Generation of Directly Reprogrammed Human Endothelial Cells.

Direct reprogramming provides a novel breakthrough for generating functional endothelial cells (ECs) without the need for intermediate stem or progenitor states, offering a promising resource for cardiovascular research and treatment. ETV2 is a key transcription factor that has been identified as a pioneering factor for specifying endothelial lineage. Achieving precise ETV2 induction is essential for effective endothelial reprogramming, and maintaining the reprogrammed cellular phenotype relies on a specific combination of growth factors and small molecules. Thus, we hereby provide a straightforward and comprehensive protocol for generating two distinct types of reprogrammed ECs (rECs) from human dermal fibroblasts (HDFs). Early rECs demonstrate a robust neovascularization property but lack the mature EC phenotype, while late rECs exhibit phenotypical similarity to human postnatal ECs and have a neovascularization capacity similar to early rECs. Both cell types can be derived from human somatic source cells, making them suitable for personalized disease investigations, drug discovery, and disease therapy.

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

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.

Forward programming of hiPSCs via the transcription factor ETV2: rapid, reproducible, and cost-effective generation of highly enriched, functional endothelial cells.

AIMS: Endothelial cell (EC) dysfunction plays a key role in the initiation and progression of cardiovascular disease. However, studying these disorders in ECs from patients is challenging, hence the use of human induced pluripotent stem cells (hiPSCs) and their in vitro differentiation into ECs represents a very promising approach. Still, the generation of hiPSC-derived ECs (hECs) remains demanding as a cocktail of growth factors and an intermediate purification step are required for hEC enrichment. Therefore, we probed the utility of a forward programming approach using transgenic hiPSC lines. METHODS AND RESULTS: We have used the transgenic hiPSC line PGP1 ETV2 iso2 to explore the in vitro differentiation of hECs via doxycycline-dependent induction of the transcription factor ETV2 and compared these with a standard differentiation protocol for hECs using non-transgenic control hiPSCs. The transgenic hECs were highly enriched without an intermediate purification step and expressed - as non-transgenic hECs and HUVECs - characteristic EC markers. The viability and yield of transgenic hECs were strongly improved by applying EC growth medium during differentiation. This protocol was successfully applied in two more transgenic hiPSC lines yielding reproducible results with low line-to-line variability. Transgenic hECs displayed typical functional properties, such as tube formation and LDL uptake, and a more mature phenotype than non-transgenic hECs. Transgenic hiPSCs preferentially differentiated into the arterial lineage, this was further enhanced by adding a high VEGF concentration to the medium. We also demonstrate that complexing lentivirus with magnetic nanoparticles and application of a magnetic field enables efficient transduction of transgenic hECs. CONCLUSIONS: We have established a highly efficient, cost-effective, and reproducible differentiation protocol for the generation of functional hECs via forward programming. The transgenic hECs can be genetically modified and are a powerful tool for disease modelling, tissue engineering, and translational purposes.

Overexpression of ETV2 in BMSCs promoted wound healing in cutaneous wound mice by triggering the differentiation of BMSCs into endothelial cells and modulating the transformation of M1 phenotype macrophages to M2 phenotype macrophages.

This study aimed to investigate the effects of E26-transformation-specific variant-2 (ETV2) overexpression on wound healing in a cutaneous wound (CW) model and clarify associated mechanisms. pLVX-ETV2 lentivirus expressing ETV2 was constructed and infected into BMSCs to generate ETV2-overexpressed BMSCs (BMSCs+pLVX+ETV2). The RT-PCR assay was applied to amplify ETV2, VE-cadherin, vWF, ARG-1, IL-6, iNOS, TGF-ß, IL-10, TNF-α. Western blot was used to determine expression of VE-cadherin and vWF. ETV2 induced differentiation of BMSCs into ECs by increasing CDH5/CD31, triggering tube-like structures, inducing Dil-Ac-LDL positive BMSCs. ETV2 overexpression increased the gene transcription and expression of VE-cadherin and vWF (P<0.01). Transcription of M1 phenotype specific iNOS gene was lower and transcription of M2 phenotype specific ARG-1 gene was higher in the RAW264.7+BMSCs+ETV2 group compared to the RAW264.7+BMSCs+pLVX group (P<0.01). ETV2 overexpression (RAW264.7+BMSCs+ETV2) downregulated IL-6 and TNF-α, and upregulated IL-10 and TGF-ß gene transcription compared to RAW264.7+BMSCs+pLVX group (P<0.01). ETV2-overexpressed BMSCs promoted wound healing in CW mice and triggered the migration of BMSCs to the wound region and macrophage activation. ETV2-overexpressed BMSCs promoted collagen fibers and blood vessel formation in the wound region of CW mice. In conclusion, this study revealed a novel biofunction of ETV2 molecule in the wound healing process. ETV2 overexpression in BMSCs promoted wound healing in CW mice by triggering BMSCs differentiation into endothelial cells and modulating the transformation of M1 pro-inflammatory and M2 anti-inflammatory macrophages in vitro and in vivo.

SOX17/ETV2 improves the direct reprogramming of adult fibroblasts to endothelial cells.

An autologous source of vascular endothelial cells (ECs) is valuable for vascular regeneration and tissue engineering without the concern of immune rejection. The transcription factor ETS variant 2 (ETV2) has been shown to directly convert patient fibroblasts into vascular EC-like cells. However, reprogramming efficiency is low and there are limitations in EC functions, such as eNOS expression. In this study, we directly reprogram adult human dermal fibroblasts into reprogrammed ECs (rECs) by overexpressing SOX17 in conjunction with ETV2. We find several advantages to rEC generation using this approach, including improved reprogramming efficiency, increased enrichment of EC genes, formation of large blood vessels carrying blood from the host, and, most importantly, expression of eNOS in vivo. From these results, we present an improved method to reprogram adult fibroblasts into functional ECs and posit ideas for the future that could potentially further improve the reprogramming process.

ETV2 regulating PHD2-HIF-1α axis controls metabolism reprogramming promotes vascularized bone regeneration.

The synchronized development of mineralized bone and blood vessels is a fundamental requirement for successful bone tissue regeneration. Adequate energy production forms the cornerstone supporting new bone formation. ETS variant 2 (ETV2) has been identified as a transcription factor that promotes energy metabolism reprogramming and facilitates the coordination between osteogenesis and angiogenesis. In vitro molecular experiments have demonstrated that ETV2 enhances osteogenic differentiation of dental pulp stem cells (DPSCs) by regulating the ETV2- prolyl hydroxylase 2 (PHD2)- hypoxia-inducible factor-1α (HIF-1α)- vascular endothelial growth factor A (VEGFA) axis. Notably, ETV2 achieves the rapid reprogramming of energy metabolism by simultaneously accelerating mitochondrial aerobic respiration and glycolysis, thus fulfilling the energy requirements essential to expedite osteogenic differentiation. Furthermore, decreased α-ketoglutarate release from ETV2-modified DPSCs contributes to microcirculation reconstruction. Additionally, we engineered hydroxyapatite/chitosan microspheres (HA/CS MS) with biomimetic nanostructures to facilitate multiple ETV2-DPSC functions and further enhanced the osteogenic differentiation. Animal experiments have validated the synergistic effect of ETV2-modified DPSCs and HA/CS MS in promoting the critical-size bone defect regeneration. In summary, this study offers a novel treatment approach for vascularized bone tissue regeneration that relies on energy metabolism activation and the maintenance of a stable local hypoxia signaling state.

Endocardium gives rise to blood cells in zebrafish embryos.

Previous studies have suggested that the endocardium contributes to hematopoiesis in murine embryos, although definitive evidence to demonstrate the hematopoietic potential of the endocardium is still missing. Here, we use a zebrafish embryonic model to test the emergence of hematopoietic progenitors from the endocardium. By using a combination of expression analysis, time-lapse imaging, and lineage-tracing approaches, we demonstrate that myeloid cells emerge from the endocardium in zebrafish embryos. Inhibition of Etv2/Etsrp or Scl/Tal1, two known master regulators of hematopoiesis and vasculogenesis, does not affect the emergence of endocardial-derived myeloid cells, while inhibition of Hedgehog signaling results in their reduction. Single-cell RNA sequencing analysis followed by experimental validation suggests that the endocardium is the major source of neutrophilic granulocytes. These findings will promote our understanding of alternative mechanisms involved in hematopoiesis, which are likely to be conserved between zebrafish and mammalian embryos.

Single-nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium.

Cardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identify a population of cardiac lymphatic endothelial cells (LECs) that reside in the epicardium. Single-nuclei multiomic analysis of the human fetal heart reveals the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we find that VEGFC is highly expressed in arterial endothelial cells and epicardium-derived cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identify a population of cardiac lymphatic endothelial cells marked by the PROX1 and the lymphangiocrine RELN and enriched in binding motifs of erythroblast transformation specific (ETS) variant (ETV) transcription factors. We report the in vivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment.

Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial progenitor population. Accessible ETV2-binding sites are active at the Etv2 locus but not at other hematoendothelial regulator genes. Hematoendothelial commitment coincides with the activation of a small repertoire of previously accessible ETV2-binding sites at hematoendothelial regulators. Hematoendothelial differentiation accompanies activation of a large repertoire of new ETV2-binding sites and upregulation of hematopoietic and endothelial gene regulatory networks. This work distinguishes specification, commitment, and sublineage differentiation phases of ETV2-dependent transcription and suggests that the shift from ETV2 binding to ETV2-bound enhancer activation, not ETV2 binding to target enhancers, drives hematoendothelial fate commitment.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

SeATAC: a tool for exploring the chromatin landscape and the role of pioneer factors.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) reveals chromatin accessibility across the genome. Currently, no method specifically detects differential chromatin accessibility. Here, SeATAC uses a conditional variational autoencoder model to learn the latent representation of ATAC-seq V-plots and outperforms MACS2 and NucleoATAC on six separate tasks. Applying SeATAC to several pioneer factor-induced differentiation or reprogramming ATAC-seq datasets suggests that induction of these factors not only relaxes the closed chromatin but also decreases chromatin accessibility of 20% to 30% of their target sites. SeATAC is a novel tool to accurately reveal genomic regions with differential chromatin accessibility from ATAC-seq data.

The Role of SCL Isoforms in Embryonic Hematopoiesis.

Three waves of hematopoiesis occur in the mouse embryo. The primitive hematopoiesis appears as blood islands in the extra embryonic yolk sac at E7.5. The extra embryonic pro-definitive hematopoiesis launches in late E8 and the embryonic definitive one turns on at E10.5 indicated by the emergence of hemogenic endothelial cells on the inner wall of the extra embryonic arteries and the embryonic aorta. To study the roles of SCL protein isoforms in murine hematopoiesis, the SCL-large (SCL-L) isoform was selectively destroyed with the remaining SCL-small (SCL-S) isoform intact. It was demonstrated that SCL-S was specifically expressed in the hemogenic endothelial cells (HECs) and SCL-L was only detected in the dispersed cells after budding from HECs. The SCLΔ/Δ homozygous mutant embryos only survived to E10.5 with normal extra embryonic vessels and red blood cells. In wild-type mouse embryos, a layer of neatly aligned CD34+ and CD43+ cells appeared on the endothelial wall of the aorta of the E10.5 fetus. However, the cells at the same site expressed CD31 rather than CD34 and/or CD43 in the E10.5 SCLΔ/Δ embryo, indicating that only the endothelial lineage was developed. These results reveal that the SCL-S is sufficient to sustain the primitive hematopoiesis and SCL-L is necessary to launch the definitive hematopoiesis.

ETV2/ER71, the key factor leading the paths to vascular regeneration and angiogenic reprogramming.

Extensive efforts have been made to achieve vascular regeneration accompanying tissue repair for treating vascular dysfunction-associated diseases. Recent advancements in stem cell biology and cell reprogramming have opened unforeseen opportunities to promote angiogenesis in vivo and generate autologous endothelial cells (ECs) for clinical use. We have, for the first time, identified a unique endothelial-specific transcription factor, ETV2/ER71, and revealed its essential role in regulating endothelial cell generation and function, along with vascular regeneration and tissue repair. Furthermore, we and other groups have demonstrated its ability to directly reprogram terminally differentiated non-ECs into functional ECs, proposing ETV2/ER71 as an effective therapeutic target for vascular diseases. In this review, we discuss the up-to-date status of studies on ETV2/ER71, spanning from its molecular mechanism to vasculo-angiogenic role and direct cell reprogramming toward ECs. Furthermore, we discuss future directions to deploy the clinical potential of ETV2/ER71 as a novel and potent target for vascular disorders such as cardiovascular disease, neurovascular impairment and cancer.

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis.

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1.

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

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 2 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 2 important cardiac lineages.

Xenotransplantation and exotransplantation: Strategies to expand the number of donor organs.

Cardiovascular disease is common and has a high mortality. Due to the limited number of organs available for orthotopic heart transplantation, alternative therapies have received intense interest. In this commentary we contrast xenotransplantation and blastocyst complementation to produce pigs that will serve as donors for organ transplantation. These strategies hold tremendous promise and have the potential to provide an unlimited number of organs for chronic, terminal diseases.

Blastocyst complementation and interspecies chimeras in gene edited pigs.

The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.

ETV2 promotes osteogenic differentiation of human dental pulp stem cells through the ERK/MAPK and PI3K-Akt signaling pathways.

BACKGROUND: The repair of cranio-maxillofacial bone defects remains a formidable clinical challenge. The Ets variant 2 (ETV2) transcription factor, which belongs to the E26 transformation-specific (ETS) family, has been reported to play a key role in neovascularization. However, the role of ETV2 in the osteogenesis of human dental pulp stem cells (hDPSCs) remains unexplored. METHODS: Transgenic overexpression of ETV2 was achieved using a lentiviral vector, based on a Dox-inducible system. The effects of Dox-induced overexpression of ETV2 on the osteogenesis of hDPSCs were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence staining, alkaline phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. Additionally, RNA-sequencing (RNA-Seq) analysis was performed to analyze the underlying mechanisms of ETV2-induced osteogenesis. Additionally, the role of ETV2 overexpression in bone formation in vivo was validated by animal studies with a rat calvarial defect model and a nude mice model. RESULTS: Our results demonstrated that ETV2 overexpression significantly upregulated the mRNA and protein expression levels of osteogenic markers, markedly enhanced ALP activity, and promoted matrix mineralization of hDPSCs. Moreover, the results of RNA-Seq analysis and western blot showed that the ERK/MAPK and PI3K-Akt signaling pathways were activated upon transgenic overexpression of ETV2. The enhanced osteogenic differentiation of hDPSCs due to ETV2 overexpression was partially reversed by treatment with inhibitors of ERK/MAPK or PI3K-AKT signaling. Furthermore, the results of in vivo studies demonstrated that ETV2 overexpression improved bone healing in a rat calvarial defect model and increased ectopic bone formation in nude mice. CONCLUSIONS: Collectively, our results indicated that ETV2 overexpression exerted positive effects on the osteogenesis of hDPSCs, at least partially via the ERK/MAPK and PI3K/AKT signaling pathways.

The regulatory role of pioneer factors during cardiovascular lineage specification - A mini review.

Cardiovascular disease (CVD) remains the number one cause of death worldwide. Ischemic heart disease contributes to heart failure and has considerable morbidity and mortality. Therefore, alternative therapeutic strategies are urgently needed. One class of epigenetic regulators known as pioneer factors has emerged as an important tool for the development of regenerative therapies for the treatment of CVD. Pioneer factors bind closed chromatin and remodel it to drive lineage specification. Here, we review pioneer factors within the cardiovascular lineage, particularly during development and reprogramming and highlight the implications this field of research has for the future development of cardiac specific regenerative therapies.