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A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation

ABSTRACT: The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263058 | GEO | 2024/07/05

REPOSITORIES: GEO

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Project description:The vascular endothelium constitutes the inner lining of the blood vessel and is directly involved in the control of blood fluidity, vascular tone, platelet aggregation, regulation of immunity, inflammation, and angiogenesis. Malfunction and injuries of the endothelium can cause cardiovascular diseases, which are the leading causes of death globally. The impairment of the endothelium is a hallmark in other diseases as well such as sepsis, stroke, tumor growth, insulin resistance, venous thrombosis, and chronic kidney failure. Generation of effective sources to replace injured endothelial cells (ECs) could have significant clinical impact and somatic cell sources like peripheral or cord blood cannot credibly supply enough endothelial cell progenitors for multitude of treatments. Pluripotent stem cells are a promising source for a reliable EC supply that have the potential to repair, reconstruct, replace damaged cells, and revascularize ischemic areas in order to restore tissue function and treat vascular diseases. We have developed methods to differentiate induced pluripotent stem cells (iPSCs) efficiently and robustly across multiple iPSC lines into non-tissue-specific pan vascular ECs (iECs) with high purity. These iECs present with canonical endothelial cell markers and exhibit measures of endothelial cell functionality with uptake of acetylated low-density lipoprotein (Dil-Ac-LDL) and tube formation. Using proteomic analysis, we revealed the iECs are more proteomically similar to established umbilical vein ECs (HUVECs) than to iPSCs. Post-translational modifications (PTMs) were most shared between HUVECs and iECs, and potential targets for increasing the proteomic similarity of iECs to HUVECs were identified. PTM analysis can be used in the future to glean additional insights and generate novel hypotheses. Here we demonstrate an efficient robust method to differentiate iPSCs into functional ECs, and for the first time provide a comprehensive protein expression profile of iECs, which indicates their similarities with a widely used immortalized HUVECs, allowing for further mechanistic studies of EC development, signaling and metabolism for future regenerative applications.

Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To understand the role of ARF6 in human ECs, first we compared GFP-AAV treated human iECs differentiated from ARF6-KO vs WT iPSCs, next we compared ARF6-GFP-AAV vs GFP-AAV treated iECs differentiated from WT iPSCs. During the ARF6-KO vs WT iEC comparison we found 983 vs 741 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins revealed mRNA processing among the most significantly affected biological processes. During the ARF6-GFP-AAV vs GFP-AAV treated WT iEC comparison we found 1106 vs 1218 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed endocytic recycling, retrograde transport (endosome to Golgi), and ER-Golgi vesicle-mediated transport among the most significantly affected biological processes. Specifically, ARF6 and its binding-protein GGA2, DNM1L and several subunits of the Conserved oligomeric Golgi complex (COG) were upregulated. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe, a major regulator of brain lipid metabolism. Proteomic analysis of BECs from Apoe deficient mice revealed a signature of accelerated aging. To explore the role of APOE in human endothelial cells, in this experiment we compared human iECs differentiated from APOE-KO and WT iPSCs and found 326 significantly altered proteins. Enrichment analyses of significantly downregulated proteins revealed vesicle-mediated transport and vesicle fusion to be among the most significantly affected biological processes. Specifically, among the 34 significantly altered vesicle-mediated transport proteins 26 were downregulated. Accordingly, we found reduced levels of endocytosis of FM1-43FX in newly formed vesicles in APOE-KO iECs.

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A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation

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