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Three days of differentiation by induced ETV2 overexpression yields iETV2‐EPCs with gene expression similarity to meso‐EPCs. (A) Differentiation and expansion of iETV2‐ECs from hPSCs was carried out in a two‐stage protocol. In stage 1, hPSCs were seeded on Matrigel‐coated plates. 1 µg/mL doxycycline was supplied for three days in a differentiation medium to initiate transient expression of ETV2 to obtain iETV2‐EPCs. In stage 2, iETV2‐EPCs were seeded on collagen IV in an endothelial medium, yielding high‐purity ETV2‐ECs. (B) Flow cytometry analysis showing the time‐course co‐expression of endothelial progenitor markers CD31 and CD34 after 1 day, 2 days, or 3 days of ETV2 overexpression in mTeSR medium during Stage 1 of the differentiation. Geometric means of CD31 at these time points were quantified. N = 3 independent differentiations for each condition quantified. **: p < 0.01; ****: p < 0.0001 in One‐way ANOVA followed by Tukey's test. (C) Immunofluorescent images showing CD31, CD144, and ZO‐1 expression in Day 3 cells differentiated from IMR90‐4 iETV2 iPSCs. Scale bars are 50 μm. (D) PACNet analysis of transcriptome profiles of iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, and meso‐EPCs. EPC populations were MACS‐purified based on CD31 expression before <t>sequencing.</t> (E) Heatmap indicating the expression of a panel of pluripotent stem cell markers ( NANOG, POU5F1, PROM1, SOX2, SOX3 ) and a panel of endothelial markers ( ANGPT2, CD34, CDH5, ESM1, FLT1, FLT4, KDR, PECAM1, TEK, TIE1 ) in iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, meso‐EPCs, HUVECs, primary lymphatic ECs and primary microvascular ECs. HUVEC data is from Grath and Dai . Primary lymphatic and microvascular EC data is from Lim et al. . (F) Principal component analysis of gene expression profiles of IMR90‐4 iPSCs, iETV2‐EPCs, meso‐EPCs, and a variety of cultured EC cell lines, including human aortic EC, lymphatic EC, microvascular EC, and HUVEC.
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Three days of differentiation by induced ETV2 overexpression yields iETV2‐EPCs with gene expression similarity to meso‐EPCs. (A) Differentiation and expansion of iETV2‐ECs from hPSCs was carried out in a two‐stage protocol. In stage 1, hPSCs were seeded on Matrigel‐coated plates. 1 µg/mL doxycycline was supplied for three days in a differentiation medium to initiate transient expression of ETV2 to obtain iETV2‐EPCs. In stage 2, iETV2‐EPCs were seeded on collagen IV in an endothelial medium, yielding high‐purity ETV2‐ECs. (B) Flow cytometry analysis showing the time‐course co‐expression of endothelial progenitor markers CD31 and CD34 after 1 day, 2 days, or 3 days of ETV2 overexpression in mTeSR medium during Stage 1 of the differentiation. Geometric means of CD31 at these time points were quantified. N = 3 independent differentiations for each condition quantified. **: p < 0.01; ****: p < 0.0001 in One‐way ANOVA followed by Tukey's test. (C) Immunofluorescent images showing CD31, CD144, and ZO‐1 expression in Day 3 cells differentiated from IMR90‐4 iETV2 iPSCs. Scale bars are 50 μm. (D) PACNet analysis of transcriptome profiles of iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, and meso‐EPCs. EPC populations were MACS‐purified based on CD31 expression before <t>sequencing.</t> (E) Heatmap indicating the expression of a panel of pluripotent stem cell markers ( NANOG, POU5F1, PROM1, SOX2, SOX3 ) and a panel of endothelial markers ( ANGPT2, CD34, CDH5, ESM1, FLT1, FLT4, KDR, PECAM1, TEK, TIE1 ) in iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, meso‐EPCs, HUVECs, primary lymphatic ECs and primary microvascular ECs. HUVEC data is from Grath and Dai . Primary lymphatic and microvascular EC data is from Lim et al. . (F) Principal component analysis of gene expression profiles of IMR90‐4 iPSCs, iETV2‐EPCs, meso‐EPCs, and a variety of cultured EC cell lines, including human aortic EC, lymphatic EC, microvascular EC, and HUVEC.
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Three days of differentiation by induced ETV2 overexpression yields iETV2‐EPCs with gene expression similarity to meso‐EPCs. (A) Differentiation and expansion of iETV2‐ECs from hPSCs was carried out in a two‐stage protocol. In stage 1, hPSCs were seeded on Matrigel‐coated plates. 1 µg/mL doxycycline was supplied for three days in a differentiation medium to initiate transient expression of ETV2 to obtain iETV2‐EPCs. In stage 2, iETV2‐EPCs were seeded on collagen IV in an endothelial medium, yielding high‐purity ETV2‐ECs. (B) Flow cytometry analysis showing the time‐course co‐expression of endothelial progenitor markers CD31 and CD34 after 1 day, 2 days, or 3 days of ETV2 overexpression in mTeSR medium during Stage 1 of the differentiation. Geometric means of CD31 at these time points were quantified. N = 3 independent differentiations for each condition quantified. **: p < 0.01; ****: p < 0.0001 in One‐way ANOVA followed by Tukey's test. (C) Immunofluorescent images showing CD31, CD144, and ZO‐1 expression in Day 3 cells differentiated from IMR90‐4 iETV2 iPSCs. Scale bars are 50 μm. (D) PACNet analysis of transcriptome profiles of iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, and meso‐EPCs. EPC populations were MACS‐purified based on CD31 expression before sequencing. (E) Heatmap indicating the expression of a panel of pluripotent stem cell markers ( NANOG, POU5F1, PROM1, SOX2, SOX3 ) and a panel of endothelial markers ( ANGPT2, CD34, CDH5, ESM1, FLT1, FLT4, KDR, PECAM1, TEK, TIE1 ) in iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, meso‐EPCs, HUVECs, primary lymphatic ECs and primary microvascular ECs. HUVEC data is from Grath and Dai . Primary lymphatic and microvascular EC data is from Lim et al. . (F) Principal component analysis of gene expression profiles of IMR90‐4 iPSCs, iETV2‐EPCs, meso‐EPCs, and a variety of cultured EC cell lines, including human aortic EC, lymphatic EC, microvascular EC, and HUVEC.

Journal: Biotechnology and Bioengineering

Article Title: ETV2 Overexpression Promotes Efficient Differentiation of Pluripotent Stem Cells to Endothelial Cells

doi: 10.1002/bit.28979

Figure Lengend Snippet: Three days of differentiation by induced ETV2 overexpression yields iETV2‐EPCs with gene expression similarity to meso‐EPCs. (A) Differentiation and expansion of iETV2‐ECs from hPSCs was carried out in a two‐stage protocol. In stage 1, hPSCs were seeded on Matrigel‐coated plates. 1 µg/mL doxycycline was supplied for three days in a differentiation medium to initiate transient expression of ETV2 to obtain iETV2‐EPCs. In stage 2, iETV2‐EPCs were seeded on collagen IV in an endothelial medium, yielding high‐purity ETV2‐ECs. (B) Flow cytometry analysis showing the time‐course co‐expression of endothelial progenitor markers CD31 and CD34 after 1 day, 2 days, or 3 days of ETV2 overexpression in mTeSR medium during Stage 1 of the differentiation. Geometric means of CD31 at these time points were quantified. N = 3 independent differentiations for each condition quantified. **: p < 0.01; ****: p < 0.0001 in One‐way ANOVA followed by Tukey's test. (C) Immunofluorescent images showing CD31, CD144, and ZO‐1 expression in Day 3 cells differentiated from IMR90‐4 iETV2 iPSCs. Scale bars are 50 μm. (D) PACNet analysis of transcriptome profiles of iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, and meso‐EPCs. EPC populations were MACS‐purified based on CD31 expression before sequencing. (E) Heatmap indicating the expression of a panel of pluripotent stem cell markers ( NANOG, POU5F1, PROM1, SOX2, SOX3 ) and a panel of endothelial markers ( ANGPT2, CD34, CDH5, ESM1, FLT1, FLT4, KDR, PECAM1, TEK, TIE1 ) in iPSC line IMR90‐4, edited iPSC line IMR90‐4 iETV2, iETV2‐EPCs, meso‐EPCs, HUVECs, primary lymphatic ECs and primary microvascular ECs. HUVEC data is from Grath and Dai . Primary lymphatic and microvascular EC data is from Lim et al. . (F) Principal component analysis of gene expression profiles of IMR90‐4 iPSCs, iETV2‐EPCs, meso‐EPCs, and a variety of cultured EC cell lines, including human aortic EC, lymphatic EC, microvascular EC, and HUVEC.

Article Snippet: PolyA mRNA enrichment and sequencing library preparation was performed by Novogene (Sacramento, CA).

Techniques: Over Expression, Gene Expression, Expressing, Flow Cytometry, Purification, Sequencing, Cell Culture