Reprogramming of human PBMCs into iPSCs for the In-vitro manufacture of engineered vascular grafts based on biodegradable synthetic polymers

Abstract

Cardiovascular diseases (CVD) remains to be the leading cause of morbidity and mortality worldwide. Replacement of affected vascular tissues has been widely used to treat CVD such as coronary heart disease, aortic aneurysm and peripheral vascular disease. However, successful treatment of CVD is often limited by the lack of suitable autologous replacement tissue. Therefore, tissue engineering (TE) represents a promising solution to replace diseased vessels. TE aims at the development of constructs that integrate with the patient’s native tissue to restore physiologic function. The success of any TE approach is dependent on three main factors: (i) the cell source, (ii) the scaffold matrix, and (iii) the ambient biochemical and physical factors. During the last years several different starter materials and cell sources have been investigated. On the one hand, biodegradable scaffold matrixes form the basis of any in vitro tissue engineering approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until the scaffold is replaced by neo-tissue. The present study systematically compares three frequently used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA) under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymer structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGApoly- 4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly–caprolactone (PCL) patches. Later, these patches were analyzed qualitatively and quantitatively. We found that PGA-P4HB and PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds. Polymer remnants were visualized by polarization microscopy. In addition, biomechanical properties of the tissue engineered patches were determined in comparison to native tissue. This study may allow future studies to specifically select certain polymer starter matrices aiming at specific tissue properties of the bioengineered constructs in vitro. On the other hand, an ideal cell source for human therapeutic and disease modeling applications should be easily accessible and possess unlimited differentiation and expansion potential. Human induced pluripotent stem cells (hiPSCs) derived from peripheral blood mononuclear cells (PBMCs) represent a promising source given their ease of harvest combined with their pluripotent nature. Therefore, hiPSCs were generated based on PBMCs and differentiated into smooth muscle cells (SMCs) as well as endothelial cells (ECs). These cells were seeded onto PGA-P4HB starter matrices and cultured under static or dynamic conditions to induce tissue formation in vitro. Resulting tissue-engineered vascular grafts (TEVGs) showed abundant amounts of extracellular matrix, containing an αSMApositive layer in the interstitium and a thin luminal layer of vWF-positive cells approximating native vessels. These results pave the way for developing autologous PBMC-derived hiPSC-based vascular constructs for therapeutic applications or disease modelling.