α smooth muscle actin α sma

Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature and washed with PBS. Continuously, cells were treated with PBS containing 5% normal goat or rabbit serum (Nichirei Biosciences Inc., Tokyo, Japan) and 0.1% Triton X-100 at room temperature. Then cells were fixed and stained with antibodies to Oct4 (1/200 Millipore, Billerica, MA), Tra-1-60 (1/200, Stemgent^®, Cambridge, MA), Tra-1-81 (1/200, Stemgent^®), SSEA-1 (1/100, Stemgent^®), SSEA-4 (1/100, Stemgent^®), MAP2 (1/200, Millipore), Nestin (1/200, Millipore), α-smooth muscle actin (α-SMA: pre-diluted, DAKO Cytomation, Glostrup, Denmark), α-fetoprotein (1/100, R&D Systems Minneapolis, MN), and to the SeV nucleocapsid protein (mouse monoclonal antibody, clone #2E4, 1.6 mg/mL). These primary antibodies were visualized with Alexa Fluor^® 488-conjugated goat anti-rabbit IgG, or Alexa Fluor^® 488-conjugated rabbit anti-mouse IgG, or Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/200, Invitrogen, Carlsbad, CA). Cell nuclei were stained with 4', 6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Fluorescence images were taken using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Germany). Alkaline phosphate (ALP) staining was performed using a Fast Red substrate kit (Nichirei) according to manufacturer’s instruction.

Corneal fibroblasts (n = 4) and myofibroblasts (n = 4) were seeded at a density of 3 × 10³ cells/cm² in a 96-well plate in serum-free DMEM/F-12 media (SFM) overnight. AlexaFluor 488-tagged MSC-exo (4 μg protein) was added and incubated in an IncuCyte ZOOM imaging system (Essen BioScience, Ann Arbor, MI, USA) for 120 h. At 0, 4, 8, 24, 72, and 120 h, the cells were stained with mouse monoclonal anti-CD90/Thy-1 (BD Biosciences, Franklin Lakes, NJ, USA) and α-smooth muscle actin (α-SMA; Agilent, Santa Clara, CA, USA) antibodies, respectively, followed by goat anti-mouse Red-X conjugated IgG secondary antibody (Jackson ImmunoResearch, Wet Grove, PA, USA) to contrast the internalized AlexaFluor 488-tagged MSC-exo.

Human iPSC–EC+MC were harvested from culture by dissociation with TypLE^TM Select for 5 min at 37°C. Afterward, single cells were washed twice with DPBS, and 5 × 10⁵ cells were processed as described in section “Flow Cytometry of hiPSC–CM.” Primary antibodies used were CD31 (Agilent, diluted 1:50 in DPBS), VE-cadherin (R&D Systems, diluted 1:13 in DPBS), vimentin (Abcam, diluted 1:100 in InsidePerm), or α-smooth muscle actin (α-SMA; Agilent, diluted 1:100 in InsidePerm). Secondary antibody used was anti–mouse IgG Alexa Fluor 488 (diluted 1:200 in InsidePerm). Cells were analyzed by a CyFlow^® space (Partec GmbH). Ten thousand events were analyzed per sample.