Journal: bioRxiv

Article Title: A Long-lived Avatar for Modeling Age-Related Vascular Disease

doi: 10.64898/2026.04.29.721776

Figure Lengend Snippet: A , Representative images of iPSC-ECs in low serum (3%) and treated with VEGF, SB, or VEGF+SB (VSL) at the indicated time points reveals that VSL further improves EC morphology. The above treatments were performed using EC medium (Lonza, EGM-2MV) with 3% FBS. Ctrl* = EC medium with 3% FBS; VEGF = VEGF (10 ng/mL) added to EC medium with 3% FBS; SB = SB (SB 431542, 10 µM) added to EC medium with 3% FBS; VEGF + SB = VEGF (10 ng/mL) and SB (SB 431542, 10 µM) added to EC medium with 3% FBS, referred to as VSL. (scale bar: 100 µm) B , Quantification of cell circularity from ( A ) reveals that VSL reduces cell circularity. C , Quantification of cell area from ( A ) reveals that VSL reduces cell area. D , UMAP analysis of iPSC-ECs treated with different viability factors or combinatorial treatments of viability factors at day 40 shows that VSL restores the transcriptional profile of HAECs at day 40 to that at day 0 (n=3). The prophylactic antibiotic (Lonza Walkersville MycoZap, 0.2%; AB) was used to prevent mycoplasma contamination. D0_Ctrl = iPSC-ECs at day 0; D40_Ctrl = iPSC-ECs treated with 3%FBS EC medium (Lonza, EGM-2MV) for 40 days; D40_VEGF = iPSC-ECs treated with the 3%FBS EC medium with the addition of VEGF (10 ng/mL) for 40 days; D40_AB = iPSC-ECs treated with 3%FBS EC medium + prophylactic antibiotic (Lonza Walkersville MycoZap, 0.2%; AB) for 40 days; D40_VSL = iPSC-ECs treated with VSL for 40 days; D40_VSL+cAMP = iPSC-ECs treated with VSL and cAMP (10 µM) for 40 days; D40_VSL+cGMP = iPSC-ECs treated with VSL and cGMP (10 µM) for 40 days; D40_VSL+Camp + cGMP = iPSC-ECs treated with VSL, cAMP (10 µM), and cGMP (10 µM) for 40 days; D40_VSL + cAMP + cGMP + AB = iPSC-ECs treated with VSL, cAMP (10 µM), cGMP (10 µM) and AB (Lonza Walkersville MycoZap, 0.2%;) for 40 days. E , Representative images of β-gal staining for iPSC-ECs treated with or without VSL at day 40 are shown (scale bar: 200 µm). F , Quantification of (E) shows that VSL reduces the percentage of β-gal positive cells (n-=3). Each dot represents one single field. G , The expression of sICAM-1 and sVCAM-1 in culture media from iPSC-ECs treated with VEGF alone or VSL at day 0 or day 20 detected by ELISA assay shows that VSL effectively suppresses inflammatory cytokines (n=3). D0_Ctrl = iPSC-ECs at day 0; D20_ Ctrl = iPSC-ECs treated with standard EC medium (Lonza, EGM-2MV) for 20 days; D20_VEGF = iPSC-ECs treated with standard EC medium and addition of VEGF (10 ng/mL) for 20 days; D20_VSL = iPSC-ECs treated with VSL for 20 days. Each dot represents one technical repeat. H , Heatmap analysis of EC marker genes and fibroblast marker genes expressed in iPSC-ECs treated with or without VSL at day 60 reveals that VSL preserves EC identity during long-term culture. Each group includes triplicate in this analysis. Ctrl_D60 = iPSC-ECs treated with 3%FBS EC medium (Lonza, EGM-2MV) for 60 days. VSL_D60 = iPSC-ECs treated with VSL. Data between two groups were analyzed by Student’s t-test. Data between multiple groups are analyzed by one-way ANOVA. Results are considered statistically significant with P<0.05(*), P<0.01(**), P<0.001(***), and P<0.0001(****).

Article Snippet: HAECs were purchased from ATCC (Cat# PCS-100-011). iPSC-ECs were generated using established methods in our laboratory .

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Marker

Journal: bioRxiv

Article Title: A Long-lived Avatar for Modeling Age-Related Vascular Disease

doi: 10.64898/2026.04.29.721776

Figure Lengend Snippet: A , mVSL+ maintains co-culture of human iPSC-ECs and iPSC-VSMCs for over 180 days, with endothelial cells aligning with the lumen direction throughout the 180-day culture (scale bar: 40 µm). B , Quantification of ( A ) shows that mVSL+ preserves lumen surface integrity throughout 180-day culture. C, Immunofluorescence staining of VE-Cadherin and α-SMA in the avatars at the indicated time points (scale bar: 40 µm). D , mVSL+ maintains the expression of sICAM-1 and sVCAM-1 in the vascular avatars at a relatively low level compared to day 5 throughout the 180-day culture. (n=3). E , Bio-plex analysis shows that mVSL+ maintains the vascular avatars at a low inflammatory state compared to day 5 throughout the 180-day culture . F , The duration of prior 3D models of vascular lumens using monoculture (in red) or co-culture (in blue). Each dot represents the duration noted in one paper. 17 papers are cited in total. The large red dot indicates our monoculture duration, and the large blue dot indicates our co-culture duration.

Article Snippet: HAECs were purchased from ATCC (Cat# PCS-100-011). iPSC-ECs were generated using established methods in our laboratory .

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Expressing