Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A) Schematic of directed differentiation protocol to generate hiEndos. (B) Phase contrast images during the indicated day of differentiation. White dashed square represents region magnified in insets. Scale bar: 50um. (C-E) RT-qPCR for the indicated primers using RNA isolated from cells on each day of differentiation, purified hiEndos at P0 and P5, and HUVECs. N=4 experimental replicates from independent wells of the same differentiation, representative results from two independent differentiations. (F) Flow cytometry of hiEndos using antibodies against the human endothelial lineage markers CD31 and CD144 (right) and isotype controls (left), N=4 biological replicates from distinct differentiations. (G) Immunostaining of hiEndos for human CD31 (green, left) and CD144 (red, right). Hoechst stains nuclei (blue). Scale bar 10um. N=3 biological replicates. (H) Capillary tube formation assay on 3D Matrigel. Scale bar 50um. N=4 biological replicates. (I-J) Human CD31 and acetylated LDL uptake in hiEndos measured by flow cytomtetry (I) or fluorescence microscopy (J). Scale bar 50um. N=8 biological replicates. (K) Schematic of experiment to test the impact of Notch (DAPT) and TGF-beta (SB431542) inhibition on hiEndo expansion (top). Quantification of CD31 and CD144 double-positive cells by flow cytometry (left) and cumulative hiEndo yield (right) at each passage grown in the indicated media conditions. N=2 experimental replicates from independent wells of the same differentiation, representative data shown from 3 independent differentiations. (L) Quantification of total hiEndo yield per input hiEndo in each media. ** p<0.01 using one-way Anova and Tukey’s multiple comparison test. (M) RT-qPCR of arterial and venous markers after serial passaging in SD medium. N=4 experimental replicates from independent wells of the same differentiation, representative results from two independent differentiations. ** p<0.01, *** p<0.001, **** p<0.0001, one-way Anova and Tukey’s multiple comparison test. Abbreviations: hiPSC (human induced pluripotent stem cell), CHIR (CHIR99021), P (passage), HUVEC (human umbilical vein endothelial cells), AcLDL (acetylated low-density lipoprotein), SB (SB431542).

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Quantitative RT-PCR, Isolation, Purification, Flow Cytometry, Immunostaining, Capillary Tube Formation Assay, Fluorescence, Microscopy, Inhibition, Comparison, Passaging

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A-C) Fold change in the expression of indicated transcripts in hiPSC-derived cells (BU3 clone) on each day of hiEndo differentiation, in purified hiEndos at P0 and P5, and in HUVECs (RT-qPCR). N=4 experimental replicates from independent wells of the same differentiation. (D) Quantitation of hiEndo differentiation efficiency on day 5 of differentiation across 12 distinct differentiations. (E) Flow cytometry of human CD31, human CD144, and isotype controls pre- and post-MACs purification of hiEndos on day 5 of the differentiation. N=1. (F) Phase contrast microscopy of BU3 hiEndos showing cobblestone morphology. Scale bar 50um. (G) Flow cytometry of human CD31, human CD44, and isotype controls in BU3 hiEndos, the plot is representative of 4 experimental replicates of independent wells from the same differentiation. (H) Quantification of the percent of CD31/CD144 double positive hiEndos by flow cytometry at each passage. N=1 differentiation (BU1). (I) Phase contrast microscopy of BU1 hiEndos at sequential passages. Scale bar 50um. Results are representative of 2 experimental replicates of independent wells from the same differentiation. (J) RT-qPCR analysis of fold change in transcript expression of markers of fibroblast and smooth muscle identity in hiEndos at sequential passages. N=2 experimental replicates of independent wells from the same differentiation. **** p<0.0001 by one-way Anova with Tukey’s multiple comparisons.

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Expressing, Derivative Assay, Purification, Quantitative RT-PCR, Quantitation Assay, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: DOT1L-AF10–mediated H3K79me3 promotes NF-κB p65–dependent inflammatory activation in endothelial cells

doi: 10.64898/2026.03.20.713137

Figure Lengend Snippet: (A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by CD144 (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.

Article Snippet: For tissue sections, permeabilization was performed using 0.5% Triton X-100, followed by blocking with 10% goat serum supplemented with anti-mouse IgG (1:1000) for 1 h. Cells and tissue sections were incubated overnight at 4°C with the following primary antibodies: DOT1L Rabbit mAb (1:500; #90878), H3K79me3 Rabbit mAb (1:500; #74073), eNOS Rabbit mAb (1:100; #32027), ICAM1 Rabbit pAb (1:500; #4915), NF-κB p65 Rabbit mAb (1:800; #8242, Cell Signaling Technology, Danvers, USA), H3K79me3 Rabbit pAb (1:50; #PA5-96121), AF10 Rabbit pAb (1:500; #BS-3696R), AF17 Rabbit pAb (1:1000; #A302-198A, Invitrogen), and CD144 Mouse mAb (1:25; #SC-9989, Santa Cruz Biotechnology, Dallas, USA).

Techniques: Immunofluorescence, Staining, Control, Expressing, Fluorescence, Marker, Software