counterstained with Hoechst (33342), scale bar is 10µm, and F) immunoblotted for the indicated proteins and quantified as in B.

Article Snippet: Primary human dermal lymphatic endothelial cells LEC (Promocell; C-12216) were maintained in Microvascular MV-2 (Promocell; C-22121) medium supplemented with 5% fetal bovine serum, basic fibroblast growth factor, insulin-like growth factor 3, epidermal growth factor, gentamicin sulfate/amphotericin, ascorbic acid, and hydrocortisone; VEGF was not added.

Techniques: Expressing, Dominant Negative Mutation, Binding Assay, Control, Infection, Labeling, Construct, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Oncogenic virus hijacks SOX18 pioneer function to enhance viral persistence

doi: 10.1101/2025.06.28.662102

Figure Lengend Snippet: A-C. A schematic of the inhibitor mode of action is shown in the top panels. CTG viability assay of uninfected LECs (LEC) or LECs infected with rKSHV.219 (KLEC) for 72h and treated with the indicated, increasing concentrations of BRG1 inhibitors A) ACBI1, B) FHT-1015 and C) PFI-3 (n=3), arrows indicate the selected concentration for following inhibitor assays. D. LECs and KLECs were treated with ACBI1, FHT-1015 and PFI-3 and treated with EdU for 4h before subjecting to EdU Click-It, imaged with Opera Phenix 20x and quantified from (n=6 independent replicates, and from each n=100 nuclei). Statistical significance was determined by one-way ANOVA with Dunnett correction for multiple comparisons, ns = non-significant. E-F. LECs infected with ΔORF50 and treated with Sm4, FHT-1015, or DMSO for 72h and processed for ATAC-seq. E. Peaks and heatmaps of the top 1000 genomic regions with reduced overall accessibility (dark blue maps) showing shared (dark blue line), unique to Sm4 (turquoise) and unique to FHT-1015 (purple) loss sites. F. Pearson’s analysis of the replicate (n = 3) samples. ns = non-significant.

Article Snippet: Primary human dermal lymphatic endothelial cells LEC (Promocell; C-12216) were maintained in Microvascular MV-2 (Promocell; C-22121) medium supplemented with 5% fetal bovine serum, basic fibroblast growth factor, insulin-like growth factor 3, epidermal growth factor, gentamicin sulfate/amphotericin, ascorbic acid, and hydrocortisone; VEGF was not added.

Techniques: Viability Assay, Infection, Concentration Assay

Journal: APL Bioengineering

Article Title: Interstitial fluid flow in an engineered human lymph node stroma model modulates T cell egress and stromal change

doi: 10.1063/5.0247363

Figure Lengend Snippet: Lymph node stroma model development. We sought to develop a model of immune cell egress from the lymph node (a). A representative immunofluorescence image of a portion of a normal human lymph node is shown. In human lymph nodes, lymphatics (PDPN+, CD31+, visualized in red and green) and fibroblastic reticular cells (PDPN+, in green) form the structure of the T cell zone (b). Schematic of an intended structure of the T cell zone model (c). LECs were seeded on the underside of a tissue culture inset. PhotoHA-collagen gels laden with FRCs are crosslinked above the LECs and then incubated for 30 min (d). After thermal cross-linking, the final gel consists of hyaluronic acid and collagen (e). With this methodology, FRCs formed networks (f) and LECs formed an intact monolayer (g). Scale bars are 50 μ m. Magnetic resonance imaging (MRI) demonstrates altered fluid transport in the presence of LN stroma (h), (i). Divergence of fluid was significantly decreased in the presence of LN stroma (j). Each data point represents a biological replicate (n = 3). Significance was determined by Students' t-test, with significant p values (<0.05) reported on the graph.

Article Snippet: Human lymph node lymphatic endothelial cells (LECs) (Sciencell) were cultured on fibronectin coated flasks in VascuLife® VEGF-Mv Endothelial Complete media, containing 5% fetal bovine serum and additional supplements as provided by the supplier (Lifeline Cell Technology).

Techniques: Immunofluorescence, Incubation, Magnetic Resonance Imaging

Journal: APL Bioengineering

Article Title: Interstitial fluid flow in an engineered human lymph node stroma model modulates T cell egress and stromal change

doi: 10.1063/5.0247363

Figure Lengend Snippet: LEC barrier integrity is decreased under IFF. Pressure heads of media are utilized to drive IFF (a). To create the highest magnitude flow rate, PE50 tubing was secured onto the tissue culture inset with no leaks (b). 3D printed lid raisers to the specifications of a 12 well plate (c) maintain sterility in the incubator. LEC proliferation is unchanged by IFF, as quantified by % of EdU+ cells (d). Representative images show LECs visualized with CD31 (gray). Nuclei are stained with DAPI (blue). Scale bars are 100 μ m (e). LEC coverage (f) and disrupted junctions (g) are quantified. Each point represents a biological replicate from an independent experiment, for n = 3. Each data point represents a biological replicate (n = 3). Significance was determined by two-way ANOVA followed by Tukey's t-test, with significant p values (<0.05) reported on each graph.

Article Snippet: Human lymph node lymphatic endothelial cells (LECs) (Sciencell) were cultured on fibronectin coated flasks in VascuLife® VEGF-Mv Endothelial Complete media, containing 5% fetal bovine serum and additional supplements as provided by the supplier (Lifeline Cell Technology).

Techniques: Sterility, Staining

Journal: APL Bioengineering

Article Title: Interstitial fluid flow in an engineered human lymph node stroma model modulates T cell egress and stromal change

doi: 10.1063/5.0247363

Figure Lengend Snippet: Presence of T cells disrupts LEC junctions regardless of flow and ameliorates flow-induced changes to FRCs. LECs are visualized in representative images with CD31 in gray and DAPI in blue (scale bar 100 μ m), and FRCs are visualized with F-actin in green (scale bar 50 μ m), in the presence of naïve CD8+ T cells (blue) (a). Quantification of LEC monolayer coverage (b) and disrupted junctions (c). FRC coverage in the presence of T cells and IFF (d). Scale bar is 50 μ m. Each data point represents a biological replicate (n = 3). Significance was determined by two-way ANOVA followed by Tukey's t-test, with significant p values (<0.05) reported on each graph.

Article Snippet: Human lymph node lymphatic endothelial cells (LECs) (Sciencell) were cultured on fibronectin coated flasks in VascuLife® VEGF-Mv Endothelial Complete media, containing 5% fetal bovine serum and additional supplements as provided by the supplier (Lifeline Cell Technology).

Techniques: