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.

Combined use of magnetic microbeads for endothelial cell isolation and enhanced cell engraftment in myocardial repair.

Background: The regenerative potential of the heart after injury is limited. Therefore, cell replacement strategies have been developed. However, the engraftment of transplanted cells in the myocardium is very inefficient. In addition, the use of heterogeneous cell populations precludes the reproducibility of the outcome. Methods: To address both issues, in this proof of principle study, we applied magnetic microbeads for combined isolation of eGFP+ embryonic cardiac endothelial cells (CECs) by antigen-specific magnet-associated cell sorting (MACS) and improved engraftment of these cells in myocardial infarction by magnetic fields. Results: MACS provided CECs of high purity decorated with magnetic microbeads. In vitro experiments revealed that the angiogenic potential of microbead-labeled CECs was preserved and the magnetic moment of the cells was strong enough for site-specific positioning by a magnetic field. After myocardial infarction in mice, intramyocardial CEC injection in the presence of a magnet resulted in a strong improvement of cell engraftment and eGFP+ vascular network formation in the hearts. Hemodynamic and morphometric analysis demonstrated augmented heart function and reduced infarct size only when a magnetic field was applied. Conclusion: Thus, the combined use of magnetic microbeads for cell isolation and enhanced cell engraftment in the presence of a magnetic field is a powerful approach to improve cell transplantation strategies in the heart.

Inhibition of Vascular Growth by Modulation of the Anandamide/Fatty Acid Amide Hydrolase Axis.

OBJECTIVE: Pathological angiogenesis is a hallmark of various diseases characterized by local hypoxia and inflammation. These disorders can be treated with inhibitors of angiogenesis, but current compounds display a variety of side effects and lose efficacy over time. This makes the identification of novel signaling pathways and pharmacological targets involved in angiogenesis a top priority. Approach and Results: Here, we show that inactivation of FAAH (fatty acid amide hydrolase), the enzyme responsible for degradation of the endocannabinoid anandamide, strongly impairs angiogenesis in vitro and in vivo. Both, the pharmacological FAAH inhibitor URB597 and anandamide induce downregulation of gene sets for cell cycle progression and DNA replication in endothelial cells. This is underscored by cell biological experiments, in which both compounds inhibit proliferation and migration and evoke cell cycle exit of endothelial cells. This prominent antiangiogenic effect is also of pathophysiological relevance in vivo, as laser-induced choroidal neovascularization in the eye of FAAH-/- mice is strongly reduced. CONCLUSIONS: Thus, elevation of endogenous anandamide levels by FAAH inhibition represents a novel antiangiogenic mechanism.

Local anti-angiogenic therapy by magnet-assisted downregulation of SHP2 phosphatase.

Anti-angiogenic therapies are promising options for diseases with enhanced vessel formation such as tumors or retinopathies. In most cases, a site-specific local effect on vessel growth is required, while the current focus on systemic distribution of angiogenesis inhibitors may cause severe unwanted side-effects. Therefore, in the current study we have developed an approach for the local inhibition of vascularization, using complexes of lentivirus and magnetic nanoparticles in combination with magnetic fields. Using this strategy in the murine embryonic stem cell (ESC) system, we were able to site-specifically downregulate the protein tyrosine phosphatase SHP2 by RNAi technology in areas with active vessel formation. This resulted in a reduction of vessel development, as shown by reduced vascular tube length, branching points and vascular loops. The anti-angiogenic effect could also be recapitulated in the dorsal skinfold chamber of mice in vivo. Here, site-specific downregulation of SHP2 reduced re-vascularization after wound induction. Thus, we have developed a magnet-assisted, RNAi-based strategy for the efficient local inhibition of angiogenesis in ESCs in vitro and also in vivo.

PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease.

For the monitoring of vascular growth as well as adaptive or therapeutic (re)vascularization endothelial-specific reporter mouse models are valuable tools. However, currently available mouse models have limitations, because not all endothelial cells express the reporter in all developmental stages. We have generated PECAM/eGFP embryonic stem (ES) cell and mouse lines where the reporter gene labels PECAM+ endothelial cells and vessels with high specificity. Native eGFP expression and PECAM staining were highly co-localized in vessels of various organs at embryonic stages E9.5, E15.5 and in adult mice. Expression was found in large and small arteries, capillaries and in veins but not in lymphatic vessels. Also in the bone marrow arteries and sinusoidal vessel were labeled, moreover, we could detect eGFP in some CD45+ hematopoietic cells. We also demonstrate that this labeling is very useful to monitor sprouting in an aortic ring assay as well as vascular remodeling in a murine injury model of myocardial infarction. Thus, PECAM/eGFP transgenic ES cells and mice greatly facilitate the monitoring and quantification of endothelial cells ex vivo and in vivo during development and injury.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

Improvement of vascular function by magnetic nanoparticle-assisted circumferential gene transfer into the native endothelium.

Gene therapy is a promising approach for chronic disorders that require continuous treatment such as cardiovascular disease. Overexpression of vasoprotective genes has generated encouraging results in animal models, but not in clinical trials. One major problem in humans is the delivery of sufficient amounts of genetic vectors to the endothelium which is impeded by blood flow, whereas prolonged stop-flow conditions impose the risk of ischemia. In the current study we have therefore developed a strategy for the efficient circumferential lentiviral gene transfer in the native endothelium under constant flow conditions. For that purpose we perfused vessels that were exposed to specially designed magnetic fields with complexes of lentivirus and magnetic nanoparticles thereby enabling overexpression of therapeutic genes such as endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). This treatment enhanced NO and VEGF production in the transduced endothelium and resulted in a reduction of vascular tone and increased angiogenesis. Thus, the combination of MNPs with magnetic fields is an innovative strategy for site-specific and efficient vascular gene therapy.

Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets.

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Transduction of murine embryonic stem cells by magnetic nanoparticle-assisted lentiviral gene transfer.

Genetic modification of embryonic stem (ES) cells is a valuable technique when combined with cell replacement strategies. Obtaining stable transgene expression and low-cytotoxicity lentiviral transduction of ES cells is advantageous. It has been shown that the efficiency of transfection and transduction approaches can be increased by magnetic nanoparticles (MNPs). Here, we present a protocol for MNP-assisted lentiviral transduction of adherent mouse ES cells. The application of MNPs increased transduction efficiency and provided the opportunity of cell positioning by a magnetic field.

Identification of magnetic nanoparticles for combined positioning and lentiviral transduction of endothelial cells.

PURPOSE: The combination of magnetic nanoparticles (MNPs) with a magnetic field is a powerful approach to enable cell positioning and/or local gene therapy. Because physical requirements for MNPs differ between these two applications we have explored whether the use of different MNPs can provide site-specific positioning combined with efficient viral transduction of endothelial cells (ECs). METHODS: A variety of MNPs was screened for magnetic cell labeling and lentivirus binding. Then two different MNPs were chosen and their combined application was evaluated regarding EC magnetization and transduction efficiency. RESULTS: The combined use of PEI-Mag2 and NDT-Mag1 particles provided both efficient lentiviral transduction and high magnetic responsiveness of ECs that could be even retained within the vascular wall under flow conditions. The use of these MNPs did not affect biological characteristics of ECs like surface marker expression and vascular network formation. Importantly, with this method we could achieve an efficient functional overexpression of endothelial nitric oxide synthase in ECs. CONCLUSIONS: The application of two different MNPs provides optimal results for magnetic labeling of ECs in combination with viral transduction. This novel approach could be very useful for targeted gene therapy ex vivo and site-specific cell replacement in the vascular system.