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High efficient serum free differentiation of endothelial cells from human iPS cells without sorting

ABSTRACT: Introduction: Endothelial cells (ECs) form the inner lining of all blood vessels of the body, play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in-vitro investigations of ECs’ physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. Method: hiPSC are cultured under serum free conditions, and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 hours. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro), and melatonin (Mel) for 48 hours. Result: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two dimensional (2D) mono layer into three dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von-Willebrand-factor, and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). Conclusion: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at an efficiency eliminating the need for further enrichment strategies, hence providing a scalable source of hiPSC-ECs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200399 | GEO | 2022/04/08

REPOSITORIES: GEO

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Project description:Although differentiation of mice embryonic stem cells into vascular endothelial cells (ECs) gives a model for investigating molecular mechanisms of vascular development in vivo, temporal dynamics of gene expressions and chromatin modifications have not been studied until now. Here, we interrogated transcriptome and two histone modifications, H3K4me3 and H3K27me3, with a genome-wide scale during ECs differentiation and elucidated epigenetic switch peculiar to ECs. We find Gata2, Fli1, Sox7, and Sox18 are master regulators from genetic and epigenetic data, these genes were induced after Etv2 activation. These genes have specific histone modification pattern which is repressed by H3K27me3 modification at Flk-sorted mesoderm and changed to the bivalent (H3K4me3 and H3K27me3 both positive) state rapidly after vascular endothelial cells growth factor (VEGF) stimuli. Using a previously reported ECs differentiation model, we demonstrate that four transcription factors are critical for ECs specific gene expressions and efficient differentiation. Moreover, from knockdown experiments using si-RNA, we discovered these factors inhibited not only TGFÎ² signaling pathway, that is endothelial mesenchymal transition pathway, but also other near lineage commitment, including blood cells, skeletal muscle cells, vascular smooth muscle cells, and cardiomyocytes. We further identify each factor specific target genes during ECs differentiation by microarray, including both activating and repressing genes. Together, our findings from a detailed epigenetic approach provide a basic understanding temporal regulated chromatin signatures and resulting gene expression profile during ECs commitment, which is applicable to other models of differentiation and production of mature and long lasting ECs for regenerative medicine. Total 17 samples were derived from [1] ES cells, Flk-sorted mesoderm cells, and in the absense or presence of VEGF (6, 12, 24, and 48h) to determine VEGF activated genes during endothelial cells differentiation, [2] control si-RNA, si-Gata2, si-Fli1, si-Sox7, or si-Sox18 transfected cells under VEGF stimuli, [3] control si-RNA or si-Mix (si-Gata2, si-Fli1, si-Sox7, and si-Sox18) transfected cells under VEGF stimuli for the identification of each transcription factor dependent genes during endothelial cells differentiation.

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High efficient serum free differentiation of endothelial cells from human iPS cells without sorting

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