The STEMdiff Endothelial Differentiation Kit (STEMCELL Technologies) was used according to the manufacturer’s instructions for the differentiation of iPSCs into eMPCs and ECs. Initially, iPSCs were detached with StemPro Accutase (Thermo Fisher Scientific) and seeded at a density of 100,000 cells per 6-well in Essential 8 Flex medium supplemented with 10 µM ROCK inhibitor Y-27632 (STEMCELL Technologies). On day 7, differentiated ECs were subcultured at a density of 150,000 cells per 6-well. The cells were expanded in STEMdiff Endothelial Expansion Medium (STEMCELL Technologies) for four days, then split on passage #1, and cultured for an additional four days. RNA was extracted for eMPCs on day 3 of the differentiation protocol and at passage #1 for expanded differentiated ECs.
The PSC 4-Marker Immunocytochemistry Kit (Thermo Fisher Scientific) was used to analyze SSEA4, OCT4, SOX2, and TRA-1-60 expression for iPSCs. An anti-mouse Alexa Fluor 555 antibody (ab150114, 1:500, Abcam, Cambridge, UK) and an anti-rat Alexa Fluor 555 antibody (A-21434, 1:500, Thermo Fisher Scientific) were used as alternative secondary antibodies for SSEA4 and SOX2 staining. Unless otherwise specified, the Immunofluorescence Application Solutions Kit (Cell Signaling, Danvers, MA, USA) was used according to the manufacturer’s instructions for marker staining after the differentiation of iPSCs into eMPCs and ECs. For eMPCs, immunofluorescence analysis for α-SMA (ab7817, 1:100, Abcam) and Brachyury (AF2085, 1:20, Bio-Techne, Minneapolis, MN, USA) was performed. For Brachyury staining, cells were permeabilized and blocked in 1% bovine serum albumin, 0.3% Triton X-100, and 10% normal donkey serum. For ECs, endothelial marker expression at passage #1 was evaluated by the staining of CD31 (3528S, 1:800, Cell Signaling), VE-Cadherin (2500S, 1:400, Cell Signaling), and VWF (MA5-14029, 1:66, Thermo Fisher Scientific). An anti-mouse Alexa Fluor 555 antibody (ab150114, 1:200, Abcam), an anti-rabbit Alexa Fluor 555 antibody (A-21429, 1:500, Thermo Fisher Scientific), and an anti-goat Alexa Fluor 555 antibody (ab150130, 1:200, Abcam) were used as secondary antibodies. DAPI or Hoechst 33342 (Thermo Fisher Scientific) were used for nuclei staining.
The PSC 4-Marker Immunocytochemistry Kit (Thermo Fisher Scientific) was used to analyze SSEA4, OCT4, SOX2, and TRA-1-60 expression for iPSCs. An anti-mouse Alexa Fluor 555 antibody (ab150114, 1:500, Abcam, Cambridge, UK) and an anti-rat Alexa Fluor 555 antibody (A-21434, 1:500, Thermo Fisher Scientific) were used as alternative secondary antibodies for SSEA4 and SOX2 staining. Unless otherwise specified, the Immunofluorescence Application Solutions Kit (Cell Signaling, Danvers, MA, USA) was used according to the manufacturer’s instructions for marker staining after the differentiation of iPSCs into eMPCs and ECs. For eMPCs, immunofluorescence analysis for α-SMA (ab7817, 1:100, Abcam) and Brachyury (AF2085, 1:20, Bio-Techne, Minneapolis, MN, USA) was performed. For Brachyury staining, cells were permeabilized and blocked in 1% bovine serum albumin, 0.3% Triton X-100, and 10% normal donkey serum. For ECs, endothelial marker expression at passage #1 was evaluated by the staining of CD31 (3528S, 1:800, Cell Signaling), VE-Cadherin (2500S, 1:400, Cell Signaling), and VWF (MA5-14029, 1:66, Thermo Fisher Scientific). An anti-mouse Alexa Fluor 555 antibody (ab150114, 1:200, Abcam), an anti-rabbit Alexa Fluor 555 antibody (A-21429, 1:500, Thermo Fisher Scientific), and an anti-goat Alexa Fluor 555 antibody (ab150130, 1:200, Abcam) were used as secondary antibodies. DAPI or Hoechst 33342 (Thermo Fisher Scientific) were used for nuclei staining.
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