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For EBs formation, ESCs were induced to the primed state in serum-medium for two days. Then, 500,000 ESCs were suspended in 5 mL KSR medium [Dulbecco’s Modified Essential Medium (DMEM) (SH30243.01; Hyclone, Logan, UT, USA) supplemented with 8% KSR (10828028; Gibco, Carlsbad, CA, USA), penicillin/streptomycin (Gibco, Carlsbad, CA, USA), and β-mercaptoethanol (Gibco, Carlsbad, CA, USA)] to allow them to form EBs. The EBs were fed every 2 days. At day 4 of EBs formation, the EBs were transferred to a 0.2% gelatin-coated culture dish containing KSR medium and allowed to attach. The following day, EBs were fed with differentiation medium [DMEM (Hyclone, Logan, UT, USA) supplemented with 15% FBS (S001-01; Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea), Minimum Essential Medium (MEM) non-essential amino acids (Gibco, Carlsbad, CA, USA), penicillin/streptomycin (Gibco, Carlsbad, CA, USA), sodium pyruvate, β-mercaptoethanol (Gibco, Carlsbad, CA, USA), and BMP4 (314-BP-010; R&D Systems, Minneapolis, MN, USA)] with 0.5 µM 4-OHTM (H6278; Sigma-Aldrich, St. Louis, MO, USA), and cultured for 7 days.

HDFs originating from neonatal skin (PCS-201-010; ATCC) were used in this study. Cells were cultured in Dulbecco’s modified Eagle’s medium [containing 25 mM glucose and supplemented with 10% fetal bovine serum (FBS; S001-01; Welgene), 100 units/ml penicillin, 10 μg/ml streptomycin, and 25 ng/ml amphotericin B (LS203-01; Welgene)] and maintained at 37 °C in 5% CO₂. Confluent cells were serially passaged at a 1:4 split ratio during low passages (up to passage 45) and a 1:3 (passages 46 and 47) or 1:2 split ratio during high passages (passages 48–50). Senescent HDF cultures were defined as populations with the following properties: >90% of the cells were positive for SA-β-gal staining, and the population doubling time (DT) of the cells was >14 days. HDFs with a DT of <1 day were defined as young cells. Cells were tested for mycoplasma contamination with a MycoAlert Mycoplasma Detection Kit (LT07-318; Lonza).

Human umbilical vein endothelial cells (HUVECs, Cat#CC-2519, Lonza), human retinal microvascular endothelial cells (HRMECs, Cat#ACBRI 181, Cell Systems) and human embryonic kidney cells 293 T (HEK293T, Cat#CRL-3216, ATCC) were authenticated according to ATCC guidelines and used within 6 months of receipt. HUVECs and HRMECs were cultured in EBM-2 (Cat#CC-3156, Lonza) supplemented with EGM-2 (Cat#CC-3162, Lonza) and 100 µg/ml penicillin/streptomycin on gelatin (Cat#G1890, Sigma-Aldrich; 0.1% in DDW) pre-coated plates. HEK293T were cultured in DMEM (Cat#LM001-5, Welgene) supplemented with 10% FBS (S001-01, Welgene) and 100 µg/ml antibiotics-antimycotics. All cells were grown at 37 °C and 5% CO₂. All experiments have been performed in accordance with the institutional guidelines.