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3D culturing of human pluripotent stem cells-derived endothelial cells for vascular regeneration


Edit Gara et al., Theranostics, 2022


Atherosclerosis is one of the most common pathophysiologies of diseases with the highest premature mortality in modern society. Given an ageing population with an increasing prevalence of diabetes mellitus, hyperlipidemia and hypertension, the burden of cardiovascular disease continues to increase globally. Peripheral arterial disease is a common manifestation of systemic atherosclerosis, affecting the infrarenal abdominal aorta, iliac, and infrainguinal arteries. Medical interventions, surgical revascularization, and endovascular therapy are the treatment options tailored to keep with individual anatomy and disease characteristics. Surgical strategies include the implantation of vessels or prosthetic grafts. However, in small-diameter artery reconstruction, the patency of synthetic grafts is particularly low due to the low flow rate and compliance mismatch between host and graft. Due to the challenges in developing polymeric grafts, efforts have been made to generate chemically and biologically modified new materials to improve clinical outcomes. However, critical issues related to thrombus formation remain unresolved; therefore, there is a need for novel vascular engineering design of functional, native-like, living conduits with the favorable properties of anti-thrombogenicity, biocompliance, and biomechanical stability.
The authors propose that tissue engineering with decellularised matrices from allogeneic or xenogeneic sources may be a promising approach for treating vascular indications. It remains unclear to what extent exogenous cell delivery can contribute to endothelial regeneration. Earlier reports showed that the fabrication of small-diameter vascular grafts using hiPSC-derived mesenchymal progenitor cells or bilayer of hiPSC-derived endothelial cells and smooth muscle cells (SMC) are feasible in vitro. The authors' preferred strategy is the complete removal of cellular components of the vessel wall to develop a structurally sound three-dimensional (3D) matrix and recellularize them with endothelial cells, differentiated from human pluripotent stem cells (hPSC), such as embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC). The main drawback of exogenously administered cells has been their limited survival and rapid clearance from the transplantation site following implantation. Adding exogenous cells to decellularised vessels may carry some costs in terms of complexity, regulatory hurdles, and the potential need for immunosuppression but would also provide decisive functional benefits. Here the authors used a rat model to select and optimize a suitable cell type, and the chosen cell type was further assessed in a canine vascular tissue engineering model. They identified viable and functional vascular cells by in vivo molecular imaging of angiogenesis and cell tracking for the small and large animal models. This cellular approach may be promoted as a potential novel means of providing reparative and disease-modifying options for patients with ischemic vascular diseases.

Results from the nanoScan PET/MRI and nanoScan SPECT/CT

PET/MRI images were recorded on the nanoScan integrated PET/MRI system. Dynamic PET scanning was initiated immediately after the injection of radioactive probes and continued for 120 min. The acquisition took place in 1-5 coincidence mode with 5 ns coincidence window, 400-600 keV energy window, and 94.7 mm scan range. A 3D expectation maximization (3D EM) PET reconstruction algorithm (Nucline-TeraTomo, version: 3.00.021, Mediso, Hungary) was applied to produce PET images, including corrections for attenuation and scatter, dead time, decay, and randoms. After 8 iterations the reconstruction ended, which resulted in images with 0.1 mm voxel size and time frames of 8 × 15 min. MR scanning was performed immediately after PET scans were obtained. Supporting MR images were acquired with nanoScan PET/MRI 1T magnet with a horizontal bore magnet, using a solenoid Tx/Rx coil (diameter of 35 mm), and 450 mT/m gradients. T2-weighted two dimensional and gradient echo 3D images were acquired. The images of the two modalities were automatically fused using Fusion software (Version: 3.03.089, Mediso, Hungary) and quantitatively analyzed in operator-defined 3D Volumes of Interest (VOI) with Vivoquant software (inviCRO, Boston, Massachusetts, USA). Reconstructed multi-modal animal image volumes were analyzed in the VOIs of the identified cellularized graft volumes for all radiotracers in terms of radioactivity concentration (kBq/mm3) and in Standardised Uptake Value (whereby the radioactivity concentration is normalized by the proportion between injected radioactivity and the weight of the animal.
Figure 2. shows the results from the PET/MRI scans: (C) Magnetic resonance imaging (MRI, co-registration with (D) positron emission tomography (PET, showing (E68Ga-NOTA-RDG2 uptake in the endothelial cell-implanted animals. PET and PET/MRI images feature white crosshairs at the plug implantation and increased vascularization sites. MRI image panel is shown without the same for ease of anatomical assessment. (F) [68Ga]-NOTA-RDG2 expressed as maximum standardized uptake values (SUVmax) in donor hydrogel (solid bar) and surrounding recipient tissue are also measured (stripped bar), mean ± SEM, n = 4-6, Kruskal-Wallis nonparametric test.

To measure perfusion of implanted constructs with SPECT/CT at two weeks follow-up of transplantation, animals received 99mTc-labelled human serum albumin intravenously via the tail vein (Albumon kit for radiolabeling, Medi-Radiopharma Ltd., Érd, Hungary). The integrin expression of implanted cellular plugs was monitored using a molecular imaging probe based on RGD (Arg-Gly-Asp) loop tripeptide coupled to the metal ion chelator 1,4,7-triazacyclononane-N`,N``,N``` triacetic acid (NOTA-RGD). This αvβ3 integrin affine agent NOTA-RGD2 was a generous gift from Dr Cheng, Stanford University. The chelator moiety allowed radiolabeling the molecule with 68Ga isotope for PET imaging. The intensity of radioactivity from higher perfusion levels with radiolabeled albumin is used as a surrogate marker of increased blood flow. Combining SPECT modalities with CT and MRI enables the localization of areas with different extent of blood supply. 99mTc radiolabeling of human serum albumin in the form of a lyophilized kit formulation (Albumon kit for radiolabeling, Medi-Radiopharma Ltd., Érd, Hungary) were applied following the kit instructions for use with 99mTc eluate from a GE TechneKow 99mTc generator (General Electric, Maryland Heights, MO, USA). SPECT imaging was performed on the injected animals at a 10% wide gamma energy window of 140keV for 99mTc. Rats were fitted into a heated animal bed (Multicell, Mediso, Hungary) at 37 °C, and they were imaged using a NanoSPECT/CT. Silver Upgrade is a dedicated small animal imaging system equipped with 1.2 mm diameter multiplexed multi-pinhole collimators in a helical scan mode. 40-min scans were acquired from the tail base to the nose of the animals. SPECT projections were reconstructed using a Tera-Tomo iterative algorithm. CT scans were performed with 65 kV voltage and reconstructed with a voxel size of 60 microns using a ray-tracing algorithm of the system. The parts of the implanted plugs having contact with neighboring tissues were defined as outside parts. An important endpoint in the rat studies for optimization assessment was lower limb perfusion post cell implantation. This allowed screening of the different cell type grafts for translatable, clinically verifiable effects of perfusion increase.
Figure 3.: In vivo multimodality imaging shows increased perfusion in response to hPSC-EC. Comparison of hydrogel-based endothelial plugs on 98mTc-human serum albumin (HAS) to assess perfusion of newly formed vessels. (A) Representative CT scan to show whole animal. (B) Multimodality quantitative SPECT/CT imaging revealed increased perfusion after hiPSC-EC, (C) human cell-free Matrigel, (D) hESC-EC and (E) HUVEC plugs. White crosshairs showing the site of implants. (F) Bar graph showing SUVmax values were calculated, mean ± SEM. (G-H) Grouped bar diagrams show expression of arterial (ephrin B2, Notch1, Notch2), venous (EphB4) and common (CD31) endothelial marker genes at two weeks after subcutaneous transplantation of hESC-EC (G) and hiPSC-EC (H) in athymic rats. mRNA levels are normalized to those in pre-implanted control cells, n = 6. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA.

  • In summary, the authors' findings from in vitro, small animal and preliminary large animal data suggest that exogenous administration of hPSC-EC can be used for vascular regeneration. 
  • This preclinical study may substantiate hPSC-EC-biomatrix engineering potential in vascular therapy and promote further clinical approaches for vascular reconstruction.

Full article on thno.org

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