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RNA sequencing of hiPSC-derived endothelial cells via doxycycline-induced ETV2 transcription factor overexpression

ABSTRACT: Endothelial cells are known to express tissue-specific phenotypes to support the organ functionality due to the residing tissue microenvironment niche. The objective of this study is to compare the transcriptomic profiling of the generated endothelial cells (called iETV2-ECs) in neuro maintenance medium (NMM) with endothelial cells derived from primary human brain and other organs, as well as human induced pluripotent stem cells. The iETV2-ECs were generated from human induced pluripotent stem cells (hiPSC) that were genome engineered with a doxycycline-inducible overexpression cassette for the master regulator of endothelial differentiation (i.e. ETV2). The cells were treated with doxycycline for 8 days in the NMM supplemented with bFGF, FBS and endothelial cell growth supplements and subsequently maintained in NMM up to 21 days. Cells were harvested on day 8, 14 and 21 days for total RNA extraction, and their transcriptomic profiles were analyzed by bulk RNA sequencing technique. P ublicly-available RNA sequencing datasets of different endothelial cell types were integrated into the iETV2-EC data set for differentially expressed genes analysis. The ultimate goal of this project is to develop vascularized 3D neural model to study human brain vascularization and disease modelling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE192919 | GEO | 2022/01/06

REPOSITORIES: GEO

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

Project description: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.

2024-07-05 | GSE263058 | GEO

Gene expression analysis of inducible ES cells overexpressing Etv2 (induced for 12 hours at day 3 of differentiation)

Project description:During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages, however, the precise roles of Etv2 in yolk sac development remains unclear. We carried out a transcriptome analysis using an ES cell line that can inducibly express Etv2. Cells were induced for 12 hours at day 3 of differentiation. An ES cell line (dervied from mouse of background strain 129P2/Ola) with a doxycycline inducible expression casette of Etv2 (ER71 A2Lox.cre) was differentiated in hanging drops. At 3 days of differentiation, cells were treated with doxycycline for 12 hours, and subsequently Flk1(+) cells were sorted. Experiment was done in triplicate and untreated cells were used for control.

2012-07-03 | E-GEOD-37658 | biostudies-arrayexpress

Comparison of Flk-1+/Etv2- vs Flk-1+/Etv2+ populations

Project description:Screening for genes up in Etv2+ cells within Flk-1+ ES derived mesoderm Microarray analysis performed to screen for the candidate genes regulated by Etv2. Differentiated Flk-1+ mesoderm can be devided into Etv2+ or-. Etv2+ cells are assumed to be committed to hemato/endothelial cells. Comparison of two populations can reveal genes relevant in this commitment. Extract RNA from sorted Flk-1+/Etv2- vs Flk-1+/Etv2+ populations.Etv2-Venus KI ES cells were differentiated on OP9 for 4-5 days and Flk-1+ population was separated into Etv2-Venus+ or- cells. Total RNA was purified from each population for analysis.

2011-08-30 | E-GEOD-31743 | biostudies-arrayexpress

Profiling gene expression of ETV2-induced vascular endothelial cells (ETVECs)

Project description:ETV2 induces expression of endothelial-specific genes in primary human adult skin fibroblasts (HAFs) Human unbillical vein endothelial cells (HUVECs) are used as a control 32 days after ETV2 induction into HAFs, cells are recognized as ETVECs. ETVECs were created from three independent skin donor. Each number of ETVEC is correspond to the number of HAF.

2013-07-19 | E-GEOD-48980 | biostudies-arrayexpress

Efficient direct reprogramming of c-Kit− mature amniotic cells into endothelial cells by ETS factors and TGFβ suppression

Project description:ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (PSC-ECs). However, these PSC-ECs are unstable and often drift towards non-vascular cell fates. We show that human mid-gestation c-Kit- lineage-committed amniotic cells (ACs) can be reprogrammed into induced vascular endothelial cells (rAC-VECs). Transient ETV2 expression in ACs generated immature iVECs, while co-expression with FLI1/ERG1 endowed rAC-VECs with a vascular repertoire and morphology matching mature ECs. Brief TGFb-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and non-vascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFb-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into generic rAC-VECs with clinical-scale expansion potential. Public banking of HLA-typed rAC-VECs would establish a vascular inventory for treatment of genetically diverse disorders. Transcriptome sequencing of clonal and non-clonal rAC-VECs, HUVECs, LSECs, CD34+/Lin-, BMS

2012-10-30 | E-GEOD-40291 | biostudies-arrayexpress

Comparison of Flk-1+/Etv2- vs Flk-1+/Etv2+ populations

2011-08-31 | GSE31743 | GEO

ETV2 upregulation marks the specification of early cardiomyocytes and endothelial cells during co-differentiation

Project description:The ability to differentiate human induced pluripotent stem cells (hiPSCs) efficiently into defined cardiac lineages, such as cardiomyocytes and cardiac endothelial cells, is crucial to study human heart development and model cardiovascular diseases in vitro. The mechanisms underlying the specification of these cell types during human development are not well-understood which limits fine-tuning and broader application of cardiac model systems. Here, we used the expression of ETV2, a master regulator of hematoendothelial specification in mice, to identify functionally distinct subpopulations during the co-differentiation of endothelial cells and cardiomyocytes from hiPSCs. Targeted analysis of single-cell RNA sequencing data revealed differential ETV2 dynamics in the two lineages. A newly created fluorescent reporter line allowed us to identify early lineage-predisposed states and show that a transient ETV2-high state initiates the specification of endothelial cells. We further demonstrated, unexpectedly, that functional cardiomyocytes can originate from progenitors expressing ETV2 at a low level. Our study thus sheds light on the in vitro differentiation dynamics of two important cardiac lineages.

2023-01-20 | GSE202901 | GEO

ETV2 Upregulation Marks the Specification of Early Cardiomyocytes and Endothelial Cells During Co-differentiation

2023-01-20 | GSE157954 | GEO

ER71/ETV2-mediated direct reprogramming of human dermal fibroblasts (HDFs) into endothelial-like cells

Project description:Samples 1-6: HDFs transduced with ER71/ETV2 were sorted on KDR expression at day 7. The isolated KDR+ cells together with human umbilical venous endothelial cells (HUVECs) and untransduced HDFs were subjected to genomic gene expression profiling. A significant number of genes related with vessel development and angiogenesis was significantly upregulated in KDR+ cells, compared to control HDFs. These findings strongly argue that ER71/ETV2 directly reprograms human fibroblasts to functional endothelial-like cells, which could be useful for disease investigation as well as autologous cell therapy. Samples 7-12: HDFs transduced with ER71/ETV2 were sorted on KDR expression at day 7. The isolated KDR+ cells were further cultured up to day 93. The further cultured cells together with human umbilical venous endothelial cells (HUVECs) and untransduced HDFs were subjected to genomic gene expression profiling. A significant number of genes related with vessel development and angiogenesis was significantly upregulated in the further cultured cells, compared to control HDFs. These findings strongly argue that ER71/ETV2 directly reprograms human fibroblasts to functional endothelial-like cells, which could be useful for disease investigation as well as autologous cell therapy. Samples 1-6: HDFs transduced with ER71/ETV2 were sorted on KDR expression at day 7. The isolated KDR+ cells together with human umbilical venous endothelial cells (HUVECs) and untransduced HDFs were subjected to genomic gene expression profiling. Samples 7-12: HDFs transduced with ER71/ETV2 were sorted on KDR expression at day 7. The isolated KDR+ cells were further cultured up to day 93. The further cultured cells together with human umbilical venous endothelial cells (HUVECs) and untransduced HDFs were subjected to genomic gene expression profiling.

2013-09-12 | E-GEOD-50801 | biostudies-arrayexpress

Genome-wide analysis of ETS factor ER71/ETV2 chromatin occupancy

Project description:We discover that ER71/ETV2 initiates hemangiogenic program by activating blood and endothelial cell lineage specifying genes while enhancing FLK1 expression and expanding hemangioblast population. Furthermore, ER71/ETV2 establishes an ETS hierarchy by directly activating Ets genes in hematopoietic and endothelial cell lineage development. As such, ER71/ETV2-initiated blood and endothelial cell program is maintained by ER71/ETV2 downstream ETS factors through an ETS switching mechanism. ChIP-seq analysis of ER71 in differentiated embryonic stem cells

2015-03-30 | E-GEOD-59402 | biostudies-arrayexpress

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