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THREE-DIMENSIONAL SCAFFOLDS FOR EFFICIENT ARTERIAL ENDOTHELIAL CELL DIFFERENTIATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS
2291 JACC April 5, 2016 Volume 67, Issue 13
Vascular Medicine THREE-DIMENSIONAL SCAFFOLDS FOR EFFICIENT ARTERIAL ENDOTHELIAL CELL DIFFERENTIATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS Poster Contributions Poster Area, South Hall A1 Saturday, April 02, 2016, 3:45 p.m.-4:30 p.m. Session Title: Vascular Medicine Basic Biology Abstract Category: 42. Vascular Medicine: Basic Presentation Number: 1148-214 Authors: Lei Ye, Shi-Hua Tan, Li-Ping Su, Stuart Cook, National Heart Centre Singapore, Singapore, Singapore
Background: We developed a protocol that incorporated a three-dimensional (3D) fibrin scaffold for endothelial cell (ECs) differentiation from human induced pluripotent stem cell (hiPSCs). We aim to 1) understand the underlying mechanism of 3D scaffolds for hiPSCs differentiation into ECs (hiPSC-ECs) and 2) by modifying this protocol for more efficient hiPSC-ECs differentiation.
Methods and Results: A 3D scaffold for EC differentiation protocol was developed and described previously. A remarkably more efficient hiPSC-EC differentiation protocol that replaces Activin/BMP-4 with CHIR-99021 (a GSK-3 inhibitor) using 3D fibrin scaffold is developed. With this new protocol, the differentiated efficiency increased from 26.8 ±5.2% to 56±3.5% for PCBC16 cell line, and it increased from 43.8 ±0.9% to 63.3±12.2% (the highest was 77%) for GRiPSC cell line. Replacing Activin/BMP-4 with CHIR-99021 results in higher brachyury gene expression, suggesting CHIR-99021 is more efficiently in inducing hiPSCs into mesodermal lineage cells. The gene and protein expression levels of vascular endothelial cell growth factor 2 (VEGFR 2) are significantly up-regulated in hiPSCs after loaded into 3D scaffolds. The differentiation efficiency progressively reduced to 25.6%, 14%, and 5.4% for PCBC16 cells, and to 18.8%, 11.8%, and 7.1% for GRiPSC cells, when ZM 323881, a VEGFR-2 inhibitor, is added into medium at different differentiation stages. Differentiated hiPSC-ECs are arterial ECs, which express Nocth-4 and EphrinB2 genes and proteins. They have typical EC phenotypes: significantly up-regulated gene and protein expression levels of CD31, CD144 and von Willebrand factor-8. They have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Conclusions: These data demonstrate that 3D scaffolds up-regulate VEGFR2 and may modulate hiPSC-EC differentiation through VEGFR2 signalling pathway. Combining CHIR-99021 (a GSK-3 inhibitor) with 3D fibrin scaffolds efficiently generate functional arterial ECs from hiPSCs.
Vascular Medicine THREE-DIMENSIONAL SCAFFOLDS FOR EFFICIENT ARTERIAL ENDOTHELIAL CELL DIFFERENTIATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS Poster Contributions Poster Area, South Hall A1 Saturday, April 02, 2016, 3:45 p.m.-4:30 p.m. Session Title: Vascular Medicine Basic Biology Abstract Category: 42. Vascular Medicine: Basic Presentation Number: 1148-214 Authors: Lei Ye, Shi-Hua Tan, Li-Ping Su, Stuart Cook, National Heart Centre Singapore, Singapore, Singapore
Background: We developed a protocol that incorporated a three-dimensional (3D) fibrin scaffold for endothelial cell (ECs) differentiation from human induced pluripotent stem cell (hiPSCs). We aim to 1) understand the underlying mechanism of 3D scaffolds for hiPSCs differentiation into ECs (hiPSC-ECs) and 2) by modifying this protocol for more efficient hiPSC-ECs differentiation.
Methods and Results: A 3D scaffold for EC differentiation protocol was developed and described previously. A remarkably more efficient hiPSC-EC differentiation protocol that replaces Activin/BMP-4 with CHIR-99021 (a GSK-3 inhibitor) using 3D fibrin scaffold is developed. With this new protocol, the differentiated efficiency increased from 26.8 ±5.2% to 56±3.5% for PCBC16 cell line, and it increased from 43.8 ±0.9% to 63.3±12.2% (the highest was 77%) for GRiPSC cell line. Replacing Activin/BMP-4 with CHIR-99021 results in higher brachyury gene expression, suggesting CHIR-99021 is more efficiently in inducing hiPSCs into mesodermal lineage cells. The gene and protein expression levels of vascular endothelial cell growth factor 2 (VEGFR 2) are significantly up-regulated in hiPSCs after loaded into 3D scaffolds. The differentiation efficiency progressively reduced to 25.6%, 14%, and 5.4% for PCBC16 cells, and to 18.8%, 11.8%, and 7.1% for GRiPSC cells, when ZM 323881, a VEGFR-2 inhibitor, is added into medium at different differentiation stages. Differentiated hiPSC-ECs are arterial ECs, which express Nocth-4 and EphrinB2 genes and proteins. They have typical EC phenotypes: significantly up-regulated gene and protein expression levels of CD31, CD144 and von Willebrand factor-8. They have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Conclusions: These data demonstrate that 3D scaffolds up-regulate VEGFR2 and may modulate hiPSC-EC differentiation through VEGFR2 signalling pathway. Combining CHIR-99021 (a GSK-3 inhibitor) with 3D fibrin scaffolds efficiently generate functional arterial ECs from hiPSCs.