Sashka Dimitrievska

Tissue-Engineered Vascular Grafts Created From Human Induced Pluripotent Stem Cells

The utility of human induced pluripotent stem cells (hiPSCs) to create tissue‐engineered vascular grafts was evaluated in this study. hiPSC lines were first induced into a mesenchymal lineage via a neural crest intermediate using a serum‐free, chemically defined differentiation scheme. Derived cells exhibited commonly known mesenchymal markers (CD90, CD105, and CD73 and negative marker CD45) and were shown to differentiate into several mesenchymal lineages (osteogenic, chondrogenic, and adipogenic). Functional vascular grafts were then engineered by culturing hiPSC‐derived mesenchymal progenitor cells in a pulsatile bioreactor system over 8 weeks to induce smooth muscle cell differentiation and collagenous matrix generation. Histological analyses confirmed layers of calponin‐positive smooth muscle cells in a collagen‐rich matrix. Mechanical tests revealed that grafts had an average burst pressure of 700 mmHg, which is approximately half that of native veins. Additionally, studies revealed that karyotypically normal mesenchymal stem cell clones led to generation of grafts with predicted features of engineered vascular grafts, whereas derived clones having chromosomal abnormalities generated calcified vessel constructs, possibly because of cell apoptosis during culture. Overall, these results provide significant insight into the utility of hiPS cells for vascular graft generation. They pave the way for creating personalized, patient‐specific vascular grafts for surgical applications, as well as for creating experimental models of vascular development and disease.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Improving in vivo outcomes of decellularized vascular grafts via incorporation of a novel extracellular matrix

Each year, hundreds of thousands coronary bypass procedures are performed in the US, yet there currently exists no off-the-shelf alternative to autologous vessel transplant. In the present study, we investigated the use of mouse thrombospondin-2 knockout (TSP2 KO) cells, which secrete a non-thrombogenic and pro-migratory extracellular matrix (TSP2 KO ECM), to modify small diameter vascular grafts. To accomplish this, we first optimized the incorporation of TSP2 KO ECM on decellularized rat aortas. Because MMP levels are known to be elevated in TSP2 KO cell culture, it was necessary to probe the effect of the modification process on the grafts mechanical properties. However, no differences were found in suture retention, Youngs modulus, or ultimate tensile strength between modified and unmodified grafts. Platelet studies were then performed to determine the time point at which the TSP2 KO ECM sufficiently reduced thrombogenicity. Finally, grafts modified by either TSP2 KO or WT cells or unmodified grafts, were implanted in an abdominal aortic interposition model in rats. After 4 weeks, grafts with incorporated TSP2 KO ECM showed improved endothelial and mural cell recruitment, and a decreased failure rate compared to control grafts. Therefore, our studies show that TSP2 KO ECM could enable the production of off-the-shelf vascular grafts while promoting reconstruction of native vessels.

New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx

Objective—It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators’ ability to assess the role of the glycocalyx in vascular health. Approach and Results—We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface’s functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm2 on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography–mass spectrometry analysis. Conclusions—Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Historical Perspective and Future Direction of Blood Vessel Developments

Over the past 40 years, remarkable advances have been made in our understanding of successful blood vessel regeneration, starting with the failures of early tissue-engineered vascular grafts designed using isolated components or molecules, such as collagen gels. The vascular tissue engineers are today better educated and have steered ongoing research developments toward clinical developments of more complete vascular grafts that replicate the multitude of specialized arterial aspects required for function.