Clone wm59

For cell characterization, 1 x 10⁵ primary hiPSCs or hiPSC-derived cells were seeded in 12-well plates, grown to 50 - 70% confluence and fixed with 4% formaldehyde for 10 minutes at room temperature. Cells were washed in PBS and permeabilized in citrate buffer and blocked with 1x Dako wash buffer/10% FBS (S300685-2, Agilent). Anti-human CD90-PE (40 ng/µl, clone 5E10, BD Biosciences), anti-human CD31-PE (12.5 ng/µl, clone WM59, BD Biosciences), anti-human Cytokeratin 14 (4 ng/µl, clone LL001, Santa Cruz), anti-human Cytokeratin 5 (20 ng/µl, clone 2C2, Thermo Fischer) primary antibodies and appropriately titrated isotype controls were applied overnight at 4°C. As secondary antibody, a goat anti-mouse PE (40 ng/µl, BD Biosciences) was applied for 1 hour at room temperature. Cell nuclei were stained with 4′,6-Diamidin-2-phenylindol (DAPI, 1:1000, D1306, Molecular Probes) at room temperature for 10 minutes. In situ reporter staining during endothelial cell differentiation was performed by adding 125 ng/ml of anti-human CD31-PE (clone WM59, BD Biosciences) antibody in basal medium and incubating for 30 min at 37°C. After washing the cells with basal medium, EGM-2 / 10% hPL was added back before reporter staining was analyzed by fluorescence imaging.

Single-cell suspensions from human skin samples of adult subjects obtained as described in the section “Isolation of adult primary skin LECs and BECs from biopsies” were prepared as described above. Subsequently, isolated single cells were stained with mouse anti-human CD34 biotinylated antibody (clone 581, Thermo Fisher Scientific) diluted 5 µL for 10⁶ cells in FACS buffer (DPBS with 2% FBS and 1 mM EDTA) for 30 min at 4 °C. After washing once with FACS buffer, isolated single cells were co-stained in FACS buffer with FITC-conjugated mouse anti-human CD45 antibody (1:25; clone HI30, Biolegend), PE-conjugated mouse anti-human CD31 antibody (1:25; clone WM59, BD Pharmingen), PerCP-conjugated streptavidin (1:400; Biolegend), Zombie NIR (1:500; BioLegend) for 30 min at 4 °C. After washing in FACS buffer, isolated single cells were filtered and sorted for living, CD45-CD31 + CD34low (LEC), or CD34 high (BEC) directly into test tubes containing 250 µL RLT plus lysis buffer, using a FACSAria (BD Biosciences). RNA was isolated using the RNeasy Plus Micro kit (Qiagen), according to the manufacturer’s instructions, including DNase digestion. qPCR was performed on an HT7900 system and analyzed as described above.