Journal: Journal of Advanced Research

Article Title: MTHFR variant links homocysteine metabolism and endothelial cell dysfunction by targeting mitophagy in human thoracic aortic dissection patient induced pluripotent stem cell (iPSC) models

doi: 10.1016/j.jare.2025.02.032

Figure Lengend Snippet: Differentiation and characterization of iPSC-derived human endothelial cells with MTHFR rs1801133-TT variant. a. Schematic scheme of the direct differentiation of endothelial cells from human iPSCs. b. Flow cytometry analysis of the endothelial cell markers (CD144 and CD31) in iPSC-ECs on Day 6 (before MACS sorting), IgG served as the isotype control. c. Representative ridgeline plots of CD144 positive population in cells at Day 10 (post MACS sorting) assayed with flow cytometry, IgG served as the isotype control. d. Representative immunofluorescence staining images of CD31 in human endothelial cells. Nuclei were counterstained with Hoechst 33342. Scale bar = 50 μm. e. Analysis of the LDL uptake in derived endothelial cells. The acetylated low density lipoproteins were marked in red, and the nuclei were stained with Hoechst 33,342 in cyan. Scale bar = 50 μm. f. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis on the mRNA expressions of arterial transcripts ( CXCR4 , EFNB2 , and NRP1 ) and venous transcripts ( EPHB4 , NR2F2 , and NRP2 ). Human umbilical vein endothelial cells (HUVEC) group was served as venous control. Data were presented as the mean ± s.e.m. (n = 3) and analyzed analyzed by using unpaired two-tailed Student’s t test (MTHFR-iPSC-ECs and Isogenic-iPSC-ECs) or one-way ANOVA followed by Brown-Forsythe test (multiple groups). g. Nitric oxide analysis of endothelial cells in response to acetylcholine (5 and 10 μM). For each group, n = 5. 0.1 % DMSO was a negative control. h. Quantitative real-time polymerase chain reaction (qRT-PCR) assay of cell adhesion molecules ( ICAM1 , VCAM1 and SELE ) post 2 ng/mL TNFα stimulation. Data were presented as the mean ± s.e.m. (n = 3) and analyzed analyzed by using unpaired two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 6, the cells were digested and sorted by CD144 microbeads with MACS cell separation (130–097-857, Miltenyi Biotec).

Techniques: Derivative Assay, Variant Assay, Flow Cytometry, Control, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Negative Control