Cd144

Phenotypic characterization of EOCs and ECFCs was performed by flow cytometry and compared to PBMCs and HUVECs [18 (link)]. Cells were harvested by washing cells with phenol red-free and serum-free basal RPMI 1640 medium followed by trypsinization with 0.05 % trypsin–EDTA (Gibco) for 15 min at 37 °C [38 (link)]. Cells were subsequently blocked with normal mouse serum for 15 min, washed and incubated for 30 min with PE-labeled fluorescent human monoclonal antibodies against the monocytic marker CD14 (R&D Systems, Minneapolis, MN, USA), the leukocytic marker CD45 (AbD Serotec, Oxford, UK), the progenitor marker CD34 (BD Biosciences), the angiogenic marker VEGFR2 (R&D Systems), and the endothelial markers CD31 (BD Biosciences) and CD144 (BD Biosciences). Cells were then fixed with paraformaldehyde for 30 min and analyzed on an Altra flow cytometer (Beckman Coulter, Mississauga, ON, Canada). Cells were gated by their characteristic forward and side scatter properties.

Immunohistochemistry was performed on 10-μm frozen sections of gastrocnemius muscle. After 5 minutes of fixation in 4% paraformaldehyde and rinsing in phosphate-buffered saline (PBS), immunostaining was performed as previously described.¹⁷ (link)^,29 (link) The slides were incubated overnight with vascular endothelial-cadherin (VE-cad; 1:200; CD144; BD Pharmingen; BD Biosciences, Franklin Lakes, NJ) rat anti-mouse primary antibody, followed by AlexaFluor 488 anti-rat secondary antibody, and washed and mounted in DAPI (4′,6-diamidino-2-phenylindole)-containing Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA). The sections were then scanned using an Axioscan microscope (Carl Zeiss, Oberkochen, Germany). The VE-cad–positive area of whole muscle was quantified blindly using Fiji software, version 1.53f51 (available at: http://fiji.sc/Fiji). Quantifications are expressed as a percentage of the VE-cad–positive area to the total surface area of the gastrocnemius muscle.

Cells were labeled with antibodies against cluster of differentiation (CD)31, CD146, CD144, CD90, CD45, CD73, CD105, HLA-DR, SSEA4, and TRA-1-60 (BD Biosciences) and analyzed by flow cytometry on a MoFlo Astrios system (Beckman Coulter, Fullerton, CA, USA).

For immunofluorescence staining, undifferentiated hiPSCs and hemangioblast were fixed with 4 % paraformaldehyde for 10 min and then permeabilized with 0.5 % saponin in PBS. The cells were blocked with 10 % normal goat serum (Sigma) for 30 min at room temperature. The cells were then incubated with the following primary anti-human antibodies overnight at 4 °C: OCT4 (BD Biosciences), ER-α (Santa Cruz), and CD144 (BD Biosciences). The cells were washed twice with PBS and incubated with fluorochrome-conjugated secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (Sigma) for 5 min. Fluorescent images were visualized with a fluorescence microscope (IX-71, Olympus).

Cell surface antigens of En-PSCs (passage 6) were analyzed by flow cytometer (BD Biosciences). Single-cell suspensions were harvested in 0.2% FBS/PBS. The cells were then incubated for 30 min with PE-conjugated anti-rat CD34 (BD Pharmingen, San Diego, CA, USA), CD144 (BD Pharmingen), CD56 (BD Pharmingen), CD105 (BD Pharmingen), CD13 (BD Pharmingen), HLA-DR (BD Pharmingen), FITC-conjugated anti-rat CD146 (BD Pharmingen), CD73 (BD Pharmingen), CD90 (BD Pharmingen), CD44 (BD Pharmingen), and CD45 (BD Pharmingen) at 4 °C for 30 min. The cells were then analyzed via flow cytometer (Becton Dickinson, USA).

Harvested mouse lungs were immediately minced and incubated with Type 1 Collagenase (Thermo Fisher Scientific) for 45 minutes. The solution was then triturated using 12 cm cannulas, filtered at 70 μm, and centrifuged at 300g for 8 minutes at 4°C. Cell pellets were then resuspended in fresh M199 buffer and selected for using DynaBeads (Invitrogen) conjugated to CD31 (Santa Cruz Biotechnology Inc., clone JC70) and CD144 (BD Biosciences, clone 55-7H1) for 4 hours. Beads with bound lung ECs were then plated on collagen-coated tissue culture dishes and grown for at least 3 days prior to further experimentation.