Bfgf2

Fabry disease (FD) fibroblasts were individually biopsied from four patients under the approval of the AMC Institutional Review Board (2011-0451). Studies using human materials including iPSCs were performed under the approval of the Institutional Review Board of KAIST (KH2016-52). Fibroblasts were cultured in DMEM (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin (Invitrogen) at 37°C, 5% CO₂ in air. Retroviruses expressing OCT4, SOX2, cMYC, and KLF4 were transfected into FD fibroblasts to generate iPSCs as previously described [23] (link). Wild type (WT)-iPSCs derived from foreskin fibroblasts (CRL-2097, ATCC, Manassas, VA) were used as a control. iPSCs were cultured in human embryonic stem cell culture medium containing DMEM/F12 (Invitrogen, Carlsbad, CA), 20% KnockOut™ Serum Replacement (Invitrogen), supplemented with 10 ng/mL bFGF2 (R&D systems, Minneapolis, MN), 1% non-essential amino acid (NEAA) (Invitrogen), 1% penicillin and streptomycin (Invitrogen) on mitomycin C-treated MEF-seeded culture plates at 37°C, 5% CO₂ in air. After detachment with dispase (Invitrogen), human iPSCs were passaged to fresh plates every 6 d. The GLA mutations were identified from genomic DNAs extracted from FD-iPSCs and fibroblasts by direct sequencing. The primers used for detecting the mutations are listed in Table S1.

Protein expression/levels for key vasculogenic factors including vascular endothelial growth factor A (VEGF-A), VEGF-D, and basic fibroblast growth factor (bFGF) was quantified via enzyme-linked immunosorbent assay (ELISA) using the following commercially available kits as per the manufacturer’s instructions: VEGF (reference no. DY493, R&D Systems), VEGF-D (reference no. DY469, R&D Systems), and bFGF2 (ref. no. DY3139-05, R&D Systems). Sham- and EFF-nanotransfected pMEFs and corresponding exosomes were processed to isolate total protein using a lysis buffer containing radioimmunoprecipitation assay buffer (reference no. 89900, Thermo Fisher Scientific), cOmplete Protease Inhibitor Cocktail (reference no. 4693116001, Roche), phosphatase inhibitor cocktail 2 (reference no. P5726-1, Sigma-Aldrich), and 1 mM phenylmethylsulfonyl fluoride. All samples were incubated in lysis buffer for 15 min at 4°C and overnight at −20°C; the samples were subsequently gently vortexed for 15 min and centrifuged at 10,000 rpm and 4°C for 30 min before protein isolation. Protein concentration for all samples was determined via a standard Bradford protein assay (reference no. 500006, Bio-Rad) following the vendor’s protocol, and these values were used to normalize protein loading for each ELISA assays.